Baldock C, Rafferty J B, Stuitje A R, Slabas A R, Rice D W
Krebs Institute for Biomolecular Research, Department of Molecular Biology and Biotechnology, The University of Sheffield, UK.
J Mol Biol. 1998 Dec 18;284(5):1529-46. doi: 10.1006/jmbi.1998.2271.
Enoyl acyl carrier protein reductase catalyses the last reductive step of fatty acid biosynthesis, reducing an enoyl acyl carrier protein to an acyl-acyl carrier protein with NAD(P)H as the cofactor. The crystal structure of enoyl reductase (ENR) from Escherichia coli has been determined to 2.1 A resolution using a combination of molecular replacement and isomorphous replacement and refined using data from 10 A to 2.1 A to an R-factor of 0.16. The final model consists of the four subunits of the tetramer, wherein each subunit is composed of 247 of the expected 262 residues, and a NAD+ cofactor for each subunit of the tetramer contained in the asymmetric unit plus a total of 327 solvent molecules. There are ten disordered residues per subunit which form a loop near the nucleotide binding site which may become ordered upon substrate binding. Each monomer is composed of a seven-stranded parallel beta-sheet flanked on each side by three alpha-helices with a further helix lying at the C terminus of the beta-sheet. This fold is highly reminiscent of the Rossmann fold, found in many NAD(P)H-dependent enzymes. Analysis of the sequence and structure of ENR and comparisons with the family of short-chain alcohol dehydrogenases, identify a conserved tyrosine and lysine residue as important for catalytic activity. Modelling studies suggest that a region of the protein surface that contains a number of strongly conserved hydrophobic residues and lies adjacent to the nicotinamide ring, forms the binding site for the fatty acid substrate.
烯酰基载体蛋白还原酶催化脂肪酸生物合成的最后一步还原反应，以NAD(P)H为辅因子，将烯酰基载体蛋白还原为酰基 - 酰基载体蛋白。利用分子置换和同晶置换相结合的方法，已将来自大肠杆菌的烯酰还原酶（ENR）的晶体结构解析到2.1 Å的分辨率，并使用10 Å至2.1 Å的数据进行精修，最终R因子为0.16。最终模型由四聚体的四个亚基组成，其中每个亚基由预期的262个残基中的247个组成，不对称单元中四聚体的每个亚基含有一个NAD+辅因子，外加总共327个溶剂分子。每个亚基有10个无序残基，在核苷酸结合位点附近形成一个环，该环在底物结合时可能会变得有序。每个单体由一个七链平行β - 折叠组成，两侧各有三个α - 螺旋，在β - 折叠的C末端还有一个螺旋。这种折叠与许多依赖NAD(P)H的酶中发现的Rossmann折叠非常相似。对ENR的序列和结构进行分析，并与短链醇脱氢酶家族进行比较，确定一个保守的酪氨酸和赖氨酸残基对催化活性很重要。建模研究表明，蛋白质表面的一个区域含有许多高度保守的疏水残基，且与烟酰胺环相邻，形成脂肪酸底物的结合位点。
