Primary and tertiary structure studies of p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens. Isolation and alignment of the CNBr peptides; interactions of the protein with flavin adenine dinucleotide.

J. Hofsteenge, J. Vereijken, W. Weijer, J. Beintema, R. Wierenga, J. Drenth
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引用次数: 50

Abstract

p-Hydroxybenzoate hydroxylase from Pseudomonas fluorescens contains six methionine residues, one of which is N-terminal. After CNBr cleavage five peptides, ranging from 13 to 158 residues in length, and free homoserine were isolated and purified by repeated gel filtration. The alignment of the CNBr fragments was deduced from a 0.25-nm electron density map and sequence data. The isolated fragments account for the entire polypeptide chain. The amino acid sequence of the N-terminal quarter of the polypeptide chain was determined. The X-ray results together with the sequence data yielded details of the binding of FAD. The AMP moiety was bound to a beta alpha beta unit resembling that found in the dehydrogenases. Hydrogen bonds were present between the protein and the ribityl residue and the isoalloxazine ring. Furthermore, a homology was found between the N-terminal amino acid sequence of p-hydroxybenzoate hydroxylase and another enzyme containing FAD, viz. D-amino acid oxidase. This finding suggests the presence of a mononucleotide binding fold at the N terminus of the latter.
荧光假单胞菌对羟基苯甲酸羟化酶的一级和三级结构研究。CNBr肽的分离与鉴定蛋白与黄素腺嘌呤二核苷酸的相互作用。
荧光假单胞菌对羟基苯甲酸羟化酶含有六个蛋氨酸残基,其中一个是n端。CNBr裂解后,分离出长度在13 ~ 158个残基之间的5个多肽和游离丝氨酸,经反复凝胶过滤纯化。从0.25 nm电子密度图和序列数据推断出CNBr片段的排列。分离的片段占整个多肽链。测定了多肽链n端四分之一的氨基酸序列。x射线结果与序列数据一起产生了FAD结合的细节。AMP部分与脱氢酶中发现的β - α - β单位结合。该蛋白与利比酯残基和异氧嘧啶环之间存在氢键。此外,还发现对羟基苯甲酸羟化酶的n端氨基酸序列与另一种含有FAD的酶d -氨基酸氧化酶具有同源性。这一发现表明在后者的N端存在单核苷酸结合褶。
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