Expression, Purification, and Activity of ActhiS, a Thiazole Biosynthesis Enzyme from Acremonium chrysogenum

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Zhihui Song, Jie Pan, Liping Xie, Guihua Gong, Shu Han, Wei Zhang, and Youjia Hu^*

China State Institute of Pharmaceutical Industry, Zhangjiang Institute, 201203 Shanghai, China; E-mail: bebydou@hotmail.com

^* To whom correspondence should be addressed.

Received January 12, 2017; Revision received April 10, 2017

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Thiamine pyrophosphate is an essential coenzyme in all organisms. Its biosynthesis involves independent syntheses of the precursors, pyrimidine and thiazole, which are then coupled. In our previous study with overexpressed and silent mutants of ActhiS (thiazole biosynthesis enzyme from Acremonium chrysogenum), we found that the enzyme level correlated with intracellular thiamine content in A. chrysogenum. However, the exact structure and function of ActhiS remain unclear. In this study, the enzyme-bound ligand was characterized as the ADP adduct of 5-(2-hydroxyethyl)-4-methylthiazole-2-carboxylic acid (ADT) using HPLC and ¹H NMR. The ligand-free ActhiS expressed in M9 minimal medium catalyzed conversion of NAD⁺ and glycine to ADT in the presence of iron. Furthermore, the C217 residue was identified as the sulfur donor for the thiazole moiety. These observations confirm that ActhiS is a thiazole biosynthesis enzyme in A. chrysogenum, and it serves as a sulfur source for the thiazole moiety.

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KEY WORDS: Acremonium chrysogenum, ActhiS, ADT, thiazole biosynthesis

DOI: 10.1134/S0006297917070112

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