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J. Biol. Chem., Vol. 282, Issue 30, 21973-21986, July 27, 2007

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Structure of Phenylalanine Hydroxylase from Colwellia psychrerythraea 34H, a Monomeric Cold Active Enzyme with Local Flexibility around the Active Site and High Overall Stability^*

Hanna-Kirsti S. Leiros, Angel L. Pey, Marte Innselset, Elin Moe, Ingar Leiros, Ida Helene Steen^¶, and Aurora Martinez¹

From the Norwegian Structural Biology Centre (NorStruct), Department of Chemistry, University of Tromsø, N-9037 Tromsø, the Department of Biomedicine, University of Bergen, Jonas Lies vei 91, 5009 Bergen, and the ^¶Department of Biology, University of Bergen, P. O. Box 7800, Jahnebakken 5, 5020 Bergen, Norway

Received for publication, October 31, 2006 , and in revised form, May 29, 2007.

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The characteristic of cold-adapted enzymes, high catalytic efficiency at low temperatures, is often associated with low thermostability and high flexibility. In this context, we analyzed the catalytic properties and solved the crystal structure of phenylalanine hydroxylase from the psychrophilic bacterium Colwellia psychrerythraea 34H (CpPAH). CpPAH displays highest activity with tetrahydrobiopterin (BH₄) as cofactor and at 25 °C (15 °C above the optimal growth temperature). Although the enzyme is monomeric with a single L-Phe-binding site, the substrate binds cooperatively. In comparison with PAH from mesophilic bacteria and mammalian organisms, CpPAH shows elevated [S_0.5](L-Phe) (= 1.1 ± 0.1 mM) and K_m(BH₄)(= 0.3 ± 0.1 mM), as well as high catalytic efficiency at 10 °C. However, the half-inactivation and denaturation temperature is only slightly lowered (T_m 52 °C; where T_m is half-denaturation temperature), in contrast to other cold-adapted enzymes. The crystal structure shows regions of local flexibility close to the highly solvent accessible binding sites for BH₄ (Gly⁸⁷/Phe⁸⁸/Gly⁸⁹) and L-Phe (Tyr¹¹⁴–Pro¹¹⁸). Normal mode and COREX analysis also detect these and other areas with high flexibility. Greater mobility around the active site and disrupted hydrogen bonding abilities for the cofactor appear to represent cold-adaptive properties that do not markedly affect the thermostability of CpPAH.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The strictly psychrophilic Colwellia psychrerythraea strain 34H, found in near-freezing Arctic marine sediments (1), possesses a variety of adaptations to low temperatures, as recently shown by comparative genome analysis (2). To obtain insight into the molecular determinants for cold-active enzymes of this organism, Methe et al. (2) prepared 2026 structure models based on 176 templates from the Protein Data Bank. There appears to be an extensive increase in flexibility in certain regions of the structures investigated, accompanied by high activity at low temperatures and some increase in thermolability (2).

A few enzymes from C. psychrerythraea have been isolated and characterized in detail. These included an extracellular cold-active aminopeptidase, which shows characteristic cold-adapted activity and structural properties (3) and two isocitrate dehydrogenase isozymes, one found to be monomeric and cold-active, and one found to be dimeric with a mesophilic activity profile (4).

The genome of C. psychrerythraea notably includes a phenylalanine hydroxylase (PAH)² ortholog (Fig. 1), an enzyme that had not achieved prominence in the previous comparative genomics study (2). PAH is a non-heme iron-dependent enzyme, which in mammals is found mainly in the liver catalyzing the hydroxylation of L-Phe using molecular oxygen and (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH₄) as co-substrates. In mammals this is the first step in the catabolism of L-Phe to carbon dioxide and water. In humans, excessively high levels of L-Phe are toxic to the brain, and a dysfunction of human PAH is associated with phenylketonuria (for review see Ref. 5). In fact, other severe human diseases (e.g. alcaptonuria, tyrosinemia, tyrosinosis, Richner-Hanhart syndrome, and hawkinsinuria) are also associated with enzyme deficiencies in the catabolism of L-Phe and L-Tyr.

Over the past few years, the functional and structural studies of several forms of human and rat PAH, including free and ligand-bound states and disease-related mutants, have advanced the understanding of the main catalytic features of the enzyme and genotype-phenotype relationships in phenylketonuria (6–9). In bacteria, PAH is relatively rare and is found predominantly within the phylum Proteobacteria. PAH from Chromobacterium violaceum (CvPAH) and Pseudomonas aeruginosa have been cloned, purified, biochemically characterized, and crystallized (10–15). Bacterial PAH crystal structure determination has been reported only for CvPAH, which shares the fold of the catalytic domain of human and rat PAHs (14).

View larger version (61K): [in this window] [in a new window]   FIGURE 1. PAH from CpPAH. A Sequence alignment of CpPAH with CvPAH and hPAH (residues Gly103–Gln428). The catalytic residues are indicated by triangles, and other important active site residues are outlined with circles. Identical residues are shown by black boxes, whereas similar residues are framed with boldface letters. The secondary structure elements of CpPAH (top) and hPAH (bottom; PDB 1PAH) are also given. For catalytic core (CC) residues 20–245, CpPAH shows an overall identity of 41 and 31% to CvPAH and hPAH, respectively.

Functional studies of bacterial enzymes have improved our understanding of bacterial metabolism, adaptation, and evolution and are also relevant to the understanding of the mammalian orthologs. The evolution of mammalian PAH and of the other aromatic amino acid hydroxylases, i.e. tyrosine hydroxylase and tryptophan hydroxylases, 1 and 2, appears to have proceeded via juxtaposition of bacterial PAH with a precursor of the regulatory domain of chorismate mutase-prephenate dehydratase (P-protein) (18–21), corresponding to the N-terminal regulatory domain of the hydroxylases. Tetrameric eukaryotic PAH, which is highly regulated both by phosphorylation and positive cooperativity for L-Phe, could thus have evolved from a monomeric bacterial PAH.

