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D-2-Hydroxyglutarate dehydrogenase plays a dual role in l-serine biosynthesis and d-malate utilization in the bacterium Pseudomonas stutzeri.

ABSTRACT: Pseudomonas is a very large bacterial genus in which several species can use d-malate for growth. However, the enzymes that can metabolize d-malate, such as d-malate dehydrogenase, appear to be absent in most Pseudomonas species. d-3-Phosphoglycerate dehydrogenase (SerA) can catalyze the production of d-2-hydroxyglutarate (d-2-HG) from 2-ketoglutarate to support d-3-phosphoglycerate dehydrogenation, which is the initial reaction in bacterial l-serine biosynthesis. In this study, we show that SerA of the Pseudomonas stutzeri strain A1501 reduces oxaloacetate to d-malate and that d-2-HG dehydrogenase (D2HGDH) from P. stutzeri displays d-malate-oxidizing activity. Of note, D2HGDH participates in converting a trace amount of d-malate to oxaloacetate during bacterial l-serine biosynthesis. Moreover, D2HGDH is crucial for the utilization of d-malate as the sole carbon source for growth of P. stutzeri A1501. We also found that the D2HGDH expression is induced by the exogenously added d-2-HG or d-malate and that a flavoprotein functions as a soluble electron carrier between D2HGDH and electron transport chains to support d-malate utilization by P. stutzeri These results support the idea that D2HGDH evolves as an enzyme for both d-malate and d-2-HG dehydrogenation in P. stutzeri In summary, D2HGDH from P. stutzeri A1501 participates in both a core metabolic pathway for l-serine biosynthesis and utilization of extracellular d-malate.

SUBMITTER: Guo X

PROVIDER: S-EPMC6177604 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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d-2-Hydroxyglutarate dehydrogenase plays a dual role in l-serine biosynthesis and d-malate utilization in the bacterium Pseudomonas stutzeri.

Guo Xiaoting X Zhang Manman M Cao Menghao M Zhang Wen W Kang Zhaoqi Z Xu Ping P Ma Cuiqing C Gao Chao C

The Journal of biological chemistry 20180821 40

Pseudomonas is a very large bacterial genus in which several species can use d-malate for growth. However, the enzymes that can metabolize d-malate, such as d-malate dehydrogenase, appear to be absent in most Pseudomonas species. d-3-Phosphoglycerate dehydrogenase (SerA) can catalyze the production of d-2-hydroxyglutarate (d-2-HG) from 2-ketoglutarate to support d-3-phosphoglycerate dehydrogenation, which is the initial reaction in bacterial l-serine biosynthesis. In this study, we ...[more]

PMID: 30131334

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Project description:NAD-independent L-lactate dehydrogenases (l-iLDHs) play important roles in L-lactate utilization of different organisms. All of the previously reported L-iLDHs were flavoproteins that catalyze the oxidation of L-lactate by the flavin mononucleotide (FMN)-dependent mechanism. Based on comparative genomic analysis, a gene cluster with three genes (lldA, lldB, and lldC) encoding a novel type of L-iLDH was identified in Pseudomonas stutzeri A1501. When the gene cluster was expressed in Escherichia coli, distinctive L-iLDH activity was detected. The expressed L-iLDH was purified by ammonium sulfate precipitation, ion-exchange chromatography, and affinity chromatography. SDS-PAGE and successive matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) analysis of the purified L-iLDH indicated that it is a complex of LldA, LldB, and LldC (encoded by lldA, lldB, and lldC, respectively). Purified L-iLDH (LldABC) is a dimer of three subunits (LldA, LldB, and LldC), and the ratio between LldA, LldB, and LldC is 1:1:1. Different from the FMN-containing L-iLDH, absorption spectra and elemental analysis suggested that LldABC might use the iron-sulfur cluster for the L-lactate oxidation. LldABC has narrow substrate specificity, and only L-lactate and DL-2-hydrobutyrate were rapidly oxidized. Mg(2+) could activate L-iLDH activity effectively (6.6-fold). Steady-state kinetics indicated a ping-pong mechanism of LldABC for the L-lactate oxidation. Based on the gene knockout results, LldABC was confirmed to be required for the L-lactate metabolism of P. stutzeri A1501. LldABC is the first purified and characterized L-iLDH with different subunits that uses the iron-sulfur cluster as the cofactor.Providing new insights into the diversity of microbial lactate utilization could assist in the production of valuable chemicals and understanding microbial pathogenesis. An NAD-independent L-lactate dehydrogenase (L-iLDH) encoded by the gene cluster lldABC is indispensable for the L-lactate metabolism in Pseudomonas stutzeri A1501. This novel type of enzyme was purified and characterized in this study. Different from the well-characterized FMN-containing L-iLDH in other microbes, LldABC in P. stutzeri A1501 is a dimer of three subunits (LldA, LldB, and LldC) and uses the iron-sulfur cluster as a cofactor.

Dataset Information

D-2-Hydroxyglutarate dehydrogenase plays a dual role in l-serine biosynthesis and d-malate utilization in the bacterium Pseudomonas stutzeri.

Publications

d-2-Hydroxyglutarate dehydrogenase plays a dual role in l-serine biosynthesis and d-malate utilization in the bacterium <i>Pseudomonas stutzeri</i>.

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