ABSTRACTNAD-independentl-lactate dehydrogenases (l-iLDHs) play important roles inl-lactate utilization of different organisms. All of the previously reportedl-iLDHs were flavoproteins that catalyze the oxidation ofl-lactate by the flavin mononucleotide (FMN)-dependent mechanism. Based on comparative genomic analysis, a gene cluster with three genes (lldA,lldB, andlldC) encoding a novel type ofl-iLDH was identified inPseudomonas stutzeriA1501. When the gene cluster was expressed inEscherichia coli, distinctivel-iLDH activity was detected. The expressedl-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 purifiedl-iLDH indicated that it is a complex of LldA, LldB, and LldC (encoded bylldA,lldB, andlldC, respectively). Purifiedl-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-containingl-iLDH, absorption spectra and elemental analysis suggested that LldABC might use the iron-sulfur cluster for thel-lactate oxidation. LldABC has narrow substrate specificity, and onlyl-lactate anddl-2-hydrobutyrate were rapidly oxidized. Mg2+could activatel-iLDH activity effectively (6.6-fold). Steady-state kinetics indicated a ping-pong mechanism of LldABC for thel-lactate oxidation. Based on the gene knockout results, LldABC was confirmed to be required for thel-lactate metabolism ofP. stutzeriA1501. LldABC is the first purified and characterizedl-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-independentl-lactate dehydrogenase (l-iLDH) encoded by the gene clusterlldABCis indispensable for thel-lactate metabolism inPseudomonas stutzeriA1501. This novel type of enzyme was purified and characterized in this study. Different from the well-characterized FMN-containingl-iLDH in other microbes, LldABC inP. stutzeriA1501 is a dimer of three subunits (LldA, LldB, and LldC) and uses the iron-sulfur cluster as a cofactor.