Here we report the cloning, expression, purification, and the structural and functional characterization of the monomeric bacterial PAH from the psychrophilic bacterium C. psychrerythraea (CpPAH). The structure of the enzyme has been analyzed and compared with that of ortholog PAHs from the mesophilic prokaryote C. violaceum (CvPAH; optimum growth temperature, 25 °C) and human (hPAH). The structural features help to explain the kinetic properties and particular cold-adaptive characteristics of CpPAH.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Growth of C. psycherythraea 34H and Detection of Gene Expression—C. psychrerythraea 34H was grown to stationary phase in Marine Broth (Difco 2216) at 9 °C. Pellets were resuspended in freshly prepared Tris-EDTA buffer (10 mM Tris-HCl, pH 8.0, 1 mM EDTA) with lysozyme (0.4 mg/ml) and incubated at room temperature for 5 min before the total RNA was isolated using the SV ("spin or vacuum") total RNA isolation kit according to the manufacturer's manual (Promega). PAH mRNA was subsequently detected using the following primer and the reverse transcriptase SuperScript^TM (Invitrogen): 5'-CCTTAAGCTCGAGACTGGCTTTTTTCACCGGAAATT-3'. Amplification was performed using Phusion^TM DNA polymerase (Finnzymes) in a PCR using the following primers: 5'-GGGAATTCCATATGGCAAAAGGAACAAAGTATGTT-3' and 5'-CCTTAAGCTCGAGACTGGCTTTTTTCACCGGAAATT-3' (NdeI and XhoI restriction sites are underlined). C. psychrerythraea genomic DNA was used as positive control (not shown).

Cloning, Expression, and Purification—The PAH gene from C. psycherythraea 34H (CpPAH) was amplified by PCR using the primers described above. The PCR products were purified using the Stratagene PCR purification kit, digested with NdeI and BamHI, and ligated into NdeI-BamHI-digested pET-30a using the rapid DNA ligation kit (Roche Applied Sciences). Recombinant CpPAH was overexpressed in Escherichia coli strain BL21 Star^TM(DE3) by growing transformed cells in LB broth containing kanamycin (30 µg/ml) at 37 °C and induction with 1 mM isopropyl 1-thio--D-galactopyranoside. Eight hours after induction at 22 °C (A_{600 nm} = 0.7–0.8), the cells were harvested and disrupted using a French press, and the supernatant was applied to a TALON® Superflow metal affinity resin column (Clontech). C-terminal His₆-tagged-CpPAH was purified at 4 °C by washing with 10 mM imidazole and eluting with 150 mM imidazole in 20 mM sodium phosphate, 300 mM NaCl, pH 7, followed by size-exclusion chromatography on a HiLoad Superdex 200 column, which also removed the imidazole. The enzyme was kept concentrated in 20 mM NaHepes, 200 mM NaCl, pH 7.0, in liquid nitrogen. The catalytic domain (Gly¹⁰³–Gln⁴²⁸) of human PAH (hPAH) was purified as described previously (22).

Activity Measurements—Steady-state PAH activity was measured by high pressure liquid chromatography with fluorimetric detection of L-Tyr (23, 24), with reactions mixtures containing 100 mM Hepes, pH 7, catalase (0.04 mg/ml), and the indicated concentrations of L-Phe and BH₄ (with 5 mM dithiothreitol) were used. Under the selected standard assay conditions, the amount of product was linear with time (up to 5 min) and amount of enzyme (up to 5 µg) when L-Phe and BH₄ were added simultaneously to initiate the assay. [S_0.5] and K_m values were determined at the specified temperature and concentrations of L-Phe and BH₄ (with or without 5 min preincubation with L-Phe) and customarily with 1 µg of CpPAH and a 1-min assay. The data were fitted to a Hill equation (for L-Phe) or to a Michaelis-Menten equation (for BH₄). Thermal inactivation experiments were performed by incubating the enzyme at the indicated temperatures in 20 mM NaHepes, 200 mM NaCl, pH 7, in the absence and the presence of 5 mM L-Phe for 10 min, followed by chilling of the samples on ice and immediate assay (1 min at 10 °C).

Tyrosine hydroxylase activity was measured at 10 and 20 °C as described (25), using reaction mixtures containing 0.2–1.0 µg of purified CpPAH, 0.05 mg/ml catalase, 20 µM ferrous ammonium sulfate, and 50 µM L-Tyr and initiating the reactions by adding BH₄ (up to 1 mM) with 5 mM dithiothreitol, and up to a 10-min reaction time. The L-Tyr-dependent oxidation of the cofactor was measured using a similar reaction mixture but additionally including 200 µM NADH and 0.4 units of dihydropteridine reductase (from sheep liver; Sigma). The reaction was started by adding 75 µM BH₄, and A₃₄₀ was recorded for 1 min. The rate of NADH oxidation (pterin oxidation) was calculated using _{340 nm} = 6220 M^-1·cm^-1.

CD Spectroscopy—CD was performed as described (26), with 25 µM CpPAH in freshly made and degassed 20 mM NaHepes, 200 mM NaCl, pH 7.0. Stoichiometric amounts of ferrous ammonium sulfate were added when indicated. The samples were incubated for 10 min at the indicated temperature prior to acquisition of the CD spectra at the same temperature. CD-recorded thermal denaturation was also performed in the same samples as described (26) by following the changes in ellipticity at 222 nm, with a scan rate of 1 K/min in the range 4–75 °C. The amount of -helical structure was estimated with the program CDNN, which employs a back-propagation neural network model for the quantitative analysis of protein UV CD spectra (27).

