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Aldolases catalyze the reversible reactions of aldol condensation and cleavage and have strong potential for the synthesis of chiral compounds, widely used in pharmaceuticals. Here, we investigated a new Class II metal aldolase from the p-hydroxyphenylacetate degradation pathway in Acinetobacter baumannii, 4-hydroxy-2-keto-heptane-1,7-dioate aldolase (AbHpaI), which has various properties suitable for biocatalysis, including stereoselectivity/stereospecificity, broad aldehyde utilization, thermostability, and solvent tolerance. Notably, the use of Zn2+ by AbHpaI as a native cofactor is distinct from other enzymes in this class. AbHpaI can also use other metal ion (M2+) cofactors, except Ca2+, for catalysis. We found that Zn2+ yielded the highest enzyme complex thermostability (Tm of 87 °C) and solvent tolerance. All AbHpaI•M2+ complexes demonstrated preferential cleavage of (4R)-2-keto-3-deoxy-D-galactonate ((4R)-KDGal) over (4S)-2-keto-3-deoxy-D-gluconate ((4S)-KDGlu), with AbHpaI•Zn2+ displaying the highest R/S stereoselectivity ratio (sixfold higher than other M2+ cofactors). For the aldol condensation reaction, AbHpaI•M2+ only specifically forms (4R)-KDGal and not (4S)-KDGlu and preferentially catalyzes condensation rather than cleavage by ∼40-fold. Based on 11 X-ray structures of AbHpaI complexed with M2+ and ligands at 1.85 to 2.0 Å resolution, the data clearly indicate that the M2+ cofactors form an octahedral geometry with Glu151 and Asp177, pyruvate, and water molecules. Moreover, Arg72 in the Zn2+-bound form governs the stereoselectivity/stereospecificity of AbHpaI. X-ray structures also show that Ca2+ binds at the trimer interface via interaction with Asp51. Hence, we conclude that AbHpaI•Zn2+ is distinctive from its homologues in substrate stereospecificity, preference for aldol formation over cleavage, and protein robustness, and is attractive for biocatalytic applications.
Aldolases catalyze reversible reactions of carbon–carbon bond formation (aldol condensation) and breakage (aldol cleavage). Based on their different catalytic mechanisms, aldolases can be classified into three groups, including pyridoxal 5′-phosphate (PLP)-dependent, Class I lysine-dependent, and Class II metal-dependent aldolases. PLP aldolases employ PLP as a cofactor to react with an amino-containing nucleophilic substrate to form a quasi-stable carbanion and an iminium intermediate. Class I lysine aldolases (also called Schiff base-forming aldolases) utilize an active lysine residue to form a Schiff base with an aldehyde/keto substrate to also result in an iminium intermediate. This imine intermediate is susceptible to C-C bond cleavage or formation. For Class II metal aldolases, the enzyme uses a divalent metal ion (M2+) as a cofactor for substrate binding and stabilization of an enolate intermediate (
), which allows the reaction to proceed through C-C bond formation or cleavage.
These aldolases are capable of catalyzing stereochemically-specific reactions, offering attractive and interesting routes for synthesis of rare sugars, β- and γ-hydroxy-α-amino acids, optically pure compounds, and antiviral agents to be used in pharmaceuticals (
). For PLP aldolases, two known enzymes—serine hydroxymethyltransferase (SHMT) and threonine aldolase (TA), which are capable of synthesizing nonnatural β-hydroxy-α-amino acids such as β-hydroxy-α,α-dialkyl-α-amino acids or L-threo-3,4-dihydroxyphenylserine and β-phenylserine (
), have been studied. A wide range of Class I lysine aldolases have been investigated due to their diversified reactions. For example, 2-deoxyribose 5-phosphate aldolase (DERA), N-acetylneuraminic acid aldolase (NeuA), and D-fructose 1,6-bisphosphate aldolase (FruA) have been extensively used in industrial applications to synthesize active pharmaceutical ingredients (APIs) (
). In contrast, applications of Class II metal aldolases have been much less investigated, but have recently been gaining more interest for their applications in the stereoselective synthesis of rare sugars. For example, the rare sugars L-fructose, D-sorbose, and D-psicose can be synthesized from the reaction of rhamnulose 1-phosphate aldolase (RhuA or RhaD) (
Use of a recombinant bacterial fructose-1,6-diphosphate aldolase in aldol reactions: Preparative syntheses of 1-deoxynojirimycin, 1-deoxymannojirimycin, 1,4-dideoxy-1,4-imino-D- arabinitol, and fagomine.
Reactions and properties of Class II metal aldolases are diversified as the enzymes can use a wide range of M2+ and carbonyl group substrates. Pyruvate-specific aldolases such as 4-hydroxy-2-keto-heptane-1,7-dioate aldolase (HpaI), 4-hydroxy-2-ketovalerate aldolase (BphI and DmpG) and 2-keto-3-deoxy-L-rhamnonate aldolase (YfaU) can use various octahedrally coordinated Mg2+, Co2+, or Mn2+ ions as cofactors (
). In the case of pyruvate-dependent aldolases, biophysical factors governing the ability of these enzymes to bind various types of M2+ and the mechanistic roles of these M2+ in catalysis are unclear.
The most well-studied pyruvate-specific Class II metal aldolase is HpaI (EC 220.127.116.11) found in the p-hydroxyphenylacetate (HPA) degradation pathway in Escherichia coli (EcHpaI). The enzyme catalyzes the reversible aldol cleavage of 4-hydroxy-2-keto-heptane-1,7-dioate (HKHD) to form pyruvate and succinic semialdehyde (SSA). Crystal structures, steady-state kinetics, and substrate specificity of EcHpaI indicate that the enzyme exists as a hexamer (a dimer of trimers) in which each subunit can bind to an octahedral divalent metal ion such as Mg2+, Mn2+, or Co2+ coordinated with substrates pyruvate (
). Results from quantum mechanics/molecular mechanics (QM/MM) calculations and site-directed mutagenesis studies indicate that Arg70 and His45 together with the M2+-bound apex water molecule are important for substrate specificity, C-C bond cleavage, and enolate stabilization (
). EcHpaI can also catalyze the aldol condensation of keto donors (pyruvate or 2-ketobutyrate) and various types of aldehyde acceptors of different carbon chain lengths (C2–C5) to generate the corresponding 4-hydroxy-2-ketoacids with preference toward a longer chain C5-aldehyde (pentaldehyde) rather than other short-chain aldehydes (
). Structures and catalytic properties of AbHpaI have never been investigated. In this work, we investigated the catalytic and biophysical properties of AbHpaI and found that the enzyme has biochemical and biophysical properties significantly different from EcHpaI and other enzymes in this class such as the use of Zn2+ as a cofactor. We also showed that AbHpaI can catalyze stereospecific aldol condensation to synthesize pure (4R)-2-keto-3-deoxy-D-galactonate ((4R)-KDGal) without producing the contaminating 4S-isomer, demonstrating that AbHpaI can control the stereospecificity of aldol product formation. Steady-state kinetics indicate that the turnover number of aldol condensation to synthesize (4R)-KDGal was about 35- to 40-fold faster than that the cleavage, suggesting that the aldol condensation is a more favored direction of AbHpaI catalysis. Moreover, AbHpaI is also tolerant to various solvents and highly thermostable especially in the Zn2+-bound form in which Tm is 87 °C. We solved 11 X-ray structures of AbHpaI in complex with various M2+ and substrates to elucidate the structural factors underlying the catalysis of AbHpaI. Structual analysis clearly explains why the 4R-isomer is more preferred over the 4S-isomer for cleavage and how different M2+ cofactors affect the binding features of both substrates. Arg72 is the key residue governing the stereochemistry of AbHpaI. Together, these properties, which are quite different from EcHpaI, make AbHpaI attractive as a robust biocatalyst for aldol condensation to produce the stereospecific/stereoselective 4-hydroxy-2-ketoacid synthons for further preparation of APIs.
