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反硝化假单胞菌中钴胺酸a,c-二酰胺合酶的纯化与特性分析

Purification and characterization of cobyrinic acid a,c-diamide synthase from Pseudomonas denitrificans.

作者信息

Debussche L, Thibaut D, Cameron B, Crouzet J, Blanche F

机构信息

Département de Chimie Analytique, Centre de Recherche de Vitry, Rhône-Poulenc Santé, Vitry-sur-Seine, France.

出版信息

J Bacteriol. 1990 Nov;172(11):6239-44. doi: 10.1128/jb.172.11.6239-6244.1990.

DOI:10.1128/jb.172.11.6239-6244.1990
PMID:2172209
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC526805/
Abstract

Cobyrinic acid a,c-diamide synthase, which catalyzes the conversion of cobyrinic acid to cobyrinic acid a,c-diamide via the intermediate formation of cobyrinic acid c-monoamide, was purified 155-fold to homogeneity from extracts of a recombinant strain of Pseudomonas denitrificans by high-performance liquid chromatography. The enzyme has an apparent molecular weight of 86,000 and consists of two identical subunits of Mr 45,000, as estimated by gel electrophoresis under denaturing conditions. Stepwise Edman degradation provided the N-terminal sequence of the first 15 amino acids. Glutamine was shown to be the preferred amino group donor (Km = 20.3 microM), but it could be replaced by ammonia (Km = 12 mM). The reaction was ATP dependent and exhibited a broad optimum pH around 7.3. Km values for (CN,aq)cobyrinic acid, (aq)2cobyrinic acid, and (CN,aq)cobyrinic acid c-monoamide were 160, greater than or equal to 250, and 71 microM, respectively. Hydrogenobyrinic acid and hydrogenobyrinic acid c-monoamide were shown to be much better substrates, with Km values of 0.41 and 0.21 microM, respectively.

摘要

钴胺素酸a,c - 二酰胺合酶通过钴胺素酸c - 单酰胺的中间形成催化钴胺素酸转化为钴胺素酸a,c - 二酰胺，该酶通过高效液相色谱法从反硝化假单胞菌重组菌株的提取物中纯化了155倍达到同质。在变性条件下通过凝胶电泳估计，该酶的表观分子量为86,000，由两个Mr为45,000的相同亚基组成。逐步的埃德曼降解提供了前15个氨基酸的N端序列。谷氨酰胺被证明是优选的氨基供体（Km = 20.3 microM），但它可以被氨（Km = 12 mM）替代。该反应依赖于ATP，在7.3左右表现出较宽的最适pH。(CN,aq)钴胺素酸、(aq)2钴胺素酸和(CN,aq)钴胺素酸c - 单酰胺的Km值分别为160、大于或等于250和71 microM。氢化钴胺素酸和氢化钴胺素酸c - 单酰胺被证明是更好的底物，Km值分别为0.41和0.21 microM。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/504e/526805/344514f66824/jbacter00165-0079-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/504e/526805/344514f66824/jbacter00165-0079-a.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/504e/526805/344514f66824/jbacter00165-0079-a.jpg

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