NAD-Independent L-Lactate Dehydrogenase Required for L-Lactate Utilization in Pseudomonas stutzeri A1501
NAD-Independent L-Lactate Dehydrogenase Required for L-Lactate Utilization in Pseudomonas stutzeri A1501
复制标题
施氏假单胞菌 A1501 中 L-乳酸利用所需的不依赖于 NAD 的 L-乳酸脱氢酶
DOI：
10.1128/jb.00017-15
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发表时间：
2015-07-01
影响因子：
3.2
通讯作者：
Ma, Cuiqing
中科院分区：
文献类型：
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作者：
Gao, Chao;Wang, Yujiao;Ma, Cuiqing
NAD非依赖性L-乳酸脱氢酶（L-iLDH）在不同生物体的L-乳酸利用中起着重要作用。所有先前报道的L-iLDH都是黄素蛋白，其通过黄素单核苷酸肽（FMN）依赖性机制催化L-乳酸的氧化。基于比较基因组分析，在施氏假单胞菌A1501中鉴定出编码新型L-iLDH的具有三个基因（lldA、lldB和lldC）的基因簇。当基因簇在大肠杆菌中表达时，检测到独特的L-iLDH活性。表达的L-iLDH通过硫酸铵沉淀、离子交换层析和亲和层析进行纯化。纯化的L-iLDH的SDS-PAGE和连续基质辅助激光解吸电离飞行时间质谱（MALDI-TOF MS）分析表明，它是LldA、LldB和LldC（分别由lldA、lldB和lldC编码）的复合物。纯化的L-iLDH（LldABC）是三个亚基（LldA、LldB和LldC）的二聚体，并且LldA、LldB和LldC之间的比例为1：1：1。吸收光谱和元素分析结果表明，LldABC可能利用铁硫簇合物进行L-乳酸的氧化。LldABC具有窄的底物特异性，并且仅L-乳酸和DL-2-氢丁酸被快速氧化。Mg ~（2+）能有效激活L-iLDH活性（6.6倍）。稳态动力学表明，乒乓机制的LldABC的L-乳酸氧化。根据基因敲除结果，证实LldABC是斯氏毕赤酵母A1501 L-乳酸代谢所必需的。LldABC是第一个纯化和表征的具有不同亚基的L-iLDH，其使用铁-硫簇作为辅因子。重要信息为微生物乳酸利用的多样性提供新的见解可以帮助生产有价值的化学品和理解微生物的发病机制。由基因簇lldABC编码的NAD非依赖性L-乳酸脱氢酶（L-iLDH）是施氏假单胞菌A1501中L-乳酸代谢所必需的。本研究对这种新型酶进行了纯化和表征。与其他微生物中充分表征的含FMN的L-iLDH不同，施氏毕赤酵母A1501中的LldABC是三个亚基（LldA、LldB和LldC）的二聚体，并使用铁-硫簇作为辅因子。
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. Mg2+ 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.