重组球形红细菌5-氨基乙酰丙酸合酶同工酶hemA和hemT的特性

将编码光合细菌Rhodobactersphaeroides 5- 氨基乙酰丙酸合酶(ALAS)的同工酶基因hemA、hemT转入E .coli中进行高表达,并将高表达的同工酶进行分离、纯化.纯化的hemA是可溶的,并具有催化活性,而hemT大部分是不溶的,且在体外条件下无活性.与其它重组ALAS相比,R .sphaeroides的hemA活性表达需PLP作为催化因子,除去PLP或用硼酸钠破坏与PLP的连接,hemA活性下降90 % .hemA PLP的紫外 可见光谱分析表明hemA与PLP之间形成一个醛亚胺键,而hemT与PLP之间未形成该键.hemA对修饰组氨酸、精氨酸、胱氨酸残基的试剂很敏感,对可切割Arg15 1和Ser15 2的类胰蛋白酶也很敏感,PLP也不能阻止该酶的切割作用.抗血清试验表明,hemA、hemT的抗血清均可与小鼠的ALAS杂交,并都有一个抗原决定簇.

Characteristics of Recombined Rhodobacter sphaeroides 5-Aminolevulinic Acid Synthase Isoenzymes hemA and hemT

As plant growth regulator,biogradable natural herbicide,and insecticide and photodynamic anti-cancer drug as well,5-aminolevulinic acid (ALA) has a variety of application and is synthesized by condensation of glycine and succinyl CoA.This reaction is catalyzed by ALA synthase (ALAS) isoenzymens hemA and hemT.Two genes of hemA and hemT from Rhodobacter sphaeroides were cloned,respectively,and allowed them high expressiong in E.coli. The over-expressed isoenzymes were isolated and purified. Isolated hemA was soluble and catalytically active whereas hemT was insoluble and failed to show any activity ex vivo. In common with other ALAS, the recombined R. sphaeroides hemA requires pyridoxal-5-phosphate(PLP) as a cofactor for catalysis. Removal of this cofactor or reduction the PLP-hemA complex with sodium borohydride resulted in 90% reduction activity of the enzyme. Ultraviolet-visible absorption spectrum with hemA suggested the presence of aldimine linkage between the enzyme and PLP that was not observed when hemT incubated with the cofactor. HemA was found to be sensitive to reagents that modify histidine, arginine, cysteine amino acid residues and the enzyme was also sensitive to tryptic cleavage between Arg151 and Ser152 in the presence and absence of PLP. Antisera testing indicated that all of the enzymes have conserved epitopes.