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Biochemical and Mechanistic Characterization of the Fungal Reverse N-1-Dimethylallyltryptophan Synthase DMATS1Ff.
ACS Chemical Biology ( IF 3.5 ) Pub Date : 2019-12-05 , DOI: 10.1021/acschembio.9b00828
Immo Burkhardt 1 , Zhongfeng Ye 1 , Slavica Janevska 2 , Bettina Tudzynski 2 , Jeroen S Dickschat 1
Affiliation  

Dimethylallyltryptophan synthases catalyze the regiospecific transfer of (oligo)prenylpyrophosphates to aromatic substrates like tryptophan derivatives. These reactions are key steps in many biosynthetic pathways of fungal and bacterial secondary metabolites. In vitro investigations on recombinant DMATS1Ff from Fusarium fujikuroi identified the enzyme as the first selective reverse tryptophan-N-1 prenyltransferase of fungal origin. The enzyme was also able to catalyze the reverse N-geranylation of tryptophan. DMATS1Ff was shown to be phylogenetically related to fungal tyrosine O-prenyltransferases and fungal 7-DMATS. Like these enzymes, DMATS1Ff was able to convert tyrosine into its regularly O-prenylated derivative. Investigation of the binding sites of DMATS1Ff by homology modeling and comparison to the crystal structure of 4-DMATS FgaPT2 showed an almost identical site for DMAPP binding but different residues for tryptophan coordination. Several putative active site residues were verified by site directed mutagenesis of DMATS1Ff. Homology models of the phylogenetically related enzymes showed similar tryptophan binding residues, pointing to a unified substrate binding orientation of tryptophan and DMAPP, which is distinct from that in FgaPT2. Isotopic labeling experiments showed the reaction catalyzed by DMATS1Ff to be nonstereospecific. Based on these data, a detailed mechanism for DMATS1Ff catalysis is proposed.

中文翻译:

真菌反向N-1-二甲基烯丙基色氨酸合酶DMATS1Ff的生化和机制表征。

二甲基烯丙基色氨酸合酶催化(寡聚)异戊烯基焦磷酸向芳香族底物(如色氨酸衍生物)的区域特异性转移。这些反应是真菌和细菌次生代谢产物许多生物合成途径中的关键步骤。藤本镰刀菌的重组DMATS1Ff的体外研究确定了该酶为真菌来源的第一个选择性反向色氨酸-N-1异戊二烯基转移酶。该酶还能够催化色氨酸的反向N-geranyation。已显示DMATS1Ff与真菌酪氨酸O-异戊二烯基转移酶和真菌7-DMATS在系统发育上相关。像这些酶一样,DMATS1Ff能够将酪氨酸转化为其规则的O-戊烯基化衍生物。通过同源性建模研究DMATS1Ff的结合位点并与4-DMATS FgaPT2的晶体结构进行比较,发现DMAPP结合的位点几乎相同,而色氨酸配位的位点却不同。DMATS1Ff的定点诱变验证了几个推定的活性位点残基。系统发育相关酶的同源性模型显示出相似的色氨酸结合残基,表明色氨酸和DMAPP具有统一的底物结合方向,这与FgaPT2中的不同。同位素标记实验表明,DMATS1Ff催化的反应是非立体特异性的。基于这些数据,提出了DMATS1Ff催化的详细机理。DMATS1Ff的定点诱变验证了几个推定的活性位点残基。系统发育相关酶的同源性模型显示出相似的色氨酸结合残基,表明色氨酸和DMAPP具有统一的底物结合方向,这与FgaPT2中的不同。同位素标记实验表明,DMATS1Ff催化的反应是非立体特异性的。基于这些数据,提出了DMATS1Ff催化的详细机理。DMATS1Ff的定点诱变验证了几个推定的活性位点残基。系统发育相关酶的同源性模型显示出相似的色氨酸结合残基,表明色氨酸和DMAPP具有统一的底物结合方向,这与FgaPT2中的不同。同位素标记实验表明,DMATS1Ff催化的反应是非立体特异性的。基于这些数据,提出了DMATS1Ff催化的详细机理。
更新日期:2019-12-06
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