Identification of the native metal ion cofactor for AbHpaI
We first explored the selectivity of metal ion binding in AbHpaI and identified its native cofactor. Using inductively coupled plasma–optical emission spectroscopy (ICP-OES), which can detect a wide variety of metal ions simultaneously, alkaline earth ions (Ca2+ and Mg2+) and transition metal ions (Zn2+, Mn2+, and Ni2+) were detected in the purified AbHpaI (Fig. 1A). Quantitative measurements indicated that Ca2+ ion was the most prevalent, followed by Zn2+ and Mg2+, while Mn2+ and Ni2+ were found in very low amounts (Fig. 1A). In contrast to the properties of EcHpaI, which could bind to three metal ions (Mn2+, Mg2+, Co2+) with Co2+ giving the highest activity (
As the presence of metal ions in the purified AbHpaI may not directly relate to the enzyme catalytic activity because their existence may depend on their availability in cells, we thus further investigated the effects of different metal ions on the catalysis of AbHpaI. First, the binding properties of these metal ions to apo-AbHpaI and in the presence of pyruvate substrate were determined. Apo-AbHpaI was reconstituted with each metal ion, namely Zn2+, Mn2+, Co2+, Ca2+, and Mg2+ and ability of these M2+ to bind to the apoenzyme was determined. Although Co2+ was not found in the purified AbHpaI (Fig. 1A), we included Co2+ in this study because it is a native cofactor of EcHpaI (
). The ICP-OES results (Fig. 1B) showed that the mole ratios of each of the reconstituted metal ions Zn2+, Mn2+, Co2+, Mg2+, and Ca2+ to apo-AbHpaI varied from 1.1, 0.6, 0.8, 0.6, and 0.1, respectively, suggesting that AbHpaI has different affinities and preferences toward these metal ions. Unexpectedly, Ca2+ has the lowest binding ability to AbHpaI after reconstitution, albeit Ca2+ was the most detected ion in the purified enzyme. We further investigated the binding constant (Kd) of M2+ and pyruvate in AbHpaI•M2+ and AbHpaI•M2+•pyruvate complexes using isothermal titration calorimetry (ITC). AbHpaI has three and sevenfold greater affinity for Zn2+ binding over Co2+ and Mn2+, respectively, while the Kd of Mg2+ and Ca2+ to AbHpaI could not be determined (Table 1). Together with the finding that Zn2+ has the highest mole ratios in metal ion reconstitution experiments and in the native purified enzyme, these results suggest that Zn2+ is the native cofactor for AbHpaI.
Table 1Thermodynamic and catalytic properties of AbHpaI reconstituted with different metal ion cofactors
As pyruvate alone cannot bind to apo-AbHpaI, we thus further explored the role of the five metal ions in facilitating the binding of pyruvate to AbHpaI by measuring the binding constant of pyruvate using ITC. We found that only the transition metal ions Zn2+, Co2+, and Mn2+ could support the binding of pyruvate in which AbHpaI•Co2+ has a three and fourfold higher affinity to pyruvate than AbHpaI•Zn2+ and AbHpaI•Mn2+ (Table 1). Notably, the Kd values of pyruvate binding to AbHpaI•Mg2+ and AbHpaI•Ca2+ could not be measured, indicating that pyruvate has poor affinity to these enzyme complexes. However, apparent kinetic results showed that AbHpaI•Mg2+ could catalyze the aldol condensation reaction of pyruvate and D-glyceraldehyde with 1.3-fold slower than AbHpaI•Zn2+, while AbHpaI•Ca2+ could not (Table 2). These suggest that Zn2+, Co2+, Mn2+, and Mg2+ but not Ca2+ have properties relevant to being metal ion cofactors. In addition, our work here indicates that an enzyme in the pyruvate-specific Class II metal aldolases can use Zn2+ as a catalytic cofactor.
Table 2Stereoselectivity of substrates in the aldol cleavage and stereochemistry of product formation in the condensation of AbHpaI with different metal ion cofactors
Detection of products from the aldol cleavage reactions was carried out by coupling with the reaction of lactate dehydrogenase (LDH) in buffer H containing 0.2 mM substrate ((4R)-KDGal or (4S)-KDGlu), 0.2 mM NADH, 0.5 mM metal ion, 26.2 μg/ml LDH, and 100 μM metal ion-reconstituted AbHpaI. The NADH absorbance decrease at 340 nm refers to the cleavage of the substrate to yield pyruvate for LDH reaction for 3 min.
The aldol condensation reactions were carried out for 1 h in buffer H containing 4 mM pyruvate, 30 mM D-glyceraldehyde, 0.1 mM metal ion, 0.05 μM metal ion-reconstituted AbHpaI. Product was analyzed by Agilent 6470 triple-quadrupole LC/MS.
a Detection of products from the aldol cleavage reactions was carried out by coupling with the reaction of lactate dehydrogenase (LDH) in buffer H containing 0.2 mM substrate ((4R)-KDGal or (4S)-KDGlu), 0.2 mM NADH, 0.5 mM metal ion, 26.2 μg/ml LDH, and 100 μM metal ion-reconstituted AbHpaI. The NADH absorbance decrease at 340 nm refers to the cleavage of the substrate to yield pyruvate for LDH reaction for 3 min.
b The aldol condensation reactions were carried out for 1 h in buffer H containing 4 mM pyruvate, 30 mM D-glyceraldehyde, 0.1 mM metal ion, 0.05 μM metal ion-reconstituted AbHpaI. Product was analyzed by Agilent 6470 triple-quadrupole LC/MS.
c None, no reaction occurred under this condition.
Stereochemistry of the catalytic reaction of AbHpaI
Stereoselectivity of the AbHpaI aldol cleavage
To investigate the influence of metal ions on the stereoselectivity of the substrate stereoisomer for aldol cleavage, AbHpaI•M2+ complexes of Zn2+, Co2+, Mn2+, Mg2+, and Ca2+, prepared by equilibrating the apo-AbHpaI with excess M2+, were employed for catalyzing the aldol cleavage of substrates, namely (4R)-KDGal and (4S)-KDGlu (see details of chemical structures in Fig. S2). (4R)-KDGal and (4S)-KDGlu were chosen as model substrates because these compounds are only different in stereo-isoforms (R and S) at the cleavage site of the C4-hydroxyl (C4-OH) group. The reaction rate of substrate cleavage was measured by coupling with the reaction of AbHpaI•M2+ with lactate dehydrogenase (LDH) to detect NADH oxidation upon pyruvate formation.
Results in Table 2 clearly showed that the apo-AbHpaI and AbHpaI•Ca2+ cannot catalyze reactions of both isomers. For other AbHpaI•M2+ complexes tested, they could catalyze aldol cleavage of both 4R- and 4S-isomers with higher activities toward the cleavage of (4R)-KDGal rather than (4S)-KDGlu (Table 2). The AbHpaI enzymes containing Zn2+, Co2+, or Mn2+ cleaved 80 to 90% (4R)-KDGal within 3 min with AbHpaI•Co2+ showing the fastest activity (∼90% (4R)-KDGal consumed within 1 min) to get pyruvate and D-glyceraldehyde, while only 50% of the (4R)-KDGal was utilized by AbHpaI•Mg2+ (Fig. 2A). These results indicate that AbHpaI with all metal ions has stereoselective preference toward (4R)-KDGal over (4S)-KDGlu.
Regarding the overall yield of (4S)-KDGlu cleavage (Fig. 2B), 15 to 20% (4S)-KDGlu could be cleaved by AbHpaI•Co2+ and AbHpaI•Mn2+, while only 1% cleavage could be catalyzed by AbHpaI•Zn2+. These results illustrate an interesting property of AbHpaI•Zn2+ as a biocatalyst, because this form of enzyme exhibits strong stereoselectivity toward the 4R-isomer. Altogether, the data obtained from both (4R)-KDGal and (4S)-KDGlu cleavage reactions confirmed that AbHpaI•M2+ prefers to cleave the 4R-isomer over 4S and the enzyme stereoselectivity is metal-dependent, with AbHpaI•Zn2+ exhibiting the highest R/S stereoselectivity ratio, ∼5- to 9-fold higher than the Co2+-, Mn2+-, and Mg2+-bound AbHpaI enzymes (Table 2).
Stereospecificity of AbHpaI aldol condensation
To explore whether AbHpaI•M2+ enzymes have stereoselectivity toward the synthesis of the 4R-isomer in the aldol condensation reaction similar to the aldol cleavage reaction, we examined the product stereoisomer resulting from the condensation of pyruvate and D-glyceraldehyde catalyzed by AbHpaI•M2+ enzymes. In order to differentiate between the two stereoisomeric compounds, (4R)-KDGal and (4S)-KDGlu, which have the same molecular mass of 178.0 g/mol, we used a high-sensitivity LC/MS system (Hi-Plex H cation-exchange column and triple quadrupole MS in a negative mode), which can distinguish between the two compounds (Fig. S3). Peak area of the product at m/z 177.0 ([M-H]−) was used for calculating the reaction yield.
The results obtained from aldol condensation of pyruvate (4 mM) and D-glyceraldehyde (30 mM) showed that only (4R)-KDGal was produced by all AbHpaI•M2+ complexes of Zn2+, Co2+, Mn2+, and Mg2+, while the reaction of AbHpaI•Ca2+ could not produce any product even after 60 min (Fig. 2C and Table 2). This was therefore confirmed that Ca2+ cannot promote pyruvate binding, consistent with the ITC binding result of pyruvate and AbHpaI•Ca2+. Similar to the cleavage rates, the Co2+ containing enzyme catalyzes formation of (4R)-KDGal ≥ 2-fold faster than the Mn2+, Zn2+, and Mg2+-bound enzymes (Table 2). It should be noted that ∼0.1% (4S)-KDGlu formation could also be detected after 30 h (Fig. S4). The results clearly showed that AbHpaI•M2+ prefers catalyzing stereoselective synthesis of the 4R-isomer, especially for the Zn2+-containing enzyme. Taken together, these findings show that AbHpaI can practically catalyze stereospecific product formation.
