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Rationally re-designed mutation of NAD-independent L-lactate dehydrogenase: high optical resolution of racemic mandelic acid by the engineered Escherichia coli.

ABSTRACT: BACKGROUND: NAD-independent L-lactate dehydrogenase (L-iLDH) from Pseudomonas stutzeri SDM can potentially be used for the kinetic resolution of small aliphatic 2-hydroxycarboxylic acids. However, this enzyme showed rather low activity towards aromatic 2-hydroxycarboxylic acids. RESULTS: Val-108 of L-iLDH was changed to Ala by rationally site-directed mutagenesis. The L-iLDH mutant exhibited much higher activity than wide-type L-iLDH towards L-mandelate, an aromatic 2-hydroxycarboxylic acid. Using the engineered Escherichia coli expressing the mutant L-iLDH as a biocatalyst, 40 g·L(-1) of DL-mandelic acid was converted to 20.1 g·L(-1) of D-mandelic acid (enantiomeric purity higher than 99.5%) and 19.3 g·L(-1) of benzoylformic acid. CONCLUSIONS: A new biocatalyst with high catalytic efficiency toward an unnatural substrate was constructed by rationally re-design mutagenesis. Two building block intermediates (optically pure D-mandelic acid and benzoylformic acid) were efficiently produced by the one-pot biotransformation system.

SUBMITTER: Jiang T

PROVIDER: S-EPMC3526519 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Rationally re-designed mutation of NAD-independent L-lactate dehydrogenase: high optical resolution of racemic mandelic acid by the engineered Escherichia coli.

Jiang Tianyi T Gao Chao C Dou Peipei P Ma Cuiqing C Kong Jian J Xu Ping P

Microbial cell factories 20121123

<h4>Background</h4>NAD-independent L-lactate dehydrogenase (L-iLDH) from Pseudomonas stutzeri SDM can potentially be used for the kinetic resolution of small aliphatic 2-hydroxycarboxylic acids. However, this enzyme showed rather low activity towards aromatic 2-hydroxycarboxylic acids.<h4>Results</h4>Val-108 of L-iLDH was changed to Ala by rationally site-directed mutagenesis. The L-iLDH mutant exhibited much higher activity than wide-type L-iLDH towards L-mandelate, an aromatic 2-hydroxycarboxy ...[more]

PMID: 23176608

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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

Rationally re-designed mutation of NAD-independent L-lactate dehydrogenase: high optical resolution of racemic mandelic acid by the engineered Escherichia coli.

Publications

Rationally re-designed mutation of NAD-independent L-lactate dehydrogenase: high optical resolution of racemic mandelic acid by the engineered Escherichia coli.

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