Kim C G, Yu T W, Fryhle C B, Handa S, Floss H G
Department of Chemistry, University of Washington, Seattle, Washington 98195-1700, USA.
J Biol Chem. 1998 Mar 13;273(11):6030-40. doi: 10.1074/jbc.273.11.6030.
The biosynthesis of ansamycin antibiotics, like rifamycin B, involves formation of 3-amino-5-hydroxybenzoic acid (AHBA) by a novel variant of the shikimate pathway. AHBA then serves as the starter unit for the assembly of a polyketide which eventually links back to the amino group of AHBA to form the macrolactam ring. The terminal enzyme of AHBA formation, which catalyzes the aromatization of 5-deoxy-5-amino-3-dehydroshikimic acid, has been purified to homogeneity from Amycolatopsis mediterranei, the encoding gene has been cloned, sequenced, and overexpressed in Escherichia coli. The recombinant enzyme, a (His)6 fusion protein, as well as the native one, are dimers containing one molecule of pyridoxal phosphate per subunit. Mechanistic studies showed that the enzyme-bound pyridoxal phosphate forms a Schiff's base with the amino group of 5-deoxy-5-amino-3-dehydroshikimic acid and catalyzes both an alpha, beta-dehydration and a stereospecific 1,4-enolization of the substrate. Inactivation of the gene encoding AHBA synthase in the A. mediterranei genome results in loss of rifamycin formation; production of the antibiotic is restored when the mutant is supplemented with AHBA.
安莎霉素类抗生素（如利福霉素B）的生物合成涉及通过莽草酸途径的一种新变体形成3-氨基-5-羟基苯甲酸（AHBA）。然后，AHBA作为聚酮化合物组装的起始单元，该聚酮化合物最终与AHBA的氨基连接形成大环内酯环。催化5-脱氧-5-氨基-3-脱氢莽草酸芳构化的AHBA形成的末端酶已从地中海拟无枝酸菌中纯化至同质，其编码基因已被克隆、测序并在大肠杆菌中过表达。重组酶是一种（His）6融合蛋白，与天然酶一样，都是二聚体，每个亚基含有一分子磷酸吡哆醛。机理研究表明，与酶结合的磷酸吡哆醛与5-脱氧-5-氨基-3-脱氢莽草酸的氨基形成席夫碱，并催化底物的α，β-脱水和立体特异性1,4-烯醇化。地中海拟无枝酸菌基因组中编码AHBA合酶的基因失活会导致利福霉素形成的丧失；当用AHBA补充突变体时，抗生素的产生得以恢复。