Steady-state kinetics of AbHpaI•Zn2+
To understand the kinetics properties of the enzyme, steady-state kinetic parameters for aldol cleavage and condensation of AbHpaI•Zn2+ were determined as listed in Table 3 and Fig. S5. Results indicate that the Michaelis–Menten constant (Km) of (4R)-KDGal cleavage was half of that for (4S)-KDGlu, suggesting that AbHpaI•Zn2+ requires lower concentrations of (4R)-KDGal to reach the maximum velocity of the reaction. The kcat value suggests that AbHpaI•Zn2+ catalyzes the cleavage of (4R)-KDGal 18-fold faster than that of (4S)-KDGlu. In addition, the catalytic constant (kcat/Km) of (4R)-KDGal cleavage was about 36-fold greater than that of (4S)-KDGlu, indicating that AbHpaI•Zn2+ catalyzes the aldol cleavage of (4R)-KDGal more efficiently than that of (4S)-KDGlu. These results agreed well with the activities measured in Table 2 in that AbHpaI•Zn2+ is highly stereoselective toward 4R-isomer.
Table 3Steady-state kinetics for aldol cleavage and condensation reactions of AbHpaI•Zn2+
Kinetics of the cleavage reaction was investigated in buffer H containing 0.1 mM Zn2+, 0.05 to 2 mM (4R)-KDGal and 0.1 to 3.2 mM (4S)-KDGlu, and 5 or 40 μM AbHpaI•Zn2+ (for (4R)-KDGal and (4S)-KDGlu, respectively). The amount of substrate was measured by RapidFire high-throughput mass spectrometry coupled with triple-quadrupole mass spectrometer.
The reactions were carried out in 0 to 8 mM pyruvate with a fixed concentration of 30 mM D-glyceraldehyde. As pyruvate concentrations greater than 4 mM showed inhibition, the kinetics data at >4 mM pyruvate were not included in the analysis.
Due to pyruvate inhibition at >4 mM, the reactions were carried out in 0 to 36 mM D-glyceraldehyde at 4 mM pyruvate.
122.9 ± 17.6
10.6 ± 3.6
11.7 ± 4.3
a Kinetics of the cleavage reaction was investigated in buffer H containing 0.1 mM Zn2+, 0.05 to 2 mM (4R)-KDGal and 0.1 to 3.2 mM (4S)-KDGlu, and 5 or 40 μM AbHpaI•Zn2+ (for (4R)-KDGal and (4S)-KDGlu, respectively). The amount of substrate was measured by RapidFire high-throughput mass spectrometry coupled with triple-quadrupole mass spectrometer.
b The condensation kinetics were analyzed by monitoring product formation using LC coupled with triple-quadrupole mass spectrometry.
c The reactions were carried out in 0 to 8 mM pyruvate with a fixed concentration of 30 mM D-glyceraldehyde. As pyruvate concentrations greater than 4 mM showed inhibition, the kinetics data at >4 mM pyruvate were not included in the analysis.
d Due to pyruvate inhibition at >4 mM, the reactions were carried out in 0 to 36 mM D-glyceraldehyde at 4 mM pyruvate.
For the aldol condensation kinetics, only the kinetics of (4R)-KDGal synthesis was investigated because (4S)-KDGlu could not be detected (Table 2). The results in Table 3 showed that the turnover number of (4R)-KDGal synthesis was ∼100 min−1 and the Km value of pyruvate was approximately sevenfold lower than that of D-glyceraldehyde, indicative for higher affinity of pyruvate to the enzyme. Together, the kinetics results from both aldol cleavage and condensation reactions firmly support that AbHpaI is stereoselective for the 4R-isomer. Further comparing the kinetics of (4R)-KDGal cleavage versus synthesis, it was interesting to note that the condensation reaction was much faster than the cleavage, as its turnover number was ∼40-fold greater. This property is interesting for AbHpaI application as a biocatalyst because stereospecific aldol condensation is useful for preparation of APIs.
Crystal structures of AbHpaI
The quaternary structure of AbHpaI is composed of a dimer of trimers
To gain insights into the molecular mechanism of AbHpaI reactivity, we determined 11 crystal structures of AbHpaI, including the apoenzyme, enzyme complexes Zn2+•(4R)-KDGal, Zn2+•(4S)-KDGlu and Mg2+•(4R)-KDGal (for understanding the aldol cleavage), and Co2+•pyruvate (PYR), Mn2+•PYR, Co2+•PYR•SSA, Mn2+•PYR•SSA, Zn2+•PYR•propionaldehyde (PPA), Zn2+•PYR•4-hydroxybenzaldehyde (HBA) (for understanding the aldol condensation) using molecular replacement method with EcHpaI as a search template (PDB code 2V5J). AbHpaI was crystallized in monoclinic C2 crystals, which diffracted at 1.85 to 2.0 Å resolutions. Data and refinement statistics are shown in Table 4 and electron density maps of ligands are shown in Figs. S6–S9. The crystal structure of AbHpaI contains a trimer per asymmetric unit and a native hexameric quaternary structure can be drawn by applying twofold rotational symmetry (Fig. 3A). Size-exclusion chromatography (SEC) also confirmed a hexameric form of AbHpaI (Fig. S10).
Table 4Data collection and refinement statistics of AbHpaI complexes
Data were processed with Proteum3 except datasets of Co2+•Pyr and Mn2+•Pyr using HKL2000.
Unit cell (Å)
a, b, c (Å)
a = 147.620, b = 90.163, c = 86.484
a = 147.811, b = 89.646, c = 86.464
a = 147.359, b = 90.323, c = 86.440
a = 147.724, b = 90.345, c = 86.600
a = 147.36, b = 90.31, c = 86.39
a = 147.22, b = 90.52, c = 86.41
a = 147.56, b = 90.35, c = 86.52
a = 147.25, b = 89.29, c = 86.14
a = 147.75, b = 89.69, c = 86.30
a = 147.18, b = 89.64, c = 86.31
a = 147.37, b = 90.49, c = 86.50
No. atoms/B-factor (Å2)
Bond length (Å)
Bond angle (°)
Values in parentheses are for the highest resolution shells. Rf = Σhkl||Fobs| − |Fcalc||/Σhkl|Fobs|, where Fobs and Fcalc are the observed and calculated structure-factor amplitudes, respectively. Rfree was calculated in the same manner as Rf but using only a 10% unrefined subset of the reflection data.
a Data were processed with Proteum3 except datasets of Co2+•Pyr and Mn2+•Pyr using HKL2000.
Each protomer contains eight β/α motifs of a TIM barrel fold with an additional α-helix (residues 6–11; N-helix) at the N-terminus (Fig. 3B). Three protomers are associated in a tight trimeric structure to generate three catalytic pockets, and the trimer is dimerized to form a stable and rigid hexamer with 35% buried area. The hexameric quaternary structure of AbHpaI is stabilized through extensive interactions from α-helices of the (β/α)1 (residues 27–38), (β/α)2 (residues 53–64), and (β/α)8 (residues 236–253). The α-helix (residues 236–253) of the (β/α)8 of each protomer docks on the surface of the neighboring trimer for holding the trimer of dimers. In the structures of AbHpaI, a C-terminal end at residues 254 to 266 was not built because of no electron density. Each active site, located on a side face of the trimer, was built from two protomer subunits with a main catalytic pocket located in one subunit accompanied by a loop linker of residues 110 to 136 between (β/α)4 and (β/α)5 motifs of the shared protomer as a pocket periphery (Fig. 3B).
Metal ion octahedral coordination in AbHpaI is important for AbHpaI reactivity and stability
Understanding how AbHpaI accommodates the M2+ cofactor could yield biochemical insights into substrate recognition, reactivity, and stereospecificity control by different types of metal ions. Therefore, the coordination geometry of the M2+ cofactor in each complex obtained was analyzed. The crystal structures of AbHpaI complexed with metal ions Zn2+, Co2+, or Mn2+ and pyruvate (PDB codes 7ET9, 7ETA and 7ETB) revealed all types of M2+ chelate to a carboxyl group of Asp177, a water molecule (WS1), and a carboxyl and 2-oxo groups of pyruvate in a square planar arrangement, and with a carboxyl group of Glu151 and a water molecule (WA) in an axial position, arranged in an octahedral coordination geometry (Fig. 3C). Besides Glu151 and Asp177, Glu46 and His47 on the (β/α)2 loop also provide water-mediated hydrogen bondings via WA and WS1. Superposition of all AbHpaI complex structures revealed that the bound M2+ cofactor is at the same position with six atoms in octahedral geometry.