Differential Scanning Calorimetry (DSC)—DSC was performed as described (26), using 32 µM CpPAH in 20 mM NaHepes, 200 mM NaCl, pH 7.0, in the absence and the presence of L-Phe (5 mM) at a 1 K/min scan rate, typically from 4 to 75 °C.

Crystallization, Data Collection, and Structure Determination—Crystallization was performed using the hanging drop method. Crystals of native CpPAH were grown by mixing 1-µl drops of 10 mg/ml protein (in 20 mM NaHepes, 200 mM NaCl, pH 7.0) with a 1-µl reservoir solution containing 1.6–1.8 M ammonium sulfate, 100 mM NaCl, and 20 mM NaHepes, pH 7.5. The drops were equilibrated at 4 °C, and crystals suitable for data collection purposes appeared after about 3 weeks. The crystals had overall dimensions of 120 x 100 x 80 µm³, were cryo-protected with 25% glycerol and the reservoir additives, and flash-cooled in a nitrogen stream (Oxford Instruments) operating at 100 K. One crystal was soaked in 1 mM 7,8-dihydrobiopterin (BH₂), 1.9 M ammonium sulfate, 80 mM NaCl, 20 mM Hepes, pH 7.5, and 25% glycerol for 15 min at 4 °C and then flash-cooled. This surprisingly resulted in the apo form of CpPAH with the iron atom missing in the crystal structure.

All diffraction data were collected at the European Synchrotron Radiation Facility (ESRF), Grenoble, France, at ID14-EH1 (for the native, holo-CpPAH) and at the Swiss-Norwegian Beamline (CpPAH-apo). The crystals belong to the space group P2₁ with unit cell dimensions for the native structure of a = 40.28 Å, b = 86.02 Å, c = 87.57 Å, and = 97.01°. The two molecules in the asymmetric unit (A/B) give a water content of 49% and a Matthews coefficient of 2.5 ų/Da. The native structure was solved by the molecular replacement method using MOLREP (28) and the crystal structure of PAH from C. violaceum (CvPAH, PDB 1LTV) as search model. The refinement and incorporation of solvent molecules were done using REF-MAC5 (29) interspersed with rounds of manual rebuilding in O (30). The final native model includes 536 residues, 2 iron atoms, and 847 water molecules, resulting in an R-factor of 16.4% and an R-free of 19.52%. For more details of data collection and refinement statistics, see Tables 1 and 2, respectively.

The WHAT IF Web Interface (32) was used to identify ion pairs with interatomic distances <6 or <4 Å between the side chains of Asp or Glu to Arg, Lys, or His residues. Electrostatic surfaces were calculated with DelPHi (33) and displayed with PyMOL (DeLano Scientific, San Carlos, CA).

Models of CpPAH with only BH₄ and with BH₄ and the substrate analog (2-thienyl)-L-alanine (THA) were prepared based on the hPAH·BH₄ (PDB 1J8U) and hPAH·Fe(II)·BH₄·THA (PDB 1MMK [PDB] ) crystal structures.

Computational Structural Analysis—The low frequency normal modes of CpPAH (molecules A and B), CvPAH (PDB 1LTV) and hPAH (PDB 1PAH [PDB] ), which provide information on regions with slow motions, were computed and analyzed by normal mode analysis (NMA) using the web server WebNM (34). The regional folding/unfolding conformational fluctuations and residue-specific stability constants were calculated using the COREX/BEST algorithm (35) on the COREX/BEST web server (36). A more detailed description of the algorithms and experimental details for NMA and COREX calculations is provided at the servers (34, 36). Calculations of the energies of charge-charge interactions were performed using a Tanford-Kirkwood model with the solvent accessibility correction of Gurd (at pH 7); other methodological details are given in Refs. 37, 38. Dielectric constants of 4.0 and 78.5 were used for the protein and the aqueous solvent, respectively. In this study, we calculated the energy of charge-charge interactions of group i with the rest of the ionizable groups in the protein W_i , which can be used to estimate the total charge-charge interaction energy in the proteins (W_q-q ), as shown in Equation 1.

(Eq. 1)

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Expression of PAH in C. psychrerythraea 34H; Cloning, Expression, and Purification of CpPAH—C. psychrerythraea displayed a growth optimum around 9 °C when cultivated in Marine Broth at temperatures from 0 to 15 °C. Reverse transcriptase PCR confirmed that PAH was expressed in C. psychrerythraea cultivated under these conditions (Fig. 2A). Addition of L-Phe at concentrations of up to 30 mM added to the Marine Broth medium stimulated the growth. This is opposite to what is found for E. coli and other bacteria that do not possess PAH (39) where L-Phe in high amounts appears to be toxic. Nevertheless, C. psychrerythraea 34H did not grow in minimal medium (M9), and the addition of L-Phe (up to 50 mM) to this medium did not stimulate growth.

After cloning the CpPAH gene into the vector pET-30a, the enzyme was successfully expressed in E. coli, and purified by chromatography on TALON® resin and consequent size-exclusion chromatography. CpPAH showed apparent molecular masses of 31 and 28 kDa by SDS-PAGE and analytic gel filtration chromatography, respectively (Fig. 2B and data not shown). The enzyme, with estimated molecular weight from the amino acid sequence of 30.71 kDa (31.77 kDa when the His tag is included), thus appears to be monomeric.

