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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.   IMPORTANCE 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.

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