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首页> 外文期刊>Journal of Molecular Biology >Switch of coenzyme specificity of p-hydroxybenzoate hydroxylase.
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Switch of coenzyme specificity of p-hydroxybenzoate hydroxylase.

机译：对羟基苯甲酸酯羟化酶的辅酶特异性的转换。

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摘要

p-Hydroxybenzoate hydroxylase (PHBH) is the archetype of the family of NAD(P)H-dependent flavoprotein aromatic hydroxylases. These enzymes share a conserved FAD-binding domain but lack a recognizable fold for binding the pyridine nucleotide. We have switched the coenzyme specificity of strictly NADPH-dependent PHBH from Pseudomonas fluorescens by site-directed mutagenesis. To that end, we altered the solvent exposed helix H2 region (residues 33-40) of the FAD-binding domain. Non-conservative selective replacements of Arg33 and Tyr38 weakened the binding of NADPH without disturbing the protein architecture. Introduction of a basic residue at position 34 increased the NADPH binding strength. Double (M2) and quadruple (M4) substitutions in the N-terminal part of helix H2 did not change the coenzyme specificity. By extending the replacements towards residues 38 and 40, M5 and M6 mutants were generated which were catalytically more efficient with NADH than with NADPH. It is concluded that specificity in P. fluorescens PHBH is conferred by interactions of Arg33, Tyr38 and Arg42 with the 2'-phosphate moiety of bound NADPH, and that introduction of an acidic group at position 38 potentially enables the recognition of the 2'-hydroxy group of NADH. This is the first report on the coenzyme reversion of a flavoprotein aromatic hydroxylase. Copyright 1999 Academic Press.
机译：对羟基苯甲酸酯羟化酶（PHBH）是NAD（P）H依赖的黄素蛋白芳香族羟化酶家族的原型。这些酶共享一个保守的FAD结合域，但缺乏识别吡啶核苷酸的折叠。我们已经通过定点诱变从荧光假单胞菌转变了严格NADPH依赖的PHBH的辅酶特异性。为此，我们更改了FAD结合域的溶剂暴露的螺旋H2区（33-40残基）。 Arg33和Tyr38的非保守选择性替代减弱了NADPH的结合，而不会干扰蛋白质的结构。在34位引入碱性残基增加了NADPH结合强度。螺旋H2的N端部分的双（M2）和四倍（M4）取代没有改变辅酶的特异性。通过将置换延伸至残基38和40，产生了M5和M6突变体，其用NADH比用NADPH催化更有效。结论是，荧光假单胞菌PHBH的特异性是通过Arg33，Tyr38和Arg42与结合的NADPH的2'-磷酸部分的相互作用而赋予的，并且在位置38引入酸性基团可能会识别2'- NADH的羟基。这是关于黄素蛋白芳香族羟化酶辅酶还原的首次报道。版权所有1999，学术出版社。

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