Efficient Production of 2-Oxobutyrate from 2-Hydroxybutyrate by Using Whole Cells of
            <i>Pseudomonas stutzeri</i>
            Strain SDM

Gao, Chao; Zhang, Wen; Lv, Chuanjuan; Li, Lixiang; Ma, Cuiqing; Hu, Chunhui; Xu, Ping

doi:10.1128/aem.02470-09

Cited by 24 publications

(16 citation statements)

References 10 publications

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“…Analytical Methods. The reaction mixtures mentioned above were analyzed by HPLC (Agilent 1100 series; Hewlett-Packard) using a refractive index detector, as described (48). Generally, a Bio-Rad Aminex HPX-87H column was used and the mobile phase (10 mM H 2 SO 4 ) was pumped at 0.4 mL•min −1 (55°C).…”

Section: Methodsmentioning

confidence: 99%

Coupling between d -3-phosphoglycerate dehydrogenase and d -2-hydroxyglutarate dehydrogenase drives bacterial l -serine synthesis

Zhang

Gao

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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l-Serine biosynthesis, a crucial metabolic process in most domains of life, is initiated by d-3-phosphoglycerate (d-3-PG) dehydrogenation, a thermodynamically unfavorable reaction catalyzed by d-3-PG dehydrogenase (SerA). d-2-Hydroxyglutarate (d-2-HG) is traditionally viewed as an abnormal metabolite associated with cancer and neurometabolic disorders. Here, we reveal that bacterial anabolism and catabolism of d-2-HG are involved in l-serine biosynthesis in A1501 and PAO1. SerA catalyzes the stereospecific reduction of 2-ketoglutarate (2-KG) to d-2-HG, responsible for the major production of d-2-HG in vivo. SerA combines the energetically favorable reaction of d-2-HG production to overcome the thermodynamic barrier of d-3-PG dehydrogenation. We identified a bacterial d-2-HG dehydrogenase (D2HGDH), a flavin adenine dinucleotide (FAD)-dependent enzyme, that converts d-2-HG back to 2-KG. Electron transfer flavoprotein (ETF) and ETF-ubiquinone oxidoreductase (ETFQO) are also essential in d-2-HG metabolism through their capacity to transfer electrons from D2HGDH. Furthermore, while the mutant with D2HGDH deletion displayed decreased growth, the defect was rescued by adding l-serine, suggesting that the D2HGDH is functionally tied to l-serine synthesis. Substantial flux flows through d-2-HG, being produced by SerA and removed by D2HGDH, ETF, and ETFQO, maintaining d-2-HG homeostasis. Overall, our results uncover that d-2-HG-mediated coupling between SerA and D2HGDH drives bacterial l-serine synthesis.

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Section: Methodsmentioning

confidence: 99%

Coupling between d -3-phosphoglycerate dehydrogenase and d -2-hydroxyglutarate dehydrogenase drives bacterial l -serine synthesis

Zhang

Gao

et al. 2017

Proc. Natl. Acad. Sci. U.S.A.

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“…After 4 h of reaction, the mixture was centrifuged at 20,000 ϫ g for 15 min. The supernatant was analyzed by high-performance liquid chromatography (HPLC) (Agilent 1100 series; Hewlett-Packard) equipped with an Aminex HPX-87H column (Bio-Rad) (20). The mobile phase (10 mM H 2 SO 4 ) was pumped at 0.4 ml min Ϫ1 (55°C).…”

Section: Methodsmentioning

confidence: 99%

“…Those enzymes catalyze the oxidation of L-lactate by a flavin mononucleotide (FMN)-dependent mechanism and share remarkable similarities with other flavoproteins catalyzing the oxidation of L-2-hydroxy acids, such as glycolate oxidase, long-chain ␣-hydroxy acid oxidase, and L-mandelate dehydrogenases (13)(14)(15)(16)(17)(18). Many biocatalysts and L-lactate determination techniques have been developed based on these enzymes (19)(20)(21)(22)(23).…”

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confidence: 99%

NAD-Independent l-Lactate Dehydrogenase Required for l-Lactate Utilization in Pseudomonas stutzeri A1501

Gao¹,

Wang²,

Zhang³

et al. 2015

J. Bacteriol.

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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. IMPORTANCEProviding 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.