Catalytic Properties of CpPAH—The enzyme activity is stimulated (40–70% depending on assay conditions and temperature) by the addition of 100 µM Fe(II) added as ferrous ammonium sulfate. This stimulation by iron is also found in other non-heme iron-dependent aromatic amino acid hydroxylases (40), including bacterial CvPAH (12, 41), and is probably because of the stabilization of the iron-bound holoenzyme during catalysis. 100 µM ferrous iron was thus routinely included in the activity assays.

View larger version (47K): [in this window] [in a new window]   FIGURE 2. Expression of CpPAH in Colwellia psychrerythraea 34H and expression and purification of cloned CpPAH in E. coli. A, expression of PAH in C. psychrerythraea grown in Marine Broth at 9 °C. Lane 1, 1-kb DNA ladder (Invitrogen); lanes 2 and 3, amplification of first strand synthesis reactions (reaction in lane 2 had half the starting RNA amount in first strand synthesis than that in lane 3); lane 4, amplification of first strand synthesis reaction without C. psychrerythraea RNA (negative control). B, purification of CpPAH. SDS-PAGE gel showing low molecular weight standards (lane 1), supernatant of cell extract (E. coli) after French press (lane 2), and purified CpPAH (lane 3).

View larger version (27K): [in this window] [in a new window]   FIGURE 3. Characterization of the CpPAH activity. A, effect of temperature on the enzyme activity measured with 0.5 mM BH4 and 5 mM L-Phe (1 min assay). Inset, Arrhenius plot for activity in the 0–25 °C range giving Ea = 4.9 ± 0.3 kcal/mol. B, effect of L-Phe concentration on CpPAH activity at 10 °C, 0.5 mM BH4 and 1 min assay, with () and without (•) 5-min preincubation at the indicated concentrations of L-Phe. Similar concentrations of L-Phe were used in the preincubation and in the assay. C, time course of L-Phe hydroxylation by CpPAH without (•) (simultaneous addition of L-Phe and BH4) and with () 5-min preincubation of the enzyme with 5 mM L-Phe. Note the lag phase in the time course for the L-Phe-preincubated enzyme. D, time course for the recovery of CpPAH activity after inhibition of the enzyme by preincubation for 10 min with 10 mM L-Phe. At time 0, the preincubation was stopped by 100-fold dilution of the enzyme in 100 mM Hepes, pH 7, and catalase (0.04 mg/ml), and the activity was assayed at the indicated times after dilution by simultaneous addition of L-Phe (5 mM) and BH4 (0.5 mM). Inset, inhibition of CpPAH as a function of the concentration of L-Phe used in preincubation for 5 min.

With respect to substrate specificity, CpPAH did not catalyze the hydroxylation of L-Tyr measured as L-DOPA production when analyzed at concentrations of both cofactor and L-Tyr up to 1 mM. With L-Tyr, CpPAH only catalyzed cofactor oxidation (as seen by the NADH-dependent dihydropteridine reductase activity (data not shown)), which is similar to the PAHs investigated so far (46, 47).

Preliminary activity assays indicated that maximal activity of CpPAH was obtained at higher concentrations of substrate and cofactor than for the human enzyme (hPAH) (48) and CvPAH (41). We therefore adjusted the concentrations used in the customary standard PAH assays from 1 mM L-Phe and 75 µM BH₄ to 5 mM L-Phe and 0.5 mM BH₄ in order to investigate the catalytic properties of CpPAH. At these conditions, CpPAH had an optimum temperature for activity of about 25 °C (Fig. 3A). The activation energy of the reaction catalyzed by CpPAH (E_a = 4.9 ± 0.3 kcal/mol) is lower than for mesophilic CvPAH (13), indicating that CpPAH possesses cold-adapted features. Studies of psychrophilic enzymes show that they have lowered their E_a in order to reduce the temperature dependence of k_cat, making catalysis less affected by a decrease in temperature (49, 50).

The enzyme activity was about eight times higher when simultaneously adding BH₄ and L-Phe (or by 5 min-preincubation with the BH₄ cofactor) compared with the activity obtained after preincubation with L-Phe (Fig. 3B). This contrasts what is found for the mammalian forms of PAH, which are activated only by preincubation with L-Phe (22, 51). To study this inhibitory effect of preincubation with L-Phe, we investigated the time course of L-Tyr formation by CpPAH. For the preincubated enzyme a lag phase was observed (Fig. 3C), although the time course on simultaneous addition of substrate and cofactor was linear, satisfying initial velocity and steady-state conditions. Moreover, the inhibition upon incubation with L-Phe (5 mM) is time-dependent with a calculated pseudofirst-order rate constant (k_obs) of 0.22 ± 0.03 min^-1 (data not shown). The inhibition was also dependent upon the concentration of L-Phe (Fig. 3D, inset) and appeared to be reversible (Fig. 3D). Elucidation of the kinetic mechanism of this inhibition would require the determination of the catalytic mechanism for the enzyme, including the order of binding for the reactants. However, the sigmoidal saturation curves for L-Phe obtained at all concentrations of cofactor tested (see below), in addition to the substrate inhibition, prevented detailed steady-state analyses of CpPAH. A previous study with noncooperative CvPAH with synthetic DMPH₄ as cofactor concluded with an ordered mechanism where L-Phe binds as the second substrate, after the pterin cofactor (41). Thus, assuming a similar mechanism for CpPAH, our results on the inhibition by L-Phe preincubation would be best explained by formation of long-lived dead-end complexes with the substrate, leading to different activities depending on the preincubation scheme (Fig. 3B).

