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

Abstract

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.

Coexistence of two d‐lactate‐utilizing systems in Pseudomonas putida KT2440

“…Thus, the exact functions of D-iLDH and glycolate oxidase in bacteria would require a case-by-case investigation. Furthermore, considering the important roles of iLDHs in microbial lactate utilization, the pathogenicity of several microbes lactate determination and biocatalysis processes (Iwuoha et al, 1999;Fuller et al, 2011;Gao et al, 2011;Jiang et al, 2014;Gao et al, 2015;Sheng et al, 2015), more experimental verification of iLDHs remains to be performed.…”

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

“…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).…”

Relative catalytic efficiencies and transcript levels of three d‐ and two l‐lactate dehydrogenases for optically pure d‐lactate production in Sporolactobacillus inulinus

“…coagulans L‐LDH at the highest degree. However, Gao et al., found that Mg 2+ exhibited the highest activation capacity for Pseudomonas stutzeri L‐LDH activity.…”

“…In addition, when KOH and NH 4 OH were used as neutralizers, the significant increase in ldhL1 transcription was correlated with a marked reduction in optical purity of d-lactate to 93.7% and 91.3%, which suggested that only L-LDH1 was the key factor affecting optical purity of d-lactate produced by S. inulinus YBS1-5. In addition, many previous reports have revealed that l-lactate permeases are involved in l-lactate utilization in different strains (Gao et al, 2015;Jiang, Gao, Ma, & Xu, 2014). Guided by the whole-genome sequence of S. inulinus CASD, we measured the transcription levels of l-lactate permease-encoding gene (lldP) under different neutralizers.…”