Notably, Glu151 and Asp177 in the Zn2+, Co2+, or Mn2+ complexes were more rigid than those of apo-AbHpaI, as reflected by temperature factors (B-factors) of the crystal structure. In the AbHpaI•M2+ complex structure, the B-factors of the (β/α)4 and (β/α)6 loop regions including the helix α6 where Asp177 is located are smaller than that of the apo structure. This suggests that M2+ can reduce the mobility in this region and strengthen subunit compactness, thereby stabilizing the overall structural architecture. In addition, the formation of the M2+ octahedral coordination with pyruvate in AbHpaI is important for enzyme reactivity.
Ca2+ ion neutralizing negatively charged Asp51 at the AbHpaI trimer surface facilitates dimerization of subunits
Ca2+ ion was found abundantly in purified AbHpaI (Fig. 1A). However, it does not act as a cofactor to enhance enzyme catalysis (Table 2). Therefore, the function of Ca2+ ion in AbHpaI was further investigated by analyzing the enzyme structure in complex with Ca2+.
The crystal structure of apo-AbHpaI (PDB code 7ET8) crystallized in the presence of CaCl2 only showed Ca2+ at the defined trimer center (not in the active site) in a distorted octahedral geometry with three Asp51 side chains (2.3–2.4 Å) and three water molecules as observed in all structures of AbHpaI studied here (Figs. 3C and S9). These data, together with the nonfunctional role of Ca2+ discussed above, confirmed that Ca2+ does not serve as a cofactor, but rather acts as a stabilizing factor on the dimerization surface of the AbHpaI trimer by neutralizing the negative charges of Asp51 on the trimer surface. In EcHpaI, the equivalent position to Asp51 was found to be Asn (Asn48Ec), thus abolishing the ability of this enzyme to bind to a divalent metal ion. Therefore, Ca2+ functions to prevent repulsive forces and to reduce movement of the (β/α)2 loop in AbHpaI where Asp51 sits. Consequently, by stabilizing the (β/α)2 loop where the active residues Glu46 and His47 reside (see proposed mechanisms), Ca2+ could indirectly aid in the catalysis of AbHpaI.
Insights into stereoselectivity of AbHpaI in the aldol cleavage reaction
To gain insights into why AbHpaI significantly prefers the 4R-isomer over the 4S-isomer in the aldol cleavage reaction (Table 2), crystal structures of AbHpaI•Zn2+ in complex with (4R)-KDGal (PDB code 7ETC) and (4S)-KDGlu (PDB code 7ETD) were determined at 1.95 and 1.90 Å resolutions, respectively. Superimposed structures illustrated that for the pyruvate core, the 1-carboxyl and 2-oxo groups of both substrates are directly coordinated to the Zn2+ site in an octahedral geometry similar to that found during pyruvate binding (Figs. 3C and 4A). However, the major differences are at the C4-OH, which interacts with Arg72 and at the binding site of the D-glyceraldehyde moiety. The structures revealed that the 4-OH of (4R)-KDGal forms a hydrogen bond with a guanidinium side chain of Arg72 at a 3.1 Å distance, whereas that of (4S)-KDGlu interacts with a longer distance (3.5–3.6 Å) (Fig. 4A). This therefore affected the arrangement and interactions of the D-glyceraldehyde moieties in the two compounds. The 5-OH and 6-OH functional groups of the D-glyceraldehyde moiety of (4R)-KDGal form hydrogen bonds with the main chains of Val′120 (2.6 Å) and Ala′122 (3.5 Å) from the pocket site created by the neighboring subunit, while those of (4S)-KDGlu do not form such interactions. Clearly, the observed interaction differences implied that AbHpaI•Zn2+ could preferably bind (4R)-KDGal over (4S)-KDGlu, further supported by QM/MM MD simulations. The binding energies of (4R)-KDGal and (4S)-KDGlu were calculated as −158 ± 9 and −131 ± 8 kcal/mol, respectively (Table S2). Furthermore, the configuration of (4R)-KDGal bound in the AbHpaI•Zn2+ complex can provide more suitable orientation and decreased motion of the substrate for the aldol cleavage in contrast to the bound (4S)-KDGlu. The structural analysis clearly supports the cleavage activity of the Zn2+-bound enzyme toward the 4R over the 4S substrates (Tables 2 and 3).
In addition, to understanding why the Mg2+ cofactor gave such a slow cleavage rate, the crystal structure of the AbHpaI•Mg2+•(4R)-KDGal complex (PDB code 7ETE) solved at 1.95 Å resolution was compared with the structure of the AbHpaI•Zn2+•(4R)-KDGal complex. Superimposed structures revealed a significant difference at the pyruvate core linked to the metal ion cofactors (Fig. 4B). A water WS2 replaced the carboxyl group of (4R)-KDGal to join the Mg2+ octahedral coordination. This feature gives rise to a longer distance between the 4-OH and Arg72 side chain in the Mg2+ complex (3.4−3.6 Å), causing a weaker binding interaction compared with the Zn2+ complex, thereby decelerating the C3–C4 bond breakage (Fig. 4B and Table 2). Based on structural and kinetics analyses, it could be summarized that the key binding features of the ligand important for stereoselective control in the aldol cleavage reaction of AbHpaI were (i) C4-OH anchoring by Arg72, (ii) interaction of the substrate pyruvate moiety in an octahedral M2+ complex, and (iii) interactions of the aldehyde moiety with the neighboring subunit.
Biocatalytic aspects of AbHpaI
AbHpaI catalyzes the aldol condensation reactions with broad aldehyde specificity
To investigate whether AbHpaI•Zn2+ can use various aldehydes as substrates in aldol condensation with pyruvate, we screened different categories of aliphatic and aromatic aldehydes. The corresponding aldol products were analyzed using liquid chromatography with high-resolution mass spectrometry to measure the exact m/z in a negative mode. The results showed that the selected aldehydes could be used by AbHpaI•Zn2+ in aldol condensation with pyruvate to yield various products (m/z values shown in Tables 5 and S3, and Fig. S11). The derivatives of aliphatic aldehydes with C3–C6 chain length could be successfully converted into the corresponding 4-hydroxy-2-keto aliphatic acids (Tables 5 and S3, and Fig. S11). Next, we examined with aromatic aldehydes and found that AbHpaI•Zn2+ can catalyze aldol condensation of pyruvate with various aromatic aldehydes such as benzaldehyde, HBA, and anisaldehyde, to yield the corresponding 4-hydroxy-2-keto aromatic acids (Tables 5 and S3, and Fig. S11). From our data, AbHpaI•Zn2+ can use a wide range of aldehyde substrates, suggesting that the enzyme active site is flexible enough to accommodate a variety of aldehydes for aldol condensation.
Space for accommodating various aldehydes in the active site of AbHpaI
To understand how the AbHpaI can accommodate various aldehydes for aldol condensation, we investigated the binding interactions of three aldehydes from the cocrystal structures of AbHpaI•Mn2+•PYR and AbHpaI•Co2+•PYR with SSA (PDB codes 7ETF and 7ETG, respectively), and AbHpaI•Zn2+•PYR with PPA and HBA (PDB codes 7ETH and 7ETI, respectively).
Overlaid structures of AbHpaI•Mn2+•PYR•SSA and AbHpaI•Co2+•PYR•SSA revealed that at the aldehyde binding site, the carbonyl group of SSA forms hydrogen bonds with Arg72 (2.7−2.9 Å) and the apex WA water (2.6–2.9 Å) in the M2+ octahedral coordination, while the carboxyl tail of SSA is hydrogen bonded to the Ala′123 or Ala′122 NH backbone of the nearby subunit and to a water network via W3 or W4 in the pocket tunnel filled with waters (Figs. 5A and 6A, see later). In addition, a distance between the C1 atom of SSA and the C3 methyl group of PYR was in the range of 3.1 to 4.1 Å (Fig. 6A, see later). We found that both metal ion complexes provide a similar binding of PYR but a slightly different configuration of SSA (Fig. 5A). This indicated that space for accommodating aldehyde substrate is larger than a van der Waals sphere of SSA, hence with a flexible hydrocarbon backbone, two configurations of SSA can be docked (Fig. 5A).