The activity of CpPAH as a function of L-Phe and cofactor concentration was measured both at 10 °C, close to the optimum growth temperature for C. psychrerythraea, and at 25 °C. Considering that CpPAH is a monomeric enzyme, it was remarkable that its activity as a function of substrate concentration resulted in sigmoidal curves both with and without preincubation with L-Phe (Hill coefficient (h) 2; see Fig. 3B and Table 3). The concentrations of L-Phe for half-maximal activity ([S_0.5](L-Phe)) depended on the L-Phe preincubation scheme (Table 3). The BH₄-dependent activity showed hyperbolic kinetics, and the K_m(BH₄) values are also included in Table 3. The catalytic efficiency ((V_max/K_m) and V_max/[S_0.5] values) appears to be maximal at 25 °C and also high at 10 °C. Both the K_m(BH₄) and [S_0.5](L-Phe) values were about 10 times higher than other PAHs characterized so far (at 25 °C), which show [S_0.5](L-Phe) in the range 90–300 µM and K_m(BH₄) about 20–30 µM for mammalian enzymes (48, 51, 52), and even lower for CvPAH (41).

Thermal Effect on the Conformational Stability of CpPAH—As earlier found for PAHs from other sources (55, 56), the far-UV CD spectrum of CpPAH (200–260 nm) recorded at 4 °C shows two minima at 208 and 222 nm (Fig. 4A), characteristic for proteins with high -helical content. Processing the spectra with the program CDNN (27) provided a 40% -helix content for CpPAH, a content that is not affected by addition of Fe(II) or L-Phe, up to 5 mM (data not shown). However, the CD spectrum in the presence of the substrate showed a high level of noise at <210 nm, and an accurate estimation of any change in secondary structure could therefore not be performed. On the other hand, higher temperatures induced a reduction in -helical structure (8% decrease from 4 to 37 °C; Fig. 4A). This conformational change was found to be almost reversible up to 37 °C because a helical content of 40% was also estimated by CDNN after cooling the sample back from 37 to 4 °C.

Thermal denaturation experiments monitored by either CD (ellipticity changes at 222 nm) (Fig. 4B) or DSC (Fig. 4C) resulted in one thermal transition with a T_m 52 °C. CpPAH unfolds irreversibly, but it appears that the unfolding transition obtained by DSC can be fitted to equilibrium thermodynamic models, because the T_m is not significantly affected by the scan rate from 0.25 to 1.5 K/min (data not shown) (57). The unfolding parameters obtained by DSC were T_m = 52.1 ± 0.1 °C and calorimetric enthalpy change (H) = 94.6 ± 1.3 kcal/mol. These values were similar in the presence of stoichiometric amounts of Fe(II), but when the experiments were performed with 5 mM L-Phe the T_m increased to 56.4 ± 0.1 °C (H = 96.6 ± 0.6 kcal/mol) (Fig. 4, B and C). Inactivation experiments, where CpPAH was preincubated for 10 min at temperatures from 0 to 70 °C prior to assay of remaining activity at 10 °C, showed half-maximal inactivation at 47 and 52 °C when samples were preincubated in the absence and presence of 5 mM L-Phe, respectively (data not shown). These results, together with the T_m values obtained by CD and DSC indicate a relatively high thermostability of the enzyme. A comparison of the T_m values for CpPAH with those obtained for CvPAH and the catalytic domain (Gly¹⁰³–Glu⁴²⁸) of human PAH (hPAH) are shown in Table 4.

View this table: [in this window] [in a new window]   TABLE 4 Tm values obtained by CD-monitored thermal unfolding and DSC Effect of L-Phe and comparison with Tm values for CpPAH, CvPAH, and the catalytic domain (Gly103–Glu428) of hPAH. For CpPAH, 25 and 32 µM were used for CD and DSC measurements, respectively, and for hPAH, 25 and 30 µM subunit for CD and DSC, respectively. Measurements were performed at pH 7 in the presence of stoichiometric amounts of Fe(II). The values are the average of two or three independent measurements; the difference in Tm from scans measured in different samples was 0.5 °C.

View larger version (16K): [in this window] [in a new window]   FIGURE 4. Effects of temperature and L-Phe on secondary structure and conformational stability of CpPAH. A, far-UV CD spectrum of CpPAH at 4 °C (black), 25 °C (red), 37 °C (green), and after cooling back to 4 from 37 °C (blue). CD-monitored (B) and (C) DSC-monitored thermal denaturation of CpPAH in the absence (black) or presence (red) of 5 mM L-Phe. The inset in B shows the first derivative of the CD signal versus temperature. Experimental details are found in the text.

View larger version (63K): [in this window] [in a new window]   FIGURE 5. Superposition of CpPAH-molecule A (pink) and CpPAH-molecule B (blue). Regions in molecule B deviating from molecule A are highlighted in green. The additional C-terminal His tag residues in molecule B are outlined (green), and a modeled BH4 molecule is indicated (cyan).

Fig. 5 shows the superposition of molecules A and B of CpPAH, which have an r.m.s.d. of 0.42 Å for 258 C- atoms (residues 10–267). The main differences (>0.7 Å) are found for the N-terminal residues 4–9 that adopt different conformations in the two molecules (Fig. 5). The His tag in CpPAH-molecule B is folded into the enzyme, a feature most likely reflecting a crystallization artifact with the tag probably being very mobile in solution. The tag does not appear to be tightly bound because of the following: (i) it was only found in 50% of the molecules, only in molecule B and not in A, and (ii) it was easily displaced in the BH₂-soaked crystal (CpPAH-apo) that diffracted to 1.90 Å resolution. At this resolution the tag, if ordered, should be visible in the electron density.

The iron-bound and iron-free CpPAH structures have an overall r.m.s.d. of 0.22–0.44 Å for the C- atoms (Table 5), indicative of no substantial overall structural differences between the two. On the other hand, in the active sites there are some rearrangements of residues His¹²², His¹²⁷, and Glu¹⁶⁷ in CpPAH-apo to compensate for the lack of the charged iron atom. Furthermore, the water molecules W1 and W2 are missing, and W3 is displaced relative to the CpPAH-holo as shown in Fig. 6, A and B.

