Gunsior Michele, Ravel Jacques, Challis Gregory L, Townsend Craig A
Department of Chemistry, The Johns Hopkins University, 3400 North Charles Street, Baltimore, Maryland 21218, USA.
Biochemistry. 2004 Jan 27;43(3):663-74. doi: 10.1021/bi035762w.
p-Hydroxyphenylpyruvate dioxygenase (HPD) plays a key role in the normal catabolism of tyrosine. An Fe2+/oxygen-dependent enzyme, it converts p-hydroxyphenylpyruvate into homogentisate and is part of the superfamily of alpha-ketoglutarate-dependent enzymes that couples oxidative decarboxylation of an alpha-ketoacid cofactor to oxidative modification of its substrate. In this case, the alpha-ketoacid is part of the substrate side chain. HPD shows strong homology to p-hydroxymandelate synthase (HMS), an enzyme that catalyzes the formation of p-hydroxymandelate from p-hydroxyphenylpyruvate, an early step in the biosynthesis of p-hydroxyphenylglycine, which is a nonproteinogenic amino acid incorporated into several biologically active secondary metabolites. Sequence alignment between the HPD and the HMS enzyme families and analysis of the Pseudomonas fluorescens HPD crystal structure highlighted four residues within each active site that may play roles in catalytic differentiation between the two products. We attempted to convert Streptomyces avermitilis HPD into an engineered S. avermitilis HMS by site-directed mutagenesis of these four residues individually and in combination. HPLC assay analysis of each His6-tagged mutant indicated that F337I successfully produced p-hydroxymandelate, along with homogentisate and an unknown compound. The structure of the latter was determined to be an oxepinone derived from the benzene-oxide intermediate long hypothesized in HPD catalysis.
对羟基苯丙酮酸双加氧酶（HPD）在酪氨酸的正常分解代谢中起关键作用。它是一种依赖Fe²⁺/氧气的酶，将对羟基苯丙酮酸转化为尿黑酸，是依赖α-酮戊二酸的酶超家族的一部分，该超家族将α-酮酸辅因子的氧化脱羧与其底物的氧化修饰偶联起来。在这种情况下，α-酮酸是底物侧链的一部分。HPD与对羟基扁桃酸合酶（HMS）具有很强的同源性，HMS催化从对羟基苯丙酮酸形成对羟基扁桃酸，这是对羟基苯甘氨酸生物合成的早期步骤，对羟基苯甘氨酸是一种掺入几种生物活性次生代谢物中的非蛋白质氨基酸。HPD和HMS酶家族之间的序列比对以及荧光假单胞菌HPD晶体结构分析突出了每个活性位点内的四个残基，它们可能在两种产物的催化分化中起作用。我们试图通过对这四个残基单独和组合进行定点诱变，将阿维链霉菌HPD转化为工程化的阿维链霉菌HMS。对每个His6标签突变体的HPLC分析表明，F337I成功产生了对羟基扁桃酸，以及尿黑酸和一种未知化合物。后者的结构被确定为一种氧杂环庚三酮，源自长期以来在HPD催化中假设的苯氧化物中间体。
