Allosteric Competitive Inhibitors of the Glucose-1-phosphate Thymidylyltransferase (RmlA) from Pseudomonas aeruginosa

Abstract

Glucose-1-phosphate thymidylyltransferase (RmlA) catalyzes the condensation of glucose-1-phosphate (G1P) with deoxy-thymidine triphosphate (dTTP) to yield dTDP-d-glucose and pyrophosphate. This is the first step in the l-rhamnose biosynthetic pathway. l-Rhamnose is an important component of the cell wall of many microorganisms, including Mycobacterium tuberculosis and Pseudomonas aeruginosa. Here we describe the first nanomolar inhibitors of P. aeruginosa RmlA. These thymine analogues were identified by high-throughput screening and subsequently optimized by a combination of protein crystallography, in silico screening, and synthetic chemistry. Some of the inhibitors show inhibitory activity against M. tuberculosis. The inhibitors do not bind at the active site of RmlA but bind at a second site remote from the active site. Despite this, the compounds act as competitive inhibitors of G1P but with high cooperativity. This novel behavior was probed by structural analysis, which suggests that the inhibitors work by preventing RmlA from undergoing the conformational change key to its ordered bi-bi mechanism.

Comparative genomic analysis of Myroides odoratimimus isolates

“…Glucose-1phosphate thymidylyltransferase (RmlA) is vital for bacteria survival (Mansuri et al, 2016). It participates in L-rhamnose synthesis (Alphey et al, 2013;Mansuri et al, 2016), a critical linker of peptidoglycan and arabinogalacton in bacterial cell wall (Mansuri et al, 2016), by catalyzing the generation of dTDP-D-glucose and pyrophosphate (PPi) (Alphey et al, 2013;Mansuri et al, 2016). dTDP-D-glucose 4,6-dehydratase (RmlB) is also involved in L-rhamnose biosynthesis, by catalyzing the conversion of dTDP-D-glucose into dTDP-4-keto-6-deoxy-D-glucose in cell wall (Allard et al, 2002).…”

Synthesis and Evaluation of l-Rhamnose 1C-Phosphonates as Nucleotidylyltransferase Inhibitors

“…L-Rhamnose (6-deoxy-L-mannose) serves as the linking unit between arabinogalactan and peptidoglycan 7,8 and is a necessary constituent of the bacterial cell wall in many bacterial species. 9 The biosynthesis of L-rhamnose begins with nucleotidylyltransferase enzymes (Cps2L/RmlA) responsible for generating activated sugars in the form of glycosylated nucleoside diphosphates (NDPs). These NDPs are generated from the enzymatic coupling of sugar 1-phosphates with nucleoside triphosphates (NTPs).…”

A severe leakage of intermediates to shunt products in acarbose biosynthesis

“…46). The substantial titer improvement of 1 with highly efficient heterologous G-1-PTs [31][32][33] further demonstrated that overexpression of enzymes that divert primary metabolites into secondary metabolism is a useful strategy 41 (Fig. 6e).…”

“…To further enhance the competing utilization of precursor 5 for the biosynthesis of 6, cognate G-1-PT genes [31][32][33] were individually co-expressed with AP-MB in Actinoplanes sp. SE50/110 (Fig.…”