The 6-coordinated iron in CpPAH is similar to CvPAH with two histidines, two water molecules, and a skewed bidentate coordination of the glutamate. On the other hand, in hPAH·Fe(III) (PDB 1PAH [PDB] ) and hPAH·Fe(II)·BH₄ (PDB 1J8U), the iron has the acidic residue (Glu³³⁰) binding with only one carboxyl oxygen atom, three water molecules (W1, W2, and W3), and two histidine residues in the coordination sphere (Fig. 6D). Other comparative values of the three PAHs are presented in Table 5.

BH₄ Cofactor-binding Site—Crystal structures of binary CpPAH complexes of either BH₄ (or its oxidized and stable analog BH₂) or L-Phe could not be obtained, but models of CpPAH with only BH₄ and with both BH₄ and THA (a substrate analog, see below) were prepared. Structurally, the loop Leu⁸⁵–Gly⁸⁹ (in CpPAH) includes highly different residues in the three enzymes (Figs. 1 and 6), and both residues Leu⁸⁵ and Phe⁸⁸ in CpPAH (molecule A) are poorly defined in the final electron density, with high B-factors. Furthermore, Phe⁸⁸ in CpPAH is incapable of making the same type of ionic interactions with BH₂ as are found in CvPAH·BH₂, where the corresponding residue Asp¹⁰⁴ forms two hydrogen bonds to O-1' of BH₂ (14) (Fig. 6C). Likewise, in the hPAH·BH₄ complex there is one important hydrogen bond from the corresponding residue Ser²⁵¹ O- (Phe⁸⁸ in CpPAH) to the cofactor (Fig. 6D) (59). Furthermore, Ser²⁵¹ in hPAH is more stabilized than Phe⁸⁸ in CpPAH, because the preceding and subsequent residues (Ser²⁵⁰ and Arg²⁵²) are involved in hydrogen-binding and ion pair interactions. CpPAH has two glycine residues at these positions (Gly⁸⁷ and Gly⁸⁹) making the loop more flexible and Phe⁸⁸ a poor stabilizer for BH₄ binding, in agreement with the high K_m value of CpPAH compared with the other PAHs. CpPAH should still be able to form two hydrogen bonds from Ile⁸⁶ O and N to BH₄, as observed in CvPAH·BH₂ and hPAH·BH₄ (14, 58) (Fig. 6, C and D).

View larger version (56K): [in this window] [in a new window]   FIGURE 6. Active site comparison. Structural comparison of the BH4-binding sites in CpPAH (A), CpPAH-apo (B), CvPAH·BH2 (1LTZ) (C), and hPAH·BH4 (PDB 1J8U) (D). The L-Phe-binding sites of CpPAH (E) and the hPAH·BH4·THA (PDB 1MMK) (F) crystal structure with the substrate analog THA. For A and E, BH4 and THA have been modeled by docking the ligands according to their position in the hPAH·BH4 and hPAH·BH4·THA structures, respectively. See the text for further details.

View larger version (78K): [in this window] [in a new window]   FIGURE 7. Structural comparison. Ribbon diagrams (left panels), calculated electrostatic surface potentials (middle panels), and mobility analysis (right panels). A–C are CpPAH (residues 4–267); D and E are CvPAH·BH2 (PDB 1LTZ, residues 9–283); F is CvPAH (PDB 1LTV, residues 9–283), G and H are hPAH·BH4 (PDB 1J8U), and I is hPAH (PDB 1PAH), all in similar orientations. In CvPAH·BH2 Tyr130 was poorly defined in electron density and its orientation very uncertain (14). The surface potentials are color coded in red, white, and blue for the charge -10, 0, and +10 KbT/e, respectively. The normalized squared atomic displacements were calculated for mode 8 (C, F, and I) and are color-coded from small (dark blue) to large (red) mobility on the ribbon diagrams. All figures were made with PyMOL (DeLano Scientific, San Carlos, CA).

Regional variations in local folding/unfolding equilibria were also calculated by the COREX/BEST algorithm (36, 61). The residue stability constant is the ratio of the summed probabilities of the states in the ensemble in which a particular residue is in a folded conformation, to the summed probability of the states in which a residue is in an unfolded conformation. We observed that the three PAHs regions with high mobility according to NMA are associated with low stability constants (supplemental Fig. S2). Comparative studies of the role of electrostatics in temperature adaptation have found several cold-adapted enzymes with increased negative surface charge and decompensated (destabilizing) charge-charge interactions (see Refs. 50, 62, 63 and references therein). It has been suggested that the acidic surface in psychrophilic enzymes would enhance protein solvation, whereas charge decompensation would ensure active site flexibility (63). CpPAH has a more negative surface potential, notably around the active site, which also appears to be wide and open (Fig. 7B). However, when compared with its mesophilic homologs, CpPAH does not include a greater proportion of destabilizing charged residues. In fact, as inferred from electrostatic analysis by implementation of the Tanford-Kirkwood model on the structures (37, 38), CvPAH presents more regions with destabilizing charge-charge interactions (i.e. positive energy of interaction of the charged groups with the rest of the ionizable groups in the protein (W_i > 0)) (supplemental Fig. S2, G–I), and these regions are located around the active site and present high mobility (supplemental Fig. S2, B and E). Accordingly, the total charge-charge of W_q-q (see Equation 1) of -25.3 kJ/mol for CvPAH was compared with -42.5 and -67 kJ/mol for CpPAH and hPAH, respectively. This is also consistent with the number of ion pairs found in the crystal structures with CvPAH having least ionic interactions followed by CpPAH and hPAH (Table 5). W_q-q is regarded as an approximation of the charge-charge contributions to the unfolding Gibbs energies (38), and our results indicate that CpPAH has generally stabilizing interactions, in agreement with the relatively high T_m. This also suggests that regional mobility in this enzyme is not primarily a result of unfavorable electrostatics. Other factors, such as a more open water-accessible active site and the higher proportion of Gly residues in some high mobility regions, appear to play an important role. In fact, important specific stabilizing charge-charge interactions were observed for CpPAH involving ion-pair networks with interactions <5 Å and very negative W_i values (supplemental Fig. S2G) indicative of strong links like: (i) Glu²³–Arg¹⁰⁹–Glu³⁰–Arg³⁴–Asp¹¹³, and (ii) Glu⁹⁰–Arg⁹³–Glu⁹⁷–Lys⁹⁹–Glu⁷⁸, which are not present in CvPAH nor in hPAH. These networks might also play a role in the refolding observed after cooling samples that were heated to 37 °C (Fig. 4A).

