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* This work was supported by NISR Research Grant from the Noda Institute for Scientific Research, Japan, and grants-in-aid for scientific research (B) from Japan Society for Promotion of Science. This article contains supplemental Figs. 1–5 and Tables 1–3.
An l-glucose-utilizing bacterium, Paracoccus sp. 43P, was isolated from soil by enrichment cultivation in a minimal medium containing l-glucose as the sole carbon source. In cell-free extracts from this bacterium, NAD+-dependent l-glucose dehydrogenase was detected as having sole activity toward l-glucose. This enzyme, LgdA, was purified, and the lgdA gene was found to be located in a cluster of putative inositol catabolic genes. LgdA showed similar dehydrogenase activity toward scyllo- and myo-inositols. l-Gluconate dehydrogenase activity was also detected in cell-free extracts, which represents the reaction product of LgdA activity toward l-glucose. Enzyme purification and gene cloning revealed that the corresponding gene resides in a nine-gene cluster, the lgn cluster, which may participate in aldonate incorporation and assimilation. Kinetic and reaction product analysis of each gene product in the cluster indicated that they sequentially metabolize l-gluconate to glycolytic intermediates, d-glyceraldehyde-3-phosphate, and pyruvate through reactions of C-5 epimerization by dehydrogenase/reductase, dehydration, phosphorylation, and aldolase reaction, using a pathway similar to l-galactonate catabolism in Escherichia coli. Gene disruption studies indicated that the identified genes are responsible for l-glucose catabolism.
All living organisms show homochirality toward organic compounds, such as amino acids, with l- rather than d- amino acids utilized for protein synthesis. The same is true for sugars, where d-sugars are more abundant than l-sugars in natural environments. Accordingly, most d-sugars can be utilized by many organisms, but l-sugar usage is less common, indicating that homochirality also occurs in sugar metabolism. A good example of this is glucose, where d-glucose is the most abundant sugar in nature, and most organisms use d-glucose as an energy source via well known pathways such as the Embden-Meyerhof-Parnas, the pentose phosphate, and the Entner-Doudoroff pathways. Meanwhile, l-glucose is not present in nature, and few instances of l-glucose usage have appeared since Rudney (
) reported l-glucose dehydrogenase activity in Pseudomonas caryophilli, but no further reports concerning this finding are currently available.
To determine whether there are organism(s) capable of utilizing l-glucose and to obtain further insights into homochirality in sugar metabolism, isolation of l-glucose-utilizing organisms and analysis of their catabolic pathways are necessary. Here, we describe the isolation of an l-glucose-utilizing bacterium, and its catabolic pathway for l-glucose metabolism is characterized.
Isolation of l-Glucose-utilizing Bacteria
Several l-glucose-utilizing bacteria were isolated from soil samples by enrichment cultivation in l-Glc MM, on which strain 43P showed a maximum growth rate with a concomitant decrease of reducing sugars in the medium (Fig. 1). Phylogenetic analysis based on 16S rRNA gene sequences showed that strain 43P is included in the genus Paracoccus cluster, with the closest related species being Paracoccus denitrificans (96.8%, supplemental Fig. 1), although it should be noted that the P. denitrificans strain NBRC 102528T did not grow on l-Glc MM (Fig. 1). These results indicate that strain 43P assimilates l-glucose, and this strain should be classified in the genus Paracoccus. Therefore, we sought to further analyze l-glucose catabolism in strain 43P.
l-GDH, the First Enzyme in the l-Glucose Catabolic Pathway
To determine the l-glucose catabolic pathway in strain 43P, we used cell-free extracts prepared from cells grown in l-Glc MM as an enzyme source and l-glucose as a substrate to analyze enzyme activities known to occur in the first reactions in sugar catabolism, such as kinase, isomerase, and dehydrogenase activity. No activities beyond NAD+-dependent l-GDH activity were detected, suggesting that the first reaction in this pathway is l-glucose oxidation. Because this activity was also detected when the strain was cultivated with d-galactose as the sole carbon source, we purified l-GDH from cells grown in a minimal medium containing d-galactose (supplemental Table 2). The purified enzyme showed a single band of ∼40 kDa on SDS-PAGE (supplemental Fig. 2A), and its N-terminal amino acid sequence MSNAEKALGVALIGTGFMGK exhibited 80% identity with that of P. denitrificans PD1222 Pden_1680, a putative oxidoreductase that is a member of the Gfo/Idh/MocA family. Therefore, we assumed that the gene encoding l-GDH in strain 43P is an ortholog of Pden_1680, and we then identified this gene by PCR and DNA sequencing as described under “Experimental Procedures.”
