Lah M S, Palfey B A, Schreuder H A, Ludwig M L
Department of Biological Chemistry, University of Michigan, Ann Arbor 48109.
Biochemistry. 1994 Feb 15;33(6):1555-64. doi: 10.1021/bi00172a036.
Structures of the mutant p-hydroxybenzoate hydroxylases, Tyr201Phe, Tyr385Phe, and Asn300Asp, each complexed with the substrate p-OHB have been determined by X-ray crystallography. Crystals of these three mutants of the Pseudomonas aeruginosa enzyme, which differs from the wild-type Pseudomonas fluorescens enzyme at two surface positions (228 and 249), were isomorphous with crystals of the wild-type P. fluorescens enzyme, allowing the mutant structures to be determined by model building and refinement, starting from the coordinates for the oxidized P. fluorescens PHBH-3,4-diOHB complex [Schreuder, H.A., van der Laan, J.M., Hol, W.G.J., & Drenth, J. (1988) J. Mol. Biol. 199, 637-648]. The R factors for the structures described here are: Tyr385Phe, 0.178 for data from 40.0 to 2.1 A; Tyr201Phe, 0.203 for data from 40.0 to 2.3 A; and Asn300Asp, 0.193 for data from 40.0 to 2.3 A. The functional effects of the Tyr201Phe and Tyr385Phe mutations, described earlier [Entsch, B., Palfey, B.A., Ballou, D.P., & Massey, V. (1991) J. Biol. Chem. 266, 17341-17349], were rationalized with the assumption that the mutations perturbed the hydrogen-bonding interactions of the tyrosine residues but caused no other changes in the enzyme structure. In agreement with these assumptions, the positions of the substrate, the flavin, and the modified residues are not altered in the Tyr385Phe and Tyr201Phe structures. In contrast, substitution of Asp for Asn at residue 300 has more profound effects on the enzyme structure. The side chain of Asp300 moves away from the flavin, disrupting the interactions of the carboxamide group with the flavin O(2) atom, and the alpha-helix H10 that begins at residue 297 is displaced, altering its dipole interactions with the flavin ring. The functional consequences of these changes in the enzyme structure and of the introduction of the carboxyl group at 300 are described and discussed in the accompanying paper (Palfey et al., 1994b).
已通过X射线晶体学确定了突变型对羟基苯甲酸羟化酶Tyr201Phe、Tyr385Phe和Asn300Asp与底物对羟基苯甲酸（p-OHB）形成的复合物的结构。铜绿假单胞菌酶的这三个突变体的晶体，在两个表面位置（228和249）与野生型荧光假单胞菌酶不同，它们与野生型荧光假单胞菌酶的晶体同晶型，这使得可以从氧化型荧光假单胞菌PHBH-3,4-二羟基苯甲酸复合物的坐标[施勒德，H.A.，范德·拉恩，J.M.，霍尔，W.G.J.，&德伦思，J.（1988年）《分子生物学杂志》199，637 - 648]出发，通过模型构建和精修来确定突变体结构。此处描述的结构的R因子为：Tyr385Phe，对于40.0至2.1 Å的数据，R因子为0.178；Tyr201Phe，对于40.0至2.3 Å的数据，R因子为0.203；Asn300Asp，对于40.0至2.3 Å的数据，R因子为0.193。先前已描述过Tyr201Phe和Tyr385Phe突变的功能效应[恩奇，B.，帕尔菲，B.A.，巴卢，D.P.，&梅西，V.（1991年）《生物化学杂志》266，17341 - 17349]，基于突变扰乱了酪氨酸残基的氢键相互作用但未引起酶结构的其他变化这一假设进行了合理化解释。与这些假设一致，在Tyr385Phe和Tyr201Phe结构中，底物、黄素和修饰残基的位置未改变。相比之下，在第300位残基处用天冬氨酸替代天冬酰胺对酶结构有更深远的影响。天冬氨酸300的侧链远离黄素，破坏了羧酰胺基团与黄素O(2)原子的相互作用，并且从第297位残基开始的α-螺旋H10发生位移，改变了其与黄素环的偶极相互作用。酶结构的这些变化以及在300位引入羧基的功能后果在随附论文（帕尔菲等人，1994b）中进行了描述和讨论。