Although the substrate binding pocket of AbHpaI is wide open and exposed to outside solvent on the protein surface, the opening narrows down to the bottom of the active site where the reaction takes place. The site for aldehyde docking appeared to be hydrophobic, as most of the residues lining the site are nonpolar except for Arg72 (Fig. 5). Next, we explored the crystal structure of the AbHpaI•Zn2+•PYR complex liganded with PPA, which is a more hydrophobic ligand than SSA. PPA, which has only a polar carbonyl moiety, can dock on a hydrophobic cleft, composed of Trp21, Leu214, Val236 of the active subunit, and Leu′124 from the shared subunit (Fig. 5, B and C) and arranges the carbonyl moiety to hydrogen bond with Arg72 in a reactive trajectory for condensation with PYR, while the pocket tunnel is still full of water. However, the number of long-chain hydrocarbons in the aldehydes may be limited due to substrate solubility if the reaction is carried out solely in an aqueous environment because the long-chain hydrocarbon would protrude out of the hydrophobic cleft toward the water milieu in the direction similar to the SSA trajectory (Fig. 5, A and C). Thus, the nonpolar aldehydes may not be able to move along the water tunnel to reach the catalytic site located deep within the protein. For aromatic aldehydes, HBA was chosen as a representative to explore the binding mode. The cocrystal structure of the AbHpaI•Zn2+•PYR•HBA complex shows that the Trp21 indole ring, Leu214, and Val236 stabilize the HBA benzene ring through van der Waals interactions, while the carbonyl moiety forms a hydrogen bond to Arg72, which may be crucial for aldol condensation with PYR (Fig. 5, B and C). This structural analysis confirmed that AbHpaI can accommodate aromatic aldehydes well, as long as they can pass through a polar environment to get inside the active pocket.
Proposed mechanism for aldol reactions catalyzed by AbHpaI
The results from structural analysis reveal several structural water molecules at the active site potentially involved in AbHpaI catalysis (Fig. 6). A possible model for the AbHpaI aldol cleavage mechanism is proposed in Figure 7A. The cleavage of a C3–C4 bond in the (4R)-KDGal substrate to yield pyruvate and D-glyceraldehyde products is facilitated by Glu46, His47, and Arg72 together with bound water molecules, WA, W1, and WS1, to deprotonate the 4-OH leading to the bond cleavage to form an enolate intermediate, which then abstracts a proton from WA to yield a pyruvate.
For aldol condensation, via pyruvate carboxylate mediation, a water W2 likely acts as a catalytic base to abstract a proton from the C3-methyl of pyruvate in a similar reaction to that of Glu46, His47, and Arg72 with three waters WA, W1, and WS1 to generate the enolate intermediate, which then forms a covalent linkage with the C1 atom of SSA to produce HKHD (Fig. 7B). This model is supported by a pKa value of pyruvate C3-methyl of ∼6.5 (previously estimated by a pD-profile of pyruvate C3 proton exchange reaction of EcHpaI (
). Therefore, we determined the effect of M2+ on the thermostability of AbHpaI using thermofluor stability measurements. The results showed that apo-AbHpaI is quite thermostable naturally with a protein melting temperature (Tm) value as high as 81.3 °C. The binding of transition M2+, but not alkaline earth M2+, can further increase the thermostability of apo-AbHpaI (Table 1). The Tm values of AbHpaI were enhanced by 5.7, 3.4, and 1.7 °C upon binding to Zn2+, Co2+, and Mn2+, respectively. In contrast, the change of Tm values for AbHpaI•Mg2+ and AbHpaI•Ca2+ was negligible. Consequently, the result suggested that Zn2+ enhances the thermostability of AbHpaI, the highest among all types of AbHpaI•M2+ enzymes. Apart from the Tm value, which represents the protein stability, we also measured the remaining activity of AbHpaI•Zn2+ incubated at increasing temperatures (25–85 °C) for various incubation times (0–24 h) to examine the thermostability of AbHpaI. The result in Figure 8A showed that AbHpaI•Zn2+ can tolerate a wide range of temperatures from 25 to 75 °C for 24 h (and possibly longer) and can tolerate 80 °C for up to 2 h without activity loss.
We then explored the effects of Zn2+ on solvent tolerance of AbHpaI. As most substrates of aldolase reactions such as aliphatic and aromatic aldehydes are not soluble well in aqueous phases, addition of organic solvents is required to enhance substrate solubility. Therefore, we determined the Tm of AbHpaI•Zn2+ complex in the presence of organic solvents to represent enzyme stability. The organic solvents generally used in industries were chosen in this study, namely polar-protic (methanol, MeOH; ethanol, EtOH; and isopropanol, IPA) and polar-aprotic (acetronitrile, ACN; and dimethylsulfoxide, DMSO). The Tm results showed that upon addition of 20% (v/v) MeOH, ACN, and DMSO, the protein stability of AbHpaI was perturbed by only 10% (Fig. 8B), while EtOH and IPA disrupted protein stability by about 30 to 40%. This implied that 20% (v/v) of MeOH, can, or DMSO can be used as a cosolvent to enhance substrate solubility with small perturbation in protein stability of AbHpaI. Taken together, these findings suggest that AbHpaI is thermostable and solvent-tolerant enzyme, which can be a promising robust biocatalyst for aldol reaction.
Our report here has shown that AbHpaI is distinct among pyruvate-specific Class II metal aldolases for its ability to catalyze stereospecific aldol condensation and to use Zn2+ as a cofactor. Zn2+ binding can enhance stereoselectivity in aldol reactions and enzyme thermostability. Comprehensive structural investigation of AbHpaI complexes can explain how the enzyme is more stereoselective toward (4R)-KDGal over (4S)-KDGlu and how a variety of aldehydes can be accommodated.
Zn2+ is the most abundant transition metal ion found in the purified AbHpaI, binds with the highest affinity, and significantly increases the substrate stereoselectivity and stability of AbHpaI (Figs. 1, 2, 4, 8, S4, S5 and Table 1, Table 2, Table 3, S2). In addition, the enzyme can use other divalent ions (Co2+, Mn2+, and Mg2+) as cofactors, but these metal ions do not mediate AbHpaI stereoselective aldol cleavage functions as effectively as Zn2+. This property is different from other HpaI enzymes such as those from E. coli (EcHpaI) and Sphingomonas wittichii RW1 (SwHpaI), which cannot use Zn2+, instead use Co2+, Mn2+, and Mg2+ as cofactors (
). Zn2+ binds to AbHpaI in an octahedral coordination with six chelating atoms consisting of the pyruvate core, Glu151, Asp177, and two water molecules (WA and WS1), so does the geometry of Co2+, Mn2+, and Mg2+ (Figure 3, Figure 4, Figure 5, Figure 6), which differs from Zn2+ tetrahedral coordination commonly found in aldolase and nonaldolase enzymes (
). We noted an interesting cooperative enhancement of ligand (pyruvate) binding to AbHpaI in the presence of Zn2+, Co2+, and Mn2+ (Figs. 4 and 5 and Table 1) while weaker interaction or none was observed with Mg2+ and Ca2+. This therefore suggests that the active site is more rigid upon cofactor binding, thereby enhancing the binding of pyruvate and the cleavage activities of (4R)-KDGal (Table 2).
Zn2+ cofactor also promotes the highest R/S stereoselectivity ratio in the AbHpaI aldol cleavage. The preference of (4R)-KDGal over (4S)-KDGlu cleavage by AbHpaI•Zn2+ is approximately sixfold greater than the reactions of other M2+ cofactors (Table 2 and Fig. 2, A and B). QM/MM MD calculations gave higher favorable binding energy of (4R)-KDGal leading to a more stable complex of AbHpaI•Zn2+•(4R)-KDGal better poised for cleavage, explaining the stereoselectivity of AbHpaI•Zn2+ (Table S2).
Structural analysis of AbHpaI•Zn2+•(4R)-KDGal and AbHpaI•Zn2+•(4S)-KDGlu complexes suggested that Arg72,Val′120, Ala′122, and Ala′123 on the pocket border from the nearby subunit (which also defines the pocket size) are key factors for stereoselectivity via facilitating stronger interactions with the 4-OH and D-glyceraldehyde moiety of (4R)-KDGal over (4S)-KDGlu (Fig. 4A). The preferred orientation of the C4-OH cleavage site of (4R)-KDGal binding to Zn2+ thus gives rise to a shorter distance between WA water (a catalytic base) and 4-OH of (4R)-KDGal (3.6 Å), compared with that of (4S)-KDGlu (3.9 Å) (Fig. 6A). Comparison of the catalytic pockets between AbHpaI and EcHpaI (As EcHpaI was solved as one protomer per asymmetric unit, thus the dimer was generated by symmetry operation) showed that the EcHpaI pocket was 0.3 Å wider than that of AbHapI. Moreover, HKHD bound in EcHpaI (PDB code 4B5V) was found in two isomeric forms, which both interact with Arg70 and the nearby subunit residues similar to the case of AbHpaI. A wider binding pocket in EcHpaI may be the cause of the lacking stereoselectivity in this enzyme (
). Altogether, molecular interactions between AbHpaI•M2+ and ligand, particularly hydrogen bonding with the aldehyde moiety of substrate governed by Arg72 and coordination of the pyruvate core in the M2+ cofactor geometry described above, promote stereoselectivity and stereospecificity in the AbHpaI•M2+-catalyzed aldol cleavage reaction.
