Comparison of the catalysis of ThDP-dependent enzymes AHAS, ALS, and DXPS.

Thiamine diphosphate (ThDP)-dependent enzymes are primarily classified into lyases, transferases, and oxidoreductases, catalyzing various biologically important reactions such as keto transfer, dehydrogenation, decarboxylation, and stereoselective C-C bond formation/cleavage in chiral natural products biosynthesis. They constitute one of the largest superfamilies of biocatalysts, operate basically in all forms of living systems. In this chapter, the research progress on three ThDP enzymes are reviewed, including acetohydroxyacid synthase (AHAS) which is a key enzyme in the anabolic pathway for the biosynthesis of branched chain amino acids, acetolactate synthase (ALS) that belongs to the catabolic pathway for the biosynthesis of platform compounds acetoin and 2,3-butanediol, and 1-deoxy-D-xylulose-5-phosphate synthase (DXPS) which is a rate-limiting enzyme in the MEP terpenoid biosynthetic pathway and is also involved in the biosynthesis of ThDP and pyridoxal phosphate in certain bacteria, with the emphasis on comparing the catalysis of the three enzymes. Furthermore, two precolumn derivatization-HPLC protocols using 4-nitro-o-phenylenediamine and 2,4-dinitrophenylhydrazine as respective derivatizing reagents are also described in detail. The methods can not only be utilized to measure the activities of the above enzymes, supplementing the deficiencies of the classic spectrophotometry, but also be used to comprehensively elucidate the consumption of the substrate(s) and the formation of the products in the reactions catalyzed by AHAS, ALS, or DXPS.

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