As expected, this gene, termed lgdA, showed 84% amino acid sequence identity with Pden_1680, and it was located in a cluster of putative inositol catabolic genes (Fig. 2A), the organization of which was quite similar to that of strain PD1222 with 76–89% identities, except that Pden_1673 encodes a hypothetical protein of 61 amino acids that was not present in strain 43P.
Recombinant LgdA was produced with an E. coli pET system and purified (supplemental Fig. 2C). Purified LgdA showed NAD+-dependent l-GDH activity (Table 1), which is comparable with the native enzyme (kcat = 710 ± 24 min−1 and Km = 44.4 ± 6.3 mm) and also had dehydrogenase activity toward myo-inositol, scyllo-inositol, and d-glucose (Table 1), with the highest activity observed for scyllo-inositol. The reaction products of LgdA from an l-glucose source were analyzed by NMR and HPLC, which showed 1H and 13C chemical shifts that were identical to potassium d-gluconate (supplemental Fig. 3A) and optical rotation that was identical to potassium l-gluconate (Fig. 3A). These data indicate that LgdA catalyzes dehydrogenation of the l-glucose C1-hydroxyl group to form l-glucono-1,5-lactone, which would in turn be spontaneously hydrolyzed to l-gluconate.
TABLE 1Kinetic parameters of the l-glucose catabolic enzymes
Next, we evaluated enzyme activity toward l-gluconate with cell-free extracts of strain 43P cultivated in l-Glc MM, and again an NAD+-dependent l-GnDH activity was detected. The enzyme catalyzing the reaction was partially purified by about 100-fold (supplemental Table 3) and showed a 35-kDa band on SDS-PAGE that corresponded to the activity (supplemental Fig. 2B). This protein had an N-terminal amino acid sequence of MKALIIEKEGHTVIGEISEP, which has 100% identity with strain PD1222 Pden_4931, a putative oxidoreductase that is a member of the zinc-containing alcohol dehydrogenase family. As such, the same PCR-based strategy used to identify lgdA was adopted to obtain sequence information for the gene for l-GnDH and its surrounding region.
The gene for l-GnDH was found in a nine-gene cluster corresponding to Pden_4924 to Pden_4932 in strain PD1222 with amino acid sequence identities of 71–91% (Fig. 2B), and it was designated lgnA-lgnI. At the cluster's upstream end, we also found a gene corresponding to Pden_4923, termed lgnR, which may function as a transcriptional regulator of the cluster. Based on the Pden gene annotations, the first four genes of the cluster, lgnA–lgnD, presumably encode subunits of an ABC transporter for polar amino acids, whereas lgnE–lgnI encode, respectively, a galactarate dehydratase, a KDGal kinase, a KDPGal aldolase, an alcohol dehydrogenase (l-GnDH), and a short chain dehydrogenase/reductase. This information led us to consider the possibility that l-gluconate can be assimilated by enzymes encoded in this cluster, and this was assessed by analyzing each enzyme activity using purified recombinant enzymes (supplemental Fig. 2C).
l-Gluconate Dehydrogenation by LgnH
As expected, LgnH showed l-GnDH activity (Table 1). The activity was solely dependent on NAD+, not NADP+. The enzyme also showed dehydrogenase activity toward l-galactonate, the C-4 epimer of l-gluconate, whereas no activity was observed toward other epimers, such as d-idonate (C-5) and l-mannonate (C-2) and uronic acids such as d-glucuronate and d-galacturonate. NMR analysis of the reaction product from l-gluconate showed chemical shifts identical to that of commercial d-5-keto-gluconate (supplemental Fig. 3B), whereas HPLC analysis showed opposite optical rotation to d-5-keto-gluconate (Fig. 3B). These data clearly indicate that LgnH catalyzes dehydrogenation of the l-gluconate C-5 hydroxyl group to form l-5-keto-gluconate.
LgnI-mediated l-5-Keto-gluconate Reduction
LgnI is a member of the short chain dehydrogenase/reductase family and is therefore predicted to catalyze NAD(P)+/NAD(P)H-dependent oxidoreductase activity. Consequently, reduction of the purified reaction product of LgnH, l-5-keto-gluconate, was observed with LgnI in an NADPH-dependent manner (Table 1). The reaction product showed the same chemical shifts and peak in NMR and HPLC analyses, respectively, as an authentic d-idonate, the C-5 epimer of l-gluconate (supplemental Fig. 3C and Fig. 3C). Therefore, we concluded that LgnI is responsible for the reduction of l-5-keto-gluconate to form d-idonate.