AbHpaI can catalyze the aldol condensation with stereospecificity and use broad aldehyde spectrum (Tables 2 and 5 and Fig. S11). Our kinetics demonstrated that AbHpaI•Zn2+ can synthesize only (4R)-KDGal from pyruvate and D-glyceraldehyde. This finding suggests that AbHpaI has a stereospecific control over aldol condensation reactions. The crystal structures showed that AbHpaI can bind both aliphatic and aromatic aldehydes (PPA, SSA, and HBA) with a proper chemical space to satisfy van der Waals interactions between the substrate and hydrophobic residues (Trp21, Leu214, Val236, and Leu′124) (Fig. 5). Recently, aromatic substituted aldehydes have been reported in two aldolases, YfaU (a pyruvate-specific Class II metal aldolase) and 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase (a pyruvate-specific Class I lysine aldolase); however, their crystal structures were not available (
). Nonetheless, the molecular dockings were performed and showed that N-benzyloxycarbonyl (N-Cbz)-substituted aldehydes are surrounded by hydrophobic residues Trp23, Phe174, and Leu216, similar to the aldehyde binding residues identified in AbHpaI. These properties offer an opportunity for AbHpaI to serve as a biocatalyst to catalyze formation of 4-hydroxy-2-ketoacid and 2-keto-3-deoxy-D-sugar acid precursors valuable for the synthesis of APIs (
Table 5Products obtained from the AbHpaI•Zn2+ catalyzed-aldol condensation of pyruvate and various aldehyde substrates
For product structures, the original pyruvate and aldehyde core structures are shown in blue and red colors, respectively. The asterisks indicate the stereocenter. The wavy bond at the stereocenter indicates the possibility to form either the R- or S-isomer.
Presently, AbHpaI is the only enzyme of HpaI superfamily in the pyruvate-specific Class II metal aldolases that catalyzes stereospecific aldol condensation. While most aldolases in Class II including EcHpaI, YfaU (Ni2+ cofactor) and putative bacterial HpaIs (Mg2+ cofactor) lack stereospecificity (
). The most investigated enzyme in this superfamily, EcHpaI catalyzes the condensation of pyruvate and acetaldehyde to produce two stereoisomers of 4R- and 4S-isomers of 4-hydroxy-2-oxopentanoate (HOPA) (
). The only exception previously reported to exhibit stereospecificity is Burkholderia xenovorans BphI aldolase (BxBphI), which shares 12% amino acid sequence identity with AbHpaI. BxBphI can catalyze specific formation of (4S)-HOPA using Mn2+ as a cofactor (
). However, the reaction of BxBphI is favored toward the aldol cleavage direction. A turnover number of BxBphI for aldol cleavage of (4S)-HOPA (4 s−1) is fourfold faster than the condensation (0.9 s−1) (
), suggesting that (4S)-HOPA product would be continuously cleaved during enzymatic turnovers. In contrast to BxBphI, AbHpaI shows a preferable aldol condensation over cleavage of ∼40-fold in AbHpaI•Zn2+ complex (Table 3). Therefore, AbHpaI can be a better candidate for stereospecific control to date.
Beyond promoting the aldol reactivity and the binding of pyruvate, Zn2+ also enhances the thermostability of AbHpaI. The enzymatic activity of AbHpaI•Zn2+ could be retained under very high temperature, i.e., 80 °C with a half-life (t1/2) at ∼ 3 h (Fig. 8A). Moreover, AbHpaI•Zn2+ is also stable in the presence of organic solvents such as MeOH, can, and DMSO up to 20% (v/v) (Fig. 8B). The data indicate that not only does Zn2+ serve as a catalytic cofactor, but it is also involved in the quaternary structure stabilization of AbHpaI. Apart from the catalytic Zn2+, our structural data demonstrates that Ca2+ is found at the trimer center on the dimerization interface of the AbHpaI hexamer, neutralizing the negative charges of three Asp51 carboxylate side chains (Fig. S9), which is unique for AbHpaI and is not found in EcHpaI, SwHpaI, other pyruvate-specific Class II metal aldolases (
In conclusion, our results here provide insightful mechanistic and structural understanding in stereoselectivity/stereospecificity control in aldol cleavage and condensation of AbHpaI. As the enzyme has broad aldehyde substrate specificity, high thermostability, and solvent tolerance, the insightful knowledge obtained from this study will serve as a basis for future rational protein engineering of AbHpaI and also other HpaIs in the pyruvate-specific Class II metal aldolases to achieve the capability to synthesize tailor-made, optically pure 4-hydroxy-2-ketoacid synthons required for preparation of APIs.
Chemicals and reagents
All chemicals were commercially available and of analytical, high purity, and HPLC grades. Buffers used in this work were (i) buffer A: 25 mM HEPES buffer, pH 7.0 containing 100 μM PMSF and 1 mM DTT; (ii) buffer B: 25 mM HEPES buffer, pH 7.0; (iii) buffer C: 25 mM HEPES buffer, pH 7.0 containing 150 mM NaCl; (iv) buffer D: 25 mM HEPES buffer, pH 7.0 containing 400 mM NaCl; (v) buffer E: 25 mM HEPES buffer, pH 7.0 containing 15% (w/v) (NH4)2SO4; (vi) buffer F: 50 mM HEPES buffer, pH 7.0; (vii) buffer G: 10 mM HEPES buffer, pH 7.0 containing 150 mM NaCl; (viii) buffer H: 10 mM HEPES buffer, pH 7.0; (ix) buffer I, 0.1 M sodium acetate buffer, pH 4.6.
Expression, purification, activity assay, and oligomeric state of AbHpaI
Recombinant AbHpaI was overexpressed in E. coli BL21(DE3) as previously described (
). Unless otherwise indicated, purification of AbHpaI was carried out at 4 °C. The cell paste (∼24 g obtained from 7.8 l culture) was resuspended in buffer A, and cells were then disrupted by ultrasonication. The broken-cell suspension was centrifuged at 36,000g for 40 min, and the clarified supernatant was collected as crude extract. Polyethyleneimine (PEI), at a final concentration of 0.5% (w/v), was added to the crude extract to remove nucleic acid contents. After centrifugation, the clarified supernatant was fractionated with 20 to 40% (w/v) ammonium sulfate ((NH4)2SO4) saturation. The protein pellet was resuspended in buffer B and dialyzed in the same buffer for 16 to 18 h. After dialysis, the dialysate was clarified by centrifugation before loading onto a DEAE-Sepharose column (172 ml, 2.5 cm × 35 cm) pre-equilibrated with buffer B. The column was washed with buffers B and C, respectively, and then eluted with a linear gradient of buffers C and D. Fractions containing AbHpaI were pooled and concentrated by ultrafiltration. The enzyme solution was further purified on a Phenyl-Sepharose column (45 ml, 1.5 cm × 25 cm) pre-equilibrated with buffer E. After loading the enzyme solution, the column was washed with buffer E and then eluted with a linear gradient of buffers E and A. Fractions containing AbHpaI were pooled and concentrated as described above. The concentrated enzyme solution was exchanged into buffer F using a Sephadex G-25 column. The concentration of the purified AbHpaI was determined using the molar absorption coefficient of 30,035 M−1 cm−1 at absorbance 280 nm (A280), which was calculated from the deduced amino acid sequence using the online tool on the ProtParam program of ExPaSy Proteomics Server (http://web.expasy.org/protparam/). The aliquots of enzyme solution were then stored at −80 °C until used. The amount of protein was determined by Bradford assay using BSA as a protein standard. The AbHpaI purity and subunit molecular weight (MW) were analyzed by 12% (w/v) SDS-PAGE.
SEC was used to determine the oligomeric state of AbHpaI as described previously (
). Briefly, a Superdex 200 Increase 10/300 GL gel-filtration column equipped with an ÄKTA FPLC system (GE Healthcare) was equilibrated with buffer G at a 0.5 ml/min flow rate at 25 °C and A280 was monitored for protein elution. Protein standards with known MWs (12.4–440 kDa) were used to construct a calibration curve. The elution volume (Ve) of each protein was measured, while that of blue dextran was used as the void volume (Vo). The protein mass was determined from a calibration curve of the relative ratio of Ve/Voversus the logarithm of the protein standard MWs. The oligomeric state of AbHpaI was then estimated based on the calculated subunit MW.
An LDH-coupled assay was used to determine the aldol cleavage activity of AbHpaI at 25 °C using oxaloacetate (OAA) as a substrate. Briefly, the reaction contained NADH (0.2 mM), LDH (30 μg/ml), Zn2+ (0.5 mM), OAA (1 mM) or (4R)-KDGal (0.2–0.3 mM), and AbHpaI (0.1 μM) in buffer F. The control reaction without enzyme was used for a background subtraction. The decrease of NADH absorbance at 340 nm can be used to infer pyruvate release from AbHpaI aldol cleavage. One unit of AbHpaI was defined as the amount of enzyme that consumes 1 μmol of NADH per minute.