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“…Pseudomonas is a vast bacterial genus in which various species have the ability to utilize lactate for growth (27)(28)(29). Several Pseudomonas genes related to lactate utilization have been identified, and several of these enzymes have been characterized and used in biocatalysis processes (23,(30)(31)(32)(33). Homologs of Fe-S D-iLDH seem to be present in all the identified lactate utilization operons of Pseudomonas (29).…”

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confidence: 99%

A Bacterial Multidomain NAD-Independent d -Lactate Dehydrogenase Utilizes Flavin Adenine Dinucleotide and Fe-S Clusters as Cofactors and Quinone as an Electron Acceptor for d -Lactate Oxidization

et al. 2017

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Bacterial membrane-associated NAD-independent d-lactate dehydrogenase (Fe-S d-iLDH) oxidizes d-lactate into pyruvate. A sequence analysis of the enzyme reveals that it contains an Fe-S oxidoreductase domain in addition to a flavin adenine dinucleotide (FAD)-containing dehydrogenase domain, which differs from other typical d-iLDHs. Fe-S d-iLDH from KT2440 was purified as a His-tagged protein and characterized in detail. This monomeric enzyme exhibited activities with l-lactate and several d-2-hydroxyacids. Quinone was shown to be the preferred electron acceptor of the enzyme. The two domains of the enzyme were then heterologously expressed and purified separately. The Fe-S cluster-binding motifs predicted by sequence alignment were preliminarily verified by site-directed mutagenesis of the Fe-S oxidoreductase domain. The FAD-containing dehydrogenase domain retained 2-hydroxyacid-oxidizing activity, although it decreased compared to the full Fe-S d-iLDH. Compared to the intact enzyme, the FAD-containing dehydrogenase domain showed increased catalytic efficiency with cytochrome as the electron acceptor, but it completely lost the ability to use coenzyme Q Additionally, the FAD-containing dehydrogenase domain was no longer associated with the cell membrane, and it could not support the utilization of d-lactate as a carbon source. Based on the results obtained, we conclude that the Fe-S oxidoreductase domain functions as an electron transfer component to facilitate the utilization of quinone as an electron acceptor by Fe-S d-iLDH, and it helps the enzyme associate with the cell membrane. These functions make the Fe-S oxidoreductase domain crucial for the d-lactate utilization function of Fe-S d-iLDH. Lactate metabolism plays versatile roles in most domains of life. Lactate utilization processes depend on certain enzymes to oxidize lactate to pyruvate. In recent years, novel bacterial lactate-oxidizing enzymes have been continually reported, including the unique NAD-independent d-lactate dehydrogenase that contains an Fe-S oxidoreductase domain besides the typical flavin-containing domain (Fe-S d-iLDH). Although Fe-S d-iLDH is widely distributed among bacterial species, the investigation of it is insufficient. Fe-S d-iLDH from KT2440, which is the major d-lactate-oxidizing enzyme for the strain, might be a representative of this type of enzyme. A study of it will be helpful in understanding the detailed mechanisms underlying the lactate utilization processes.

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Efficient Production of 2-Oxobutyrate from 2-Hydroxybutyrate by Using Whole Cells of Pseudomonas stutzeri Strain SDM

Cited by 24 publications

References 10 publications

Coupling between d -3-phosphoglycerate dehydrogenase and d -2-hydroxyglutarate dehydrogenase drives bacterial l -serine synthesis

Coupling between d -3-phosphoglycerate dehydrogenase and d -2-hydroxyglutarate dehydrogenase drives bacterial l -serine synthesis

NAD-Independent l-Lactate Dehydrogenase Required for l-Lactate Utilization in Pseudomonas stutzeri A1501

A Bacterial Multidomain NAD-Independent d -Lactate Dehydrogenase Utilizes Flavin Adenine Dinucleotide and Fe-S Clusters as Cofactors and Quinone as an Electron Acceptor for d -Lactate Oxidization

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