The structural basis for catalytic function of GMD and RMD, two closely related enzymes from the GDP‐d‐rhamnose biosynthesis pathway

Abstract

The rare 6-deoxysugar d-rhamnose is a component of bacterial cell surface glycans, including the d-rhamnose homopolymer produced by Pseudomonas aeruginosa, called A-band O polysaccharide. GDP-d-rhamnose synthesis from GDP-d-mannose is catalyzed by two enzymes. The first is a GDP-d-mannose-4,6-dehydratase (GMD). The second enzyme, RMD, reduces the GMD product (GDP-6-deoxy-d-lyxo-hexos-4-ulose) to GDP-d-rhamnose. Genes encoding GMD and RMD are present in P. aeruginosa, and genetic evidence indicates they act in A-band O-polysaccharide biosynthesis. Details of their enzyme functions have not, however, been previously elucidated. We aimed to characterize these enzymes biochemically, and to determine the structure of RMD to better understand what determines substrate specificity and catalytic activity in these enzymes. We used capillary electrophoresis and NMR analysis of reaction products to precisely define P. aeruginosa GMD and RMD functions. P. aeruginosa GMD is bifunctional, and can catalyze both GDP-d-mannose 4,6-dehydration and the subsequent reduction reaction to produce GDP-d-rhamnose. RMD catalyzes the stereospecific reduction of GDP-6-deoxy-d-lyxo-hexos-4-ulose, as predicted. Reconstitution of GDP-d-rhamnose biosynthesis in vitro revealed that the P. aeruginosa pathway may be regulated by feedback inhibition in the cell. We determined the structure of RMD from Aneurinibacillus thermoaerophilus at 1.8 Å resolution. The structure of A. thermoaerophilus RMD is remarkably similar to that of P. aeruginosa GMD, which explains why P. aeruginosa GMD is also able to catalyze the RMD reaction. Comparison of the active sites and amino acid sequences suggests that a conserved amino acid side chain (Arg185 in P. aeruginosa GMD) may be crucial for orienting substrate and cofactor in GMD enzymes.

ABC Transporters Involved in Export of Cell Surface Glycoconjugates

“…Both antigens have a backbone of poly-Drhamnose (not the more usual L-rhamnose), and in O99, this is modified with glucose residues (9, 113). The activated precursor for the polyrhamnose backbone is GDP-D-rhamnose, which is synthesized from GDP-D-mannose in a two-step process involving a dehydratase and a reductase (71). The serotype O99 rhamnosyltransferases share significant levels of similarity with the corresponding mannosyltransferases from E. coli O8/ O9a (113).…”

Activated Sugar Precursors: Biosynthetic Pathways and Biological Roles of an Important Class of Intermediate Metabolites in Bacteria

“…4). This conclusion is also supported by the existence of GMD proteins with bifunctional activity, which catalyzes the dehydration of GDP-D-mannose and the reduction of the 4-keto sugar nucleotide to a 6-deoxysugar nucleotide (King et al, 2009). …”

“…Currently, the analysis of the nucleotide sugars is performed by HPLC methods coupled with other methods such as diode-array detection (DAD), electrospray ionization mass spectrometry (ESI-MS) or nuclear magnetic resonance (NMR) (Ramm et al, 2004). Capillary electrophoresis (CE) has also been used to resolve closely related sugar nucleotides, together with NMR spectroscopy to identify their chemical structures (Lehmann et al, 2000;King et al, 2009). Recently, porous graphitic carbon (PGC) liquid chromatographyelectrospray ionization-mass spectrometry (LC-ESI-MS) was successfully applied to sugar nucleotide separation and analysis (Pabst et al, 2010).…”

“…The GMD enzyme activity is widespread in nature, and also catalyzes the first step in the biosynthesis of other sugars, including L-fucose, D-talose and D-perosamine (King et al, 2009). Bioinformatic analysis suggests that the closest paralog of RMD is GMD (Fig.…”

Polyene macrolide biosynthesis in streptomycetes and related bacteria: recent advances from genome sequencing and experimental studies

“…This is thought to be D-rhamnose because some GDP--D-mannose 4, 6 dehydratases further convert their GDP--D-4-keto-6-deoxymannose product to GDP--D-rhamnose in vitro (King et al, 2009). The AmphDI mycosaminyl transferase is capable of modifying the aglycone with this neutral sugar.…”