肺炎克雷伯菌3-羟基苯甲酸-6-羟化酶的纯化与特性分析

Purification and characterization of the 3-hydroxybenzoate-6-hydroxylase from Klebsiella pneumoniae.

作者信息

Suárez M, Ferrer E, Garrido-Pertierra A, Martín M

机构信息

Departamento de Bioquímica y Biología Molecular IV, Facultad de Veterinaria, Universidad Complutense de Madrid, Spain.

出版信息

FEMS Microbiol Lett. 1995 Mar 1;126(3):283-90. doi: 10.1111/j.1574-6968.1995.tb07431.x.

DOI:10.1111/j.1574-6968.1995.tb07431.x

PMID:7729672

Abstract

We isolated 3-hydroxybenzoate-6-hydroxylase (E.C.1.14.13.), an inducible enzyme that catalyzed the para-hydroxylation of 3-hydroxybenzoate (3-HBA) to 2,5-dihydroxybenzoate, from Klebsiella pneumoniae. Although the enzyme was found to be mainly induced by its substrate, a coordinated induction of 3-hydroxybenzoate hydroxylase and gentisate dioxygenase was also observed in the presence of the product of the reaction. The purified enzyme was a monomer with a molecular mass of 42,000. It contained FAD as a prosthetic group, utilized NADH or NADPH with similar efficiencies and its activity was inhibited by Cu2+, Fe2+ and Hg2+. Other properties, such as induction mechanism and kinetic parameters were also studied. Moreover, for the first time the amino acid composition of a 3-hydroxybenzoate-6-hydroxylase was determined.

摘要

我们从肺炎克雷伯菌中分离出了3-羟基苯甲酸-6-羟化酶（E.C.1.14.13.），这是一种可诱导酶，能催化3-羟基苯甲酸（3-HBA）对羟基化生成2,5-二羟基苯甲酸。尽管发现该酶主要由其底物诱导，但在反应产物存在的情况下，也观察到了3-羟基苯甲酸羟化酶和龙胆酸双加氧酶的协同诱导。纯化后的酶是一种分子量为42,000的单体。它含有黄素腺嘌呤二核苷酸（FAD）作为辅基，对烟酰胺腺嘌呤二核苷酸（NADH）或烟酰胺腺嘌呤二核苷酸磷酸（NADPH）的利用效率相似，其活性受到铜离子（Cu2+）、亚铁离子（Fe2+）和汞离子（Hg2+）的抑制。还研究了其他特性，如诱导机制和动力学参数。此外，首次测定了3-羟基苯甲酸-6-羟化酶的氨基酸组成。