Homologs of the Rml Enzymes from Salmonella enterica Are Responsible for dTDP-β- l -Rhamnose Biosynthesis in the Gram-Positive Thermophile Aneurinibacillus thermoaerophilