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


OBJECTIVE
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.
State Key Laboratory of Microbial Technology, Shandong University, Jinan, People's Republic of China.
Chao
C
Gao
State Key Laboratory of Microbial Technology, Shandong University, Jinan, People's Republic of China.
Yujiao
Y
Wang
State Key Laboratory of Microbial Technology, Shandong University, Jinan, People's Republic of China.
Yingxin
Y
Zhang
State Key Laboratory of Microbial Technology, Shandong University, Jinan, People's Republic of China.
Min
M
Lv
State Key Laboratory of Microbial Technology, Shandong University, Jinan, People's Republic of China.
Peipei
P
Dou
State Key Laboratory of Microbial Metabolism and School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, People's Republic of China.
Ping
P
Xu
State Key Laboratory of Microbial Technology, Shandong University, Jinan, People's Republic of China macq@sdu.edu.cn.
Cuiqing
C
Ma
D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D015971:Gene Expression Regulation, Enzymologic / Q000502:physiology*; D006863:Hydrogen-Ion Concentration; D042844:L-Lactate Dehydrogenase (Cytochrome) / Q000235:genetics / Q000378:metabolism*; D019344:Lactic Acid / Q000378:metabolism*; D044223:Pseudomonas stutzeri / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D013696:Temperature
D019344:Lactic Acid; D042844:L-Lactate Dehydrogenase (Cytochrome)