Measurement of metal ions in AbHpaI
The M2+ species in the purifed AbHpaI were measured by Agilent 700 Series ICP-OES (Agilent Technologies). In total, 270 μM of the purified enzyme in buffer F was subjected to ICP-OES. The emission intensity of Zn2+, Mn2+, Co2+, Ni2+, Cu2+, Fe2+, Ca2+, and Mg2+ was detected and quantified by subtraction from the background emission intensity of a buffer blank. The concentration of each M2+ was determined by a calibration curve of varying concentrations (0.1–10 mg/l) of each standard M2+ prepared in 2% (v/v) HNO3versus the emission intensity.
To determine the mole ratios of AbHpaI and M2+, the apo-AbHpaI was reconstituted with each of the M2+ ions. Briefly, the purified enzyme was treated with chelating agents, EDTA, EGTA, and Chelex 100 (Merck KGaA), to strip off the M2+ ions. The fivefold excess concentrations of EDTA and EGTA (5 mM) and 0.5 g of Chelex 100 were added into a 10-ml AbHpaI solution (1 mM). The solution mixture was thoroughly mixed and incubated at 4 °C for 16 to 18 h to complete the metal ion chelation. The excess chelating agents and the metal ion chelation complexes were then removed by a Sephadex G-25 gel-filtration column equilibrated with buffer F to obtain apo-AbHpaI. To assure that the M2+ ions were completely removed, the apo-AbHpaI was first analyzed by ICP-OES. For the reconstitution process, each M2+ ion in a chloride salt form, namely ZnCl2, MnCl2, CoCl2, CaCl2, and MgCl2 was dissolved in the Chelex 100 treated Milli-Q Type I ultrapure water, and a fivefold excess of each M2+ chloride (1.5 mM) was added into a 2-ml apo-AbHpaI solution (0.3 mM). All samples were mixed thoroughly and incubated at 4 °C for 16 to 18 h to reconstitute the apo-AbHpaI. The excess M2+ in each sample was removed by a PD-10 desalting column equilibrated with buffer F to obtain a holoenzyme. The mole ratio of AbHpaI and M2+ was then determined.
Measurement of the Kd values for the AbHpaI•ligand complex
The Kd values for the binding of AbHpaI with ligands including M2+ ions (Zn2+, Co2+, Mn2+, Mg2+, and Ca2+) and pyruvate were measured by MicroCal PEAQ-ITC technique (Malvern Panalytical). Briefly, a 10-ml solution of apo-AbHpaI (200 μM) was dialyzed in 2 l of buffer F at 4 °C for 16 to 18 h. The dialyzed buffer was used to prepare a stock solution of each ligand. To measure the Kd value for the AbHpaI•M2+ complex, a 200-μl solution of the apo-AbHpaI (40 μM) was loaded into the sample cell and the Milli-Q Type I ultrapure water was used as a reference. Three microliters of 1.5 mM ligand solution of each M2+ in a syringe was continuously titrated into the sample cell (0.3 μM per each injection for 13 injections) until the ligand binding reached an equilibrium at 25 °C. The Microcal PEAQ-ITC analysis software was used to calculate the Kd using the one-site binding model. To determine the pyruvate binding constant to AbHpaI, a 200-μl solution of the apo-AbHpaI (40 μM) was placed in the sample cell and sequentially titrated with 3 μl of 10 mM pyruvate solution from a syringe. To determine the pyruvate binding constant to the AbHpaI•M2+ complex, a 200-μl solution of the mixture of apo-AbHpaI (40 μM) and 10 Kd of each M2+ was placed in the sample cell and sequentially titrated with 3 μl of a 10 mM solution mixture of pyruvate and 10 Kd of each M2+.
Thermal and solvent tolerance assay
To examine the thermal tolerance of AbHpaI, thermal stability and activity measurements were carried out by thermofluor and LDH-coupled assays, respectively. Thermofluor stability assays were performed as previously described (
). The Tm values were determined for apo-AbHpaI and AbHpaI•M2+ complexes of Zn2+, Co2+, Mn2+, Mg2+, and Ca2+. For thermal stability measurements of the AbHpaI•Zn2+ complex, the purified AbHpaI was first incubated at various temperatures from 25 to 85 °C for 0 to 24 h. The activity was then measured by LDH-coupled assay.
To investigate the solvent tolerance, the Tm values of the purified AbHpaI in the presence of 0 to 50% (v/v) of polar-protic (MeOH, EtOH and IPA) and polar-aprotic (ACN and DMSO) solvents were determined as above. The percentage of relative protein stability was calculated from the Tm of AbHpaI in the absence of solvents as 100%.
Aldol cleavage reactions
The aldol cleavage assays were carried out by LDH-coupled assay in buffer F containing 0.2 mM NADH, 0.2 mM substrate ((4R)-KDGal or (4S)-KDGlu), 0.1 mM AbHpaI, and 0.5 mM of each M2+ ion (Zn2+, Co2+, Mn2+, Mg2+, or Ca2+). The apparent rates of (4R)-KDGal and (4S)-KDGlu cleavages catalyzed by each AbHpaI•M2+ complex were measured.
The aldol cleavage steady-state kinetics of (4R)-KDGal and (4S)-KDGlu catalyzed by AbHpaI•Zn2+ were carried out using RapidFire high-throughput mass spectrometry. The reactions contained 0.1 mM ZnCl2 and the purified AbHpaI in buffer H and varying concentrations of the substrate. For Km determination, (4R)-KDGal (0.05–2 mM) and 5 μM AbHpaI or (4S)-KDGlu (0.1–3.2 mM) and 40 μM AbHpaI were used. Before RapidFire analysis, the reaction was quenched by an equal volume of ACN at various times (0.5–30 min) and the quenched solution was centrifuged at 12,000g for 10 min and filtered by a 0.22-μm nylon membrane syringe filter (FilterBio Nylon Syringe Filter) to obtain the filtrate of the remaining substrate. The substrate control reaction without the enzyme was performed. To analyze the remaining substrate, 10 μl of each filtrate was injected into the RapidFire C18 cartridge (G9203-80105, Agilent Technologies) with the optimized conditions set up as follows. The mobile phase reagents were 0.5% formic acid in H2O (A) and 100% ACN (B). The loading and washing steps were performed with 100% A at a flow rate of 1.5 and 1.25 ml/min, respectively. The elution step was carried out with isocratic solution mixture of A:B (30:70) at 0.4 ml/min flow rate. Peak areas of the remaining substrate were measured in a negative mode with a quantitative selected ion monitoring (SIM) mode to detect the m/z 177.0 ([M-H]−) of both (4R)-KDGal and (4S)-KDGlu, and the concentrations were determined from the calibration plot of substrate standard concentrations (0.025–3.2 mM) versus peak areas. The initial velocity (νo) of the substrate depletion from each individual concentration of substrate was calculated from the slope of the plot between the remaining substrate and time. The plots of νoversus each substrate concentration were analyzed by Michaelis–Menten equation using the Levenberg–Marquardt algorithms in GraphPad Prism version 7 software (GraphPad Software, Inc) to determine Km and kcat.
Aldol condensation reactions
The aldol condensation steady-state kinetics of pyruvate and D-glyceraldehyde catalyzed by AbHpaI•Zn2+ were monitored by the formation of (4R)-KDGal and (4S)-KDGlu using a triple-quadrupole LC/MS in a negative mode. The reactions were carried out in buffer H containing 0.1 mM ZnCl2, 0.5 μM purified AbHpaI, and varying concentrations of the substrates. For Km determination, varying concentrations of pyruvate (0.25–8 mM) at 30 mM D-glyceraldehyde or varying concentrations of D-glyceraldehyde (0.25–32 mM) at 4 mM pyruvate were used. The LC condition was carried out at 30 °C using a Hi-Plex H cation exchange column (8 μm, 7.7 × 300 mm) and 0.5% (v/v) formic acid in H2O as a mobile phase at a flow rate of 0.3 ml/min. (4R)-KDGal and (4S)-KDGlu were eluted at a retention time of 17.707 and 18.632 min, respectively. The exact m/z 177.0 ([M-H]−) of both products was monitored by a SIM mode and their concentrations were determined by using a calibration curve of each product (0.005–0.4 mM). The kinetic parameters were calculated as described above.
The aldol condensation of pyruvate and D-glyceraldehyde catalyzed by AbHpaI•M2+ complexes of Zn2+, Co2+, Mn2+, Mg2+, and Ca2+ was monitored as described above. The assay reactions were carried out at 25 °C for 1 h in buffer H containing 4 mM pyruvate, 30 mM D-glyceraldehyde, 0.1 mM metal chloride, and 0.05 μM of each AbHpaI•M2+ complex. The rate of (4R)-KDGal and (4S)-KDGlu formation was determined for each AbHpaI•M2+ complex. Time-course synthesis of (4R)-KDGal was performed with 0.5 μM AbHpaI•Zn2+ and the product was monitored for 90 h.