LgnE-mediated Dehydration of d-Idonate
When LgnE was incubated with d-idonate in the presence of FeCl2 and DTT, the formation of reducing sugars was detected (Table 1). We did not detect any activity toward meso-galactarate, although LgnE showed sequence similarity to galactarate dehydratase (GarD) from E. coli K-12 (38%). NMR and HPLC analyses of the reaction products showed, respectively, the same chemical shifts and peaks as KDGal, which was prepared from d-galactonate using recombinant E. colid-galactonate dehydratase (DgoD) (supplemental Fig. 3D and Fig. 3D). These data indicate that LgnE catalyzes dehydration of d-idonate to produce KDGal.
Phosphorylation and Aldol Cleavage of KDGal by LgnF and LgnG
Because LgnF and LgnG showed amino acid sequence similarity to E. coli DgoK (33%) and DgoA (43%), which catalyze ATP-dependent phosphorylation of KDGal to form KDPGal and aldol-cleavage of KDPGal to form pyruvate and GAP, respectively, we tested their corresponding activities.
As expected, LgnF produced ADP from ATP only in the presence of KDGal, indicating that it phosphorylates KDGal (Table 1). Also, pyruvate and GAP formation was enzymatically detected when LgnG was incubated with KDPGal. These data clearly show that LgnF catalyzes phosphorylation of KDGal to form KDPGal, which is then converted to pyruvate and GAP by LgnG.
Involvement of the Identified Genes in the l-Glucose Catabolic Pathway
To determine how the identified genes participate in the l-glucose catabolic pathway, we constructed gene disruption mutants of lgdA, lgnE, lgnH, and lgnI, based on strain 43P, by inserting a Kmr cassette into the respective genes (supplemental Fig. 4). Growth of cells harboring these disrupted genes in minimal medium containing l-glucose or related compounds was observed. As expected, the ΔlgdA, ΔlgnH, and ΔlgnI mutants did not grow in the presence of “upstream” compounds of the pathway, such as l-glucose for ΔlgdA and l-glucose and l-gluconate for ΔlgnH and ΔlgnI mutants, whereas they could grow using “downstream” compounds (Fig. 4). These results strongly indicate that these genes are responsible for l-glucose catabolism.
In contrast, the ΔlgnE mutant showed rather weak but distinct growth in the presence of each compound tested. It may be possible that strain 43P possesses a paralog to lgnE, as the related strain, PD1222, possesses such a paralog, Pden_4671, which exhibits 53% identity.
Although LgnH utilized l-galactonate as a substrate as well as l-gluconate, none of the gene-disrupted mutants, including the ΔlgnH mutant, showed growth defects with l-galactonate (Fig. 4D), suggesting that these genes are not involved in l-galactonate catabolism (see below). Also, none of the mutants showed growth defects with scyllo- and myo-inositols, except the ΔlgdA mutation affected the growth with scyllo-inositol severely (Fig. 4, E and F). These results indicate that lgdA is required for both l-glucose and scyllo-inositol utilization, whereas the genes in the lgn cluster are not involved in inositol catabolism.
This study sought to describe the l-glucose catabolic pathway in Paracoccus sp. 43P, a model for which is shown in Fig. 5. Based on the gene organization, this pathway could be divided into two parts, oxidation of l-glucose by LgdA and l-gluconate catabolism by enzymes encoded by the lgn cluster.
LgdA showed sequence similarity to the Gfo/IDH/MocA family proteins, and its gene was located in a putative inositol utilization gene cluster of strain 43P. Kinetic analysis of purified LgdA showed efficient oxidation of scyllo-inositol rather than l-glucose, and the ΔlgdA mutation affected the growth with scyllo-inositol. Together, these results strongly indicate that the physiological role of LgdA involves scyllo-inositol catabolism. Among inositol dehydrogenases (IDHs), myo-IDH of Bacillus subtilis (IolG) is the best characterized enzyme, and it was reported to catalyze oxidation of the axial hydroxyl group of myo-inositol and d-chiro-inositol (
), which possess the same stereo-configuration of myo-inositol hydroxyl groups. A β-anomer of l-glucose possesses the same stereo-configuration as scyllo-inositol, i.e. hydroxyl groups occupy all equatorial positions, so it is possible that LgdA utilizes both l-glucose and scyllo-inositol as substrates. There are two scyllo-IDHs that have been characterized at an enzyme level, IolW and IolX from B. subtilis (
Dong-min Kang and Ken-ichi Yoshida, Kobe University, personal communication.
that was about 1/50th that of LgdA. These findings may indicate that LgdA has a unique substrate recognition mechanism.
However, results for the ΔlgdA mutant showed a clear involvement of this gene in l-glucose catabolism. Although l-glucose is an unnatural sugar, these results indicate that, at least for strain 43P, bacterial inositol dehydrogenases may be involved in both inositol and sugar catabolism.
