Biocatalytic preparation and characterization of alternative substrate of MshB, a mycothiol pathway enzyme

[摘要] ENGLISH ABSTRACT: Mycobacterium tuberculosis (M. tuberculosis), the causative agent of tuberculosis,utilizes mycothiol (MSH) as the major low molecular weight thiol to protect itselfagainst oxidative stress and thereby to ensure its growth and survival. MSH is apseudo-disaccharide molecule that contains an α(1→1) glycosidic bond, and isbiosynthesised in five enzymatic steps involving the enzymes MshA, MshA2, MshB,MshC and MshD. Owing to the essentiality of MSH to M. tuberculosis, variousstudies have focused on the MSH biosynthetic and other MSH-dependent enzymesviewed as potential drug targets for the development of antituberculosis agents. Inthe course of this study two practical challenges affecting the development ofinhibitors of one the MSH biosynthesis pathway enzyme, MshB, were addressed.These challenges entail the lack of a high-throughput continuous assay to determineMshB activity, and the poor availability of the natural and alternative MshBsubstrates. In this study an alternate MshB substrate was characterized and shownto undergo a rearrangement reaction upon deactylation, which allowed thedevelopment of a new continuous assay for MshB activity that uses DNTB (Ellman'sreagent). In addition, three new α-thioglycoligases were created from the α-Nacetylglucosaminidaseof Clostridium perfringens. These enzymes showed potential as biocatalysts that can be used for the enzymatic synthesis of thioglycoside-basedalternative substrates of MshB.

[发布机构] Stellenbosch University