For analysis of a broad spectrum of aldehydes, different aldehydes PPA, SSA, butyraldehyde, pentanal, glutaraldehyde, hexanal, benzaldehyde, HBA, and anisaldehyde were used as substrates. The reactions were carried out at 25 °C for 1 h in buffer H containing 10 mM pyruvate, 5 mM aldehyde, 2 mM ZnCl2, and 10 μM purified AbHpaI. The exact m/z ([M-H]−) of the product was monitored by high-resolution Compact QTOF (Bruker Daltonics) in negative mode, equipped with a Zorbax-eclipse C18 column (5 μm, 4.6 × 250 mm) ultrahigh-performance liquid chromatography (Thermo Scientific) operating at 30 °C with a flow rate of 0.5 ml/min of 0.5% (v/v) formic acid.
Crystallization and X-ray data collection and structure determination
For crystallization, apo-AbHpaI (0.7 mM) was incubated for 10 min at 25 °C in buffer F containing 52 mM pyruvate and 11 mM divalent metal chloride (ZnCl2 or CoCl2 or MnCl2). Crystals were grown at 18 °C in microbatch drops containing 1 μl of apo-AbHpaI complex with 1 μl of buffer I containing 20 mM CaCl2 and 30% (v/v) 2-methyl-2,4-pentanediol (MPD) as a crystallizing agent. For SSA soaking, crystals of apo-AbHpaI•M2+•PYR complexes were soaked in a crystallizing agent containing 18.5 mM pyruvate, 7% (v/v) glycerol, 3.6 mM divalent metal chloride (ZnCl2, CoCl2, or MnCl2) and 36 mM SSA in a microbatch well at 27 °C for 5 to 10 min. For PPA complex formation, crystal soaking was performed in a sitting drop well containing 25 μl of a crystallizing agent plus similar concentrations of pyruvate and ZnCl2, and 2.5 μl of 13.9 M PPA with 7.5 μl of 13.9 M PPA in the reservoir for 3 days at 18 °C. All soaking solutions contained 7% (v/v) glycerol for cryoprotection. Crystals of AbHpaI cocomplexed with (4R)-KDGal and (4S)-KDGlu compounds and Zn2+/Mg2+ cofactors were obtained from the wells containing 1 μl crystallizing solution (40 mM CaCl2 and 30% (v/v) MPD in buffer I) and 1 μl of 0.18 mM AbHpaI, 0.71 mM ZnCl2 and 50 mM (4R)-KDGal at 4 °C, 20 h for AbHpaI•Zn2+•(4R)-KDGal, 0.35 mM AbHpaI, 1.16 mM ZnCl2 and 66.63 mM (4S)-KDGlu at 4 °C, 20 h for AbHpaI•Zn2+•(4S)-KDGal, and 0.37 mM AbHpaI, 18.30 mM MgCl2 and 75 mM (4R)-KDGal at 4 °C, 42 h for AbHpaI•Mg2+•(4R)-KDGal. A crystal of AbHpaI•Zn2+•PYR•HBA was from a well containing 1 μl crystallizing solution (40 mM CaCl2, 30% (v/v) MPD and 5% (v/v) trifluoroethanol in buffer I) and 1 μl of 0.32 mM AbHpaI, 1.76 mM ZnCl2, 5.88 mM PYR, and 73.5 mM HBA at 15 °C, 20 h. Data were collected at 100 K on a D8 venture with a microfocus TXS rotating anode and Bruker PHOTON 100 detector at the NSTDA Characterization and Testing Center (NCTC). Data processing was carried out using either PROTEUM3 software pipeline (Bruker AXS 2017) (
To investigate the binding interaction of AbHpaI•Zn2+ with (4R)-KDGal and (4S)-KDGlu, QM/MM MD simulations were performed. The structures of AbHpaI•Zn2+•(4R)-KDGal (PDB code 7ETC) and AbHpaI•Zn2+•(4S)-KDGal (PDB code 7ETD) complexes were employed and prepared as follows. The system was truncated to a 25 Å sphere with the center on the C4 atom of (4R)-KDGal. The positions of the hydrogen atoms were located in the enzyme using the CHARMM procedure HBUILD (
). For investigation of the enzyme–substrate interactions, the system was divided into two parts, QM and MM. The QM part consisted of substrate (either (4R)-KDGal or (4S)-KDGal), which was minimized using 1000 steps of Adopted Basis Newton-Raphson (ABNR) minimization with the AM1/CHARMM27 method. Next, the AM1/CHARMM27 MD using the leapfrog Langevin dynamics with a time step of 0.001 ps was performed at 300 K. The rest of protein, Zn2+, and water molecules were treated as the MM part. The system was equilibrated with QM/MM MD for 120 ps. The structures of this equilibration were collected at every 20 ps. Distances between substrate, metal ion, and the surrounding residues were determined. The binding energies of both substrates to the AbHpaI•Zn2+ were calculated and compared.
Data of X-ray structures are available at Protein Data Bank under PDB codes indicated.
The authors declare that they have no conflicts of interest with the contents of this article.
We thank the Frontier Research Center (FRC), Vidyasirimedhi Institute of Science and Technology (VISTEC) that supports ICP-OES and UHPLC-QTOF for metal ion and product analysis, respectively. We also thank NSTDA Characterization and Testing Center (NCTC, NSTDA Thailand) for X-ray data collection on the single crystal X-ray diffraction D8 venture.
P. W., A. B., A. J., N. L., Pimchai Chaiyen, Penchit Chitnumsub, and S. M. conceptualization; P. W., A. B., A. J., N. L., J. T., J. J., Pimchai Chaiyen, Penchit Chitnumsub, and S. M. data curation; P. W., A. B., A. J., N. L., J. T., J. J., L. C., J. P., R. T., Pimchai Chaiyen, Penchit Chitnumsub, and S. M. formal analysis; P. W., A. B., N. L., J. T., J. J., Pimchai Chaiyen, Penchit Chitnumsub, and S. M. funding acquisition; P. W., A. B., A. J., J. T., J. J., L. C., J. P., R. T., Pimchai Chaiyen, Penchit Chitnumsub, and S. M. investigation; P. W., A. B., A. J., N. L., J. T., J. J., L. C., J. P., R. T., Pimchai Chaiyen, Penchit Chitnumsub, and S. M. methodology; P. W., A. B., Pimchai Chaiyen, Penchit Chitnumsub, and S. M. project administration; P. W., A. B., A. J., N. L., J. T., J. J., Pimchai Chaiyen, Penchit Chitnumsub, and S. M. resources; P. W., A. B., A. J., N. L., J. T., J. J., Pimchai Chaiyen, Penchit Chitnumsub, and S. M. software; P. W., A. B., A. J., Pimchai Chaiyen, Penchit Chitnumsub, and S. M. supervision; P. W., A. B., A. J., N. L., J. T., J. J., Pimchai Chaiyen, Penchit Chitnumsub, and S. M. validation; P. W., A. B., A. J., N. L., J. T., J. J., R. T., Pimchai Chaiyen, Penchit Chitnumsub, and S. M. visualization; P. W., A. B., A. J., N. L., J. T., J. J., Pimchai Chaiyen, Penchit Chitnumsub, and S. M. writing—original draft; P. W., A. B., A. J., N. L., J. T., J. J., Pimchai Chaiyen, Penchit Chitnumsub, and S. M. writing—review and editing.
Funding and additional information
We thank Faculty of Science, Burapha University (to S. M. and J. T.), Mahidol University (to R. T.), Walailak University (to L. C.), Chiang Mai University (to N. L.), National Science and Technology Development Agency, Thailand (to Penchit Chitnumsub and A. J.), School of Biomolecular Science and Engineering, VISTEC (to Pimchai Chaiyen, P. W., A. B., J. J., and J. P.).
This work was funded by The National Research Council of Thailand (NRCT) Grant NRCT5-RSA63012-01 (to S. M.), National Center for Genetic Engineering and Biotechnology (Thailand) P16-52034 and National Science and Technology Development Agency (Thailand) P20-50077 (to Penchit Chitnumsub and A. J.), Funding supports from Vidyasirimedhi Institute of Science and Technology (VISTEC) and Global Partnership Program from Program Management Unit-B and Royal Academy of Engineering (UK) (to Pimchai Chaiyen, P. W., A. B., J. J., and J. P.), The Thailand Science Research and Innovation Grant MRG6180151 (to R. T.), a partial funding support from Chiang Mai University (to N. L.), and The Office of the Permanent Secretary, Ministry of Higher Education, Science, Research and Innovation through grant RGNS 63-212 (to L. C.).
Use of a recombinant bacterial fructose-1,6-diphosphate aldolase in aldol reactions: Preparative syntheses of 1-deoxynojirimycin, 1-deoxymannojirimycin, 1,4-dideoxy-1,4-imino-D- arabinitol, and fagomine.