The reactions downstream of the LgdA reaction product, l-gluconate, are analogous to those of the E. colil-galactonate catabolic pathway, which is the C-4 epimer of l-gluconate (
). Both of the l-aldonates are epimerized at the C-5 position by dehydrogenase/reductase reactions (d/l conversion) to produce d-aldonates, converted to d-2-keto-3-deoxyaldonates by dehydratases, phosphorylated at C-6 position by kinases, and then converted by aldolases to the glycolysis substrates pyruvate and GAP. Comparison of the amino acid sequences of the lgn enzymes, LgnH, LgnI, LgnE, LgnF, and LgnG, with those of corresponding enzymes in the E. colil-galactonate pathway, YjjN, UxaB, UxaA, KdgK and Eda, respectively, showed limited sequence identities of about 30%, with LgnI and LgnF in particular showing no similarities to their corresponding proteins. Moreover, the mutant deficient in LgnH or LgnI could not grow on l-gluconate, and it did not show any growth defects on l-galactonate (Fig. 4D). Taken together, we conclude that the lgn cluster enzymes are specific to l-gluconate rather than l-galactonate.
The d/l conversion by the dehydrogenase/reductase reactions are also seen in the catabolism of other six-carbon l-aldonates, such as l-gulonate and l-idonate, in E. coli (
). Like the pathway described in this study, the final products of these catabolic pathways are pyruvate and GAP, where GAP is used as a key intermediate for substrate level phosphorylation in the downstream portion of the Embden-Meyerhof-Parnas pathway. Because l-glyceraldehyde 3-phosphate is not utilized as a substrate by GAPDH, but rather shows a bactericidal effect (
), the d/l conversion may be important for obtaining the GAP d-form following cleavage reactions that are catalyzed by aldolases. In contrast, the fungal l-galactonate catabolic pathway does not involve this process, with l-2-keto-3-deoxygalactonate, formed by l-galactonate dehydratase, being directly cleaved to form pyruvate and l-glyceraldehyde. However, the latter product is not utilized directly but is reduced to an achiral compound, glycerol (
). Therefore, a homochirality issue is likely present in the catabolism of glyceraldehyde, the minimum chiral compound, not in six-carbon sugars. This notion agrees with the recent findings that d-glyceraldehyde might have been selectively synthesized in the prebiotic era, via a formose reaction in the presence of l-amino acids (
Phylogenetic analyses based on the amino acid sequences of the enzymes in this pathway and their related sequences showed that they are located in deeply branched lineages that are distinct from those of enzymes in the corresponding protein families whose functions are known and from KEGG genome database sequences, except for those from P. denitrificans (supplemental Fig. 5). The lineages containing LgdA, LgnF, and LgnG were included in the clusters of Alphaproteobacteria, whereas those of LgnH, LgnI, and LgnE diverged at branch points with clusters including sequences from other bacterial affiliations, i.e. clusters containing sequences mainly from Bacteroidetes for LgnH and LgnI and Betaproteobacteria for LgnE. This divergence may indicate a unique evolutionary origin of this catabolic pathway.
There are several microorganisms in the class Alpha-, Beta-, and Gammaproteobacteria that possess sets of all six orthologs, or five orthologs corresponding to the enzymes for l-gluconate assimilation, which can be assumed as “potential” l-glucose or l-gluconate utilizing organisms from a metabolic pathway standpoint. These orthologs were widely distributed within the clusters of those from the genome sequences, and there are no organisms other than P. denitrificans that possess all such orthologs found in strain 43P in the same or nearby clusters. Most of these organisms likely cannot assimilate l-glucose or l-gluconate, because there are no previous reports on the assimilation of such compounds. Rather, it may be that either no other organisms can utilize such compounds or different catabolic pathways for l-glucose or l-gluconate exist if any of these organisms can indeed utilize them.
P. denitrificans utilized l-gluconate (Fig. 4B) but not l-glucose (Fig. 1). In our preliminary experiments, recombinant Pden_1680 from strain NBRC 102528T showed l-GDH activity similar to LgdA (kcat = 836 ± 12 min−1, Km = 78.0 ± 8.2 mm), indicating that this strain possesses the ability to utilize l-glucose. The reason why P. denitrificans cannot assimilate l-glucose is not known, but it could be because the gene cluster, including Pden_1680, is not transcribed or l-glucose cannot be incorporated into the cells, at least under our culture conditions.
l-Glucose is not found in natural environment, and therefore the physiological role of this catabolic pathway is unknown. However, our data clearly show that the lgn gene cluster is specifically responsible for l-gluconate utilization. This finding may indicate the presence of l-gluconate in nature, which is believed to not exist like l-glucose. We are now attempting to analyze the l-glucose catabolic pathway of the other isolates we obtained, which will lead to a better understanding of l-glucose catabolism.