Kim Jihoe, Darley Daniel, Selmer Thorsten, Buckel Wolfgang
Laboratorium für Mikrobiologie, Fachbereich Biologie, Philipps-Universität, D-35032 Marburg, Germany.
Appl Environ Microbiol. 2006 Sep;72(9):6062-9. doi: 10.1128/AEM.00772-06.
The strictly anaerobic pathogenic bacterium Clostridium difficile occurs in the human gut and is able to thrive from fermentation of leucine. Thereby the amino acid is both oxidized to isovalerate plus CO(2) and reduced to isocaproate. In the reductive branch of this pathway, the dehydration of (R)-2-hydroxyisocaproyl-coenzyme A (CoA) to (E)-2-isocaprenoyl-CoA is probably catalyzed via radical intermediates. The dehydratase requires activation by an ATP-dependent one-electron transfer (J. Kim, D. Darley, and W. Buckel, FEBS J. 272:550-561, 2005). Prior to the dehydration, a dehydrogenase and a CoA transferase are supposed to be involved in the formation of (R)-2-hydroxyisocaproyl-CoA. Deduced amino acid sequences of ldhA and hadA from the genome of C. difficile showed high identities to d-lactate dehydrogenase and family III CoA transferase, respectively. Both putative genes encoding the dehydrogenase and CoA transferase were cloned and overexpressed in Escherichia coli; the recombinant Strep tag II fusion proteins were purified to homogeneity and characterized. The substrate specificity of the monomeric LdhA (36.5 kDa) indicated that 2-oxoisocaproate (K(m) = 68 muM, k(cat) = 31 s(-1)) and NADH were the native substrates. For the reverse reaction, the enzyme accepted (R)- but not (S)-2-hydroxyisocaproate and therefore was named (R)-2-hydroxyisocaproate dehydrogenase. HadA showed CoA transferase activity with (R)-2-hydroxyisocaproyl-CoA as a donor and isocaproate or (E)-2-isocaprenoate as an acceptor. By site-directed mutagenesis, the conserved D171 was identified as an essential catalytic residue probably involved in the formation of a mixed anhydride with the acyl group of the thioester substrate. However, neither hydroxylamine nor sodium borohydride, both of which are inactivators of the CoA transferase, modified this residue. The dehydrogenase and the CoA transferase fit well into the proposed pathway of leucine reduction to isocaproate.
严格厌氧的病原菌艰难梭菌存在于人体肠道中，能够利用亮氨酸发酵而蓬勃生长。在此过程中，亮氨酸既被氧化为异戊酸和二氧化碳，又被还原为异己酸。在该途径的还原分支中，（R）-2-羟基异己酰辅酶A（CoA）脱水生成（E）-2-异己烯酰辅酶A可能是通过自由基中间体催化的。这种脱水酶需要通过ATP依赖的单电子转移来激活（J. Kim、D. Darley和W. Buckel，《欧洲生物化学学会联合会杂志》272:550 - 561，2005年）。在脱水之前，一种脱氢酶和一种CoA转移酶被认为参与了（R）-2-羟基异己酰辅酶A的形成。从艰难梭菌基因组中推导的ldhA和hadA的氨基酸序列分别与d-乳酸脱氢酶和III族CoA转移酶具有高度同源性。编码脱氢酶和CoA转移酶的两个假定基因都被克隆并在大肠杆菌中过表达；重组的链霉亲和素标签II融合蛋白被纯化至均一性并进行了表征。单体LdhA（36.5 kDa）的底物特异性表明，2-氧代异己酸（K(m)=68 μM，k(cat)=31 s(-1)）和NADH是天然底物。对于逆反应，该酶接受（R）-2-羟基异己酸而不接受（S）-2-羟基异己酸，因此被命名为（R）-2-羟基异己酸脱氢酶。HadA以（R）-2-羟基异己酰辅酶A作为供体，异己酸或（E）-2-异己烯酸作为受体时表现出CoA转移酶活性。通过定点诱变，保守的D171被鉴定为一个必需的催化残基，可能参与与硫酯底物的酰基形成混合酸酐。然而，作为CoA转移酶失活剂的羟胺和硼氢化钠都没有修饰这个残基。脱氢酶和CoA转移酶与所提出的亮氨酸还原为异己酸的途径非常吻合。