Despite the fact that detailed analysis of the hydrogen bonding and ion pair patterns are dependent on the resolution of the crystal structure, both CpPAH structures have more hydrogen bonds than the other PAH structures, and fewer ion pairs (<4 Å) per residue than hPAH but more than CvPAH (Table 5). This also fits with the fact that CpPAH is not cold-adapted in terms of lower global thermal stability.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
The Function of CpPAH—In some prokaryotic organisms containing PAH the initial steps in a catabolic pathway to mediate Phe and Tyr carbon recycling involve the function of the phh operon and include the reactions L-Phe L-Tyr (catalyzed by PAH) and L-Tyr 4-hydroxyphenylpyruvate (catalyzed by tyrosine aminotransferase) (64), similar to the steps for L-Phe catabolism in eukaryotes. 4-Hydroxyphenylpyruvate is then further oxidized to homogentisic acid (65). The fact that C. psychrerythraea lacks an ortholog of homogentisate dioxygenase catalyzing the oxidation of homogentisic acid to 4-maleylacetoacetate, a step needed for the complete oxidation of L-Phe to carbon dioxide and water (65), further supports the hypothesis that PAH is not involved in catabolism but in specialized synthesis of metabolites derived from L-Phe and L-Tyr in C. psychrerythraea. This was also suggested to be the function of PAH from P. aeruginosa, based on the observation that this microorganism grows very slowly on L-Phe as the sole carbon source and that the amino acid substrate induces expression of PAH in the presence of other more effective energy and carbon sources, such as glucose (17). PAH is present in only about 20% of the bacterial genomes sequenced so far. Like C. psychrerythraea, many of the species that contain PAH have been described as red-brown pigmented, and in several of them, such as Vibrio cholerae, Hyphomonas strain sp., Shewanella colwelliana, and P. aeruginosa (66, 67), it has been shown that the pigment providing this color, usually referred to as pyomelanin or alkapton, derives from the oxidation and oligomerization of homogentisate. Homogentisic acid is a derivative of L-Tyr (the product of PAH), and it seems very likely that PAH is the first enzyme in the biosynthesis of pyomelanin. Studies on the synthesis of this pigment have usually focused on enzyme activities downstream in the pathway, e.g. hydroxyphenylpyruvate dehydrogenase (67). Pyomelanin is associated with several functions, such as biofilm formation, redox activity, and protection against oxidants and radiation (67–69). To the best of our knowledge, the function of the pigment in C. psychrerythraea has not been described previously or clarified, although we have observed that pigment production increases when the bacteria grow forming biofilms. It is probable that the red pigment of C. psychrerythraea, which is responsible for the name of this organism (70), is a pyomelanin, or alkapton, formed from spontaneous dimerization and oxidation of homogentisic acid. Interestingly, C. psychrerythraea thus appears to be a prokaryotic model of the disease alkaptonuria (71), where patients also lack the enzyme homogentisate dioxygenase.

The Effect of Substrate Preincubation—The specific activity of CpPAH under standard conditions is highly affected by the order of addition of substrate and cofactor in the assay, where preincubation with L-Phe resulted in very low activity (Fig. 3B; Table 3). Our results support formation of slow-dissociating dead-end enzyme complexes with L-Phe if the substrate binds before the cofactor (Fig. 3, C and D). In combination with a possible ordered kinetic mechanism, with BH₄ binding first in the active site, L-Phe second, and dioxygen last, as earlier determined for CvPAH (41), these results may provide an explanation for the low specific activity following preincubation with L-Phe. The orientation of L-Phe when bound to free CpPAH in the absence of BH₄ is probably not optimal, and only when the substrate binds to the binary CpPAH·BH₄ complex do the reactants adopt a correct orientation relative to each other and to the iron. A structural effect of BH₄ binding might be that the loop Leu⁸⁵–Phe⁹¹ (in CpPAH) becomes more ordered when Phe⁹¹ stacks against the cofactor and hydrogen bonds to the main chain of Ile⁸⁶ are formed, as found for the corresponding residues in hPAH (58). Ordering or different folding of the N terminus of CpPAH, which is highly mobile and adjacent to the L-Phe-binding site (Fig. 7C and Fig. S2A), might also facilitate correct L-Phe binding.

Positive Cooperativity for L-Phe—It was interesting to find positive cooperativity for the activity of CpPAH as a function of L-Phe concentration (Fig. 3B and Table 3). This regulatory mechanism is characteristic of tetrameric mammalian PAHs (48, 51, 52). Positive cooperativity occurs in a few monomeric enzymes, of which the most studied is glucokinase (72). The equilibrium binding of glucose to glucokinase displays hyperbolic saturation curves, in contrast to the sigmoidal dependence on glucose in turnover (73), indicating that the positive cooperativity with the substrate originates from a kinetic property. The kinetic mechanism is, however, not completely understood, and several models have been proposed to explain the cooperative kinetics, such as a mnemonical model (74) and a slow transition model (75). Both models are based on the existence of different conformational forms of glucokinase that interconvert by substrate binding, and a slow transition leading to the catalytically competent enzyme-glucose complex relative to the catalytic step (73, 76). Similarly to what is found for glucokinase, hyperbolic binding curves were found for the binding of the substrate L-Phe to CpPAH (supplemental Fig. S1), also indicating a kinetic nature for the cooperativity. Our results are thus compatible with a slow conformational transition in CpPAH induced by L-Phe binding and leading to a catalytically competent enzyme when L-Phe binds after BH₄ (see also previous paragraph). On the other hand, sigmoidal binding curves are found for the binding of L-Phe to tetrameric mammalian PAH (26, 77).

The existence of positive cooperativity in CpPAH is remarkable. This mechanism is present at different evolutionary stages from bacteria to man, most probably as a means for the enzyme to sensitize the L-Phe concentrations and maintaining homeostatic levels of this essential amino acid. However, the elucidation of the molecular basis for the positive cooperativity as well as the nature of the regulatory and kinetic states of the enzyme, must await the structural determination of binary and ternary complexes with substrate and cofactor.

Cold Adaptation of CpPAH—Under the conditions used in this study, CpPAH shows maximal activity and catalytic efficiency at 25 °C. This temperature is about 15 °C above the optimal growth temperature for C. psychrerythraea. Other enzymes from the same bacterium and other cold-adapted organisms also show maximal activity 10–20 °C above the optimal growth temperature (2–4). The drop in activity at temperatures above 25 °C is not associated with low overall stability (Fig. 4). The K_m(BH₄) and [S_0.5](L-Phe) values might further increase at temperatures >25 °C, although definitive measurement of these values at high temperatures was hampered by a more marked substrate inhibition at these conditions. A very likely explanation of the temperature-induced inactivation might be associated with failure in the formation of a catalytically competent CpPAH·Fe(II)·BH₄·L-Phe·O₂ complex. The formation of this complex is absolutely dependent on the correct arrangement of all substrates, with the right distance and orientation with respect to each other (9, 60, 78). The temperature-dependent increased mobility around the active site may not directly affect the binding of the individual reactants but rather their spatial organization and the coupling between substrate hydroxylation and cofactor oxidation.

The increasing availability of crystal structures from other psychrophilic organisms, notably bacteria, has further revealed that cold-adapted enzymes often show high flexibility and thermolability compared with their mesophilic and thermophilic counterparts (50, 53, 79). Most cold-adapted enzymes show a global rather uniform distribution of their flexibility throughout the three-dimensional structure (50). However, there are also examples in which the flexibility is locally increased, notably in regions around the active site, resulting in a weaker binding of the substrate (high K_m values), and high k_cat values at low temperatures (see Refs. 50, 53 and references therein), as is the case of CpPAH.

Overall conformational flexibility in cold-adapted enzymes appears to be obtained by the following: (i) a lower number of prolines and arginines; (ii) more glycines; (iii) fewer hydrogen bonds and fewer ion pair interactions; (iv) higher numbers of serine and methionine; and (v) reduced hydrophobicity (particularly for internal residues) expressed as lower (Ile + Leu)/(Ile + Leu + Val) ratio; or (vi) density of charged surface residues (50, 53, 79). As can be seen in Table 5, these structural features are not distinctive for CpPAH, except for the Gly clusters Gly⁸⁷–Phe⁸⁸–Gly⁸⁹, located in the highly mobile loop 81–87 in the BH₄-binding site, and Gly¹⁸¹–Gly¹⁸²–Gly¹⁸³, at the top of the L-Phe active site (Figs. 6E and 7C). An additional cold-adaptive feature encountered in CpPAH is a more open active site (53) (Table 6), mostly due to a shorter N terminus (Fig. 7). This open, solvent-accessible active site could also contribute to local flexibility, notably in the region 105–119 (supplemental Fig. S2A), which includes the water-exposed loop. Moreover, CvPAH and hPAH have several Pro residues at the beginning or endings of -helices and loops not present in CpPAH (Fig. 1).

The distribution of charge on the surface of proteins is important for their stability, and as we move from psychrophiles to mesophiles to thermophiles, a clear trend can be observed that shows an increase in the number of ionic attractions on the protein surface (38, 80). CpPAH presents a slightly higher negative charge at neutral pH and negative surface potential around the active site (Fig. 7B), but there are no regions with very dense charge, as is the case in the other PAHs (Fig. 7). CpPAH does not have a typical cold-adapted surface with a greater negative charge or uncompensated charge-charge interactions (Fig. 7B and Fig. S2G) that can lead to structural destabilization and thermolability.

The present biochemical and structural characterization of PAH from a cold-adapted organism shows an enzyme with relatively high thermostability and low affinity for substrate and cofactor, most probably associated with high local flexibility around the active site and disrupted hydrogen-bonding capacity of the cofactor.

	FOOTNOTES

 
The atomic coordinates and structure factors (code 2v27 and 2v28) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

^* This work was supported by grants from the Research Council of Norway, Helse-Vest, and the Nanoscience Program at the University of Bergen. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1, Figs. S1 and S2, and Refs. 1 and 2.

¹ To whom correspondence should be addressed. Tel.: 47-55586427; Fax: 47-55586360; E-mail: