School of Life Sciences, KNU Creative BioResearch Group (BK21 Plus Program), Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 702-701, Republic of Korea.
Department of Biology, Teachers College, Kyungpook National University, Daehak-ro 80, Buk-ku, Daegu 702-701, Republic of Korea.
Biochem Biophys Res Commun. 2014 May 30;448(2):163-8. doi: 10.1016/j.bbrc.2014.04.101. Epub 2014 Apr 29.
3-Hydroxybutyryl-CoA dehydrogenase is an enzyme involved in the synthesis of the biofuel n-butanol by converting acetoacetyl-CoA to 3-hydroxybutyryl-CoA. To investigate the molecular mechanism of n-butanol biosynthesis, we determined crystal structures of the Ralstonia eutropha-derived 3-hydroxybutyryl-CoA dehydrogenase (RePaaH1) in complex with either its cofactor NAD(+) or its substrate acetoacetyl-CoA. While the biologically active structure is dimeric, the monomer of RePaaH1 comprises two separated domains with an N-terminal Rossmann fold and a C-terminal helical bundle for dimerization. In this study, we show that the cofactor-binding site is located on the Rossmann fold and is surrounded by five loops and one helix. The binding mode of the acetoacetyl-CoA substrate was found to be that the adenosine diphosphate moiety is not highly stabilized compared with the remainder of the molecule. Residues involved in catalysis and substrate binding were further confirmed by site-directed mutagenesis experiments, and kinetic properties of RePaaH1were examined as well. Our findings contribute to the understanding of 3-hydroxybutyryl-CoA dehydrogenase catalysis, and will be useful in enhancing the efficiency of n-butanol biosynthesis by structure based protein engineering.
3-羟丁酰辅酶 A 脱氢酶是一种参与生物燃料正丁醇合成的酶，可将乙酰乙酰辅酶 A 转化为 3-羟丁酰辅酶 A。为了研究正丁醇生物合成的分子机制，我们测定了来源于恶臭假单胞菌的 3-羟丁酰辅酶 A 脱氢酶（RePaaH1）与辅因子 NAD（+）或其底物乙酰乙酰辅酶 A 复合物的晶体结构。虽然生物活性结构是二聚体，但 RePaaH1 的单体由两个分离的结构域组成，一个 N 端罗斯曼折叠和一个 C 端螺旋束用于二聚化。在本研究中，我们表明辅因子结合位点位于罗斯曼折叠上，由五个环和一个螺旋围绕。发现乙酰乙酰辅酶 A 底物的结合模式是腺苷二磷酸部分与分子的其余部分相比没有得到高度稳定。通过定点突变实验进一步证实了催化和底物结合的残基，并研究了 RePaaH1 的动力学特性。我们的研究结果有助于理解 3-羟丁酰辅酶 A 脱氢酶的催化作用，并将有助于通过基于结构的蛋白质工程提高正丁醇生物合成的效率。
