Bayer M, Günther H, Simon H
Institute for Organic Chemistry, Technical University, Munich, Garching, Germany.
Appl Microbiol Biotechnol. 1994 Oct;42(1):40-5. doi: 10.1007/BF00170222.
An NADP(+)-dependent reversible 3-hydroxycarboxylate oxidoreductase present in Clostridium tyrobutyricum has been purified. As judged by gel electrophoresis the enzyme was pure after a 940-fold enrichment by four chromatographic steps. Its molecular mass was estimated to be 40-43 kDa. The enzyme was most active at pH 4.5 in the reduction of 3-oxobutyrate. Other substrates were 3-oxovalerate, 3-oxocaproate, 3-oxoisocaproate and 4-chloro-3-oxobutyrate. Except for the latter all substrates were converted enantioselectively to (S)-3-hydroxy acids in the presence of NADPH. 4-Chloro-3-oxobutyrate was reduced to the (R)-3-hydroxy acid. The specific activity of the enzyme was about 1400 mumol min-1 mg-1 protein for the reduction of 3-oxobutyrate at pH 5.0. The Michaelis constant (Km) values for 3-oxobutyrate, 3-oxovalerate and 3-oxocaproate were determined to be 0.22, 1.6 and 3.0 mM, respectively. The Km values for dehydrogenation of (S)-3-hydroxybutyrate, (S)-3-hydroxyvalerate and (S)-3-hydroxycaproate were found to be 2.6, 1.1 and 5.2 mM, respectively. The identity of 43 of the first 45 N-terminal amino acid residues has been determined. So far such enzyme activities have been described in eucaryotes only.
已对存在于酪丁酸梭菌中的一种依赖烟酰胺腺嘌呤二核苷酸磷酸（NADP⁺）的可逆3-羟基羧酸盐氧化还原酶进行了纯化。通过凝胶电泳判断，经四个色谱步骤富集940倍后该酶达到了纯品状态。其分子量估计为40 - 43 kDa。该酶在pH 4.5时还原3-氧代丁酸的活性最高。其他底物包括3-氧代戊酸、3-氧代己酸、3-氧代异己酸和4-氯-3-氧代丁酸。除了后者，在烟酰胺腺嘌呤二核苷酸磷酸（NADPH）存在的情况下，所有底物都能对映选择性地转化为（S）-3-羟基酸。4-氯-3-氧代丁酸被还原为（R）-3-羟基酸。在pH 5.0时，该酶还原3-氧代丁酸的比活性约为1400 μmol min⁻¹ mg⁻¹蛋白质。已确定3-氧代丁酸、3-氧代戊酸和3-氧代己酸的米氏常数（Km）值分别为0.22、1.6和3.0 mM。已发现（S）-3-羟基丁酸、（S）-3-羟基戊酸和（S）-3-羟基己酸脱氢的Km值分别为2.6、1.1和5.2 mM。已确定了前45个N端氨基酸残基中43个的序列。到目前为止，这种酶活性仅在真核生物中有过描述。
