An L-glucose catabolic pathway in Paracoccus species 43P

Abstract

Background: L-Glucose, the enantiomer of D-glucose, was believed not to be utilized by any organisms.

Results: An L-glucose-utilizing bacterium was isolated, and its L-glucose catabolic pathway was identified genetically and enzymatically.

Conclusion: L-Glucose was utilized via a novel pathway to pyruvate and D-glyceraldehyde 3-phosphate.

Significance: This might lead to an understanding of homochirality in sugar metabolism. 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.

FIGURE 1.

Growth (open symbols) and l-glucose consumption (closed symbols) of strains 43P (circles) and NBRC 102528^T (triangles) in l-Glc MM. Growth was monitored by measuring the absorbance at 600 nm. The DNS method was used to determine the l-glucose concentration by measuring the reducing sugars in the culture medium of strains 43P and NBRC 102528^T. Cultivation was conducted in three independent cultures, and average values ± S.D. are shown.

FIGURE 2.

Gene organization of the clusters containing lgdA (A) and lgn (B). Genes encoding the enzymes for l-glucose catabolism are shaded, and sequence identities to the corresponding P. denitrificans PD1222 genes are shown below.

FIGURE 3.

HPLC analysis of LgdA (A), LgnH (B), LgnI (C), and LgnE (D) reaction products detected by a refractive index (RI) and chiral detector (OR). Black and gray lines indicate chromatograms of the reaction products and authentic compounds l-gluconate (A), d-5-keto-gluconate (B), d-idonate (C), and KDGal (D), respectively.

FIGURE 4.

Growth of strain 43P and gene disruption mutants in minimal media containing l-glucose (A), l-gluconate (B), d-idonate (C), l-galactonate (D), scyllo-inositol (E), and myo-inositol (F). Open circles, strain 43P; closed circles, ΔlgdA strain; open triangles, ΔlgnE strain; closed triangles, ΔlgnH strain; open squares, ΔlgnI strain. B, the growth of strain NBRC 102528^T is also shown (closed squares). Cultivation was conducted in three independent cultures, and average values ± S.D. are shown.

FIGURE 5.

Model of the l-glucose catabolic pathway in strain 43P based on the results of this study. Altered moieties in each enzyme reaction are shown in gray.