Mechanistic studies of novel anti-tuberculosis targets

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Rv0802c from Mycobacterium tuberculosis: the first structure of a succinyltransferase with the GNAT fold

The structure of Rv0802c was determined in an unliganded form to 2.0 Å resolution utilizing single-wavelength anomalous dispersion from a samarium soak that resulted in a single bound Sm3+:citrate2 complex

The position of a key tyrosine in dTDP-4-keto-6-deoxy-D-glucose-5-epimerase (EvaD) alters the substrate profile for this RmlC-like enzyme

Vancomycin, the last line of defense antibiotic, depends upon the attachment of the carbohydrate vancosamine to an aglycone skeleton for antibacterial activity. Vancomycin is a naturally occurring secondary metabolite that can be produced by bacterial fermentation. To combat emerging resistance, it has been proposed to genetically engineer bacteria to produce analogues of vancomycin. This requires a detailed understanding of the biochemical steps in the synthesis of vancomycin. Here we report the 1.4 Angstrom structure and biochemical characterization of EvaD, an RmlC-like protein that is required for the C-5' epimerization during synthesis of dTDP-epivancosamine. EvaD, although clearly belonging to the RmlC class of enzymes, displays very low activity in the archetypal RmlC reaction ( double epimerization of dTDP-6-deoxy-4-keto-D-glucose at C-3' and C-5'). The high resolution structure of EvaD compared with the structures of authentic RmlC enzymes indicates that a subtle change in the enzyme active site repositions a key catalytic Tyr residue. A mutant designed to re-establish the normal position of the Tyr increases the RmlC-like activity of EvaD.</p

Mechanistic evidence for a front-side, SNi-type reaction in a retaining glycosyltransferase

A previously determined crystal structure of the ternary complex of trehalose-6-phosphate synthase identified a putative transition state–like arrangement based on validoxylamine A 6?-O-phosphate and uridine diphosphate in the active site. Here linear free energy relationships confirm that these inhibitors are synergistic transition state mimics, supporting front-face nucleophilic attack involving hydrogen bonding between leaving group and nucleophile. Kinetic isotope effects indicate a highly dissociative oxocarbenium ion–like transition state. Leaving group 18O effects identified isotopically sensitive bond cleavages and support the existence of a hydrogen bond between the nucleophile and departing group. Brønsted analysis of nucleophiles and Taft analysis highlight participation of the nucleophile in the transition state, also consistent with a front-face mechanism. Together, these comprehensive, quantitative data substantiate this unusual enzymatic reaction mechanism. Its discovery should prompt useful reassessment of many biocatalysts and their substrates and inhibitor

Among sadists

In the decade after the death of Edmund Campion at Tyburn on December 1, 1581, his fellow Jesuit Robert Southwell composed extraordinary verses which were circulated in secret manuscript and...</p

Site-selective chemoenzymatic construction of synthetic glycoproteins using endoglycosidases †

Combined chemical tagging followed by Endo-A catalysed elongation allows access to homogeneous, elaborated glycoproteins. A survey of different linkages and sugars demonstrated not only that unnatural linkages can be tolerated but they can provide insight into the scope of Endo-A transglycosylation activity. S-linked GlcNAc-glycoproteins are useful substrates for Endo-A extensions and display enhanced stability to hydrolysis at exposed sites. O-CH 2 -triazole-linked GlcNAc-glycoproteins derived from azidohomoalanine-tagged protein precursors were found to be optimal at sterically demanding sites