Tiffany K A, Roberts D L, Wang M, Paschke R, Mohsen A W, Vockley J, Kim J J
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, Wisconsin 53226, USA.
Biochemistry. 1997 Jul 15;36(28):8455-64. doi: 10.1021/bi970422u.
Isovaleryl-CoA dehydrogenase (IVD) belongs to an important flavoprotein family of acyl-CoA dehydrogenases that catalyze the alpha,beta-dehydrogenation of their various thioester substrates. Although enzymes from this family share similar sequences, catalytic mechanisms, and structural properties, the position of the catalytic base in the primary sequence is not conserved. E376 has been confirmed to be the catalytic base in medium-chain (MCAD) and short-chain acyl-CoA dehydrogenases and is conserved in all members of the acyl-CoA dehydrogenase family except for IVD and long-chain acyl-CoA dehydrogenase. To understand this dichotomy and to gain a better understanding of the factors important in determining substrate specificity in this enzyme family, the three-dimensional structure of human IVD has been determined. Human IVD expressed in Escherichia coli crystallizes in the orthorhombic space group P212121 with unit cell parameters a = 94.0 A, b = 97.7 A, and c = 181.7 A. The structure of IVD was solved at 2.6 A resolution by the molecular replacement method and was refined to an R-factor of 20.7% with an Rfree of 28.8%. The overall polypeptide fold of IVD is similar to that of other members of this family for which structural data are available. The tightly bound ligand found in the active site of the structure of IVD is consistent with that of CoA persulfide. The identity of the catalytic base was confirmed to be E254, in agreement with previous molecular modeling and mutagenesis studies. The location of the catalytic residue together with a glycine at position 374, which is a tyrosine in all other members of the acyl-CoA dehydrogenase family, is important for conferring branched-chain substrate specificity to IVD.
异戊酰辅酶A脱氢酶（IVD）属于酰基辅酶A脱氢酶这一重要的黄素蛋白家族，该家族催化各种硫酯底物的α,β-脱氢反应。尽管这个家族的酶具有相似的序列、催化机制和结构特性，但催化碱基在一级序列中的位置并不保守。E376已被确认为中链（MCAD）和短链酰基辅酶A脱氢酶中的催化碱基，并且在酰基辅酶A脱氢酶家族的所有成员中都保守，除了IVD和长链酰基辅酶A脱氢酶。为了理解这种二分法，并更好地了解决定该酶家族底物特异性的重要因素，已确定了人IVD的三维结构。在大肠杆菌中表达的人IVD在正交空间群P212121中结晶，晶胞参数为a = 94.0 Å，b = 97.7 Å，c = 181.7 Å。通过分子置换法以2.6 Å的分辨率解析了IVD的结构，并将其精修至R因子为20.7%，Rfree为28.8%。IVD的整体多肽折叠与该家族其他有结构数据的成员相似。在IVD结构的活性位点发现的紧密结合配体与过硫化辅酶A一致。催化碱基的身份被确认为E254，这与之前的分子建模和诱变研究一致。催化残基的位置以及374位的甘氨酸（在酰基辅酶A脱氢酶家族的所有其他成员中该位置是酪氨酸）对于赋予IVD支链底物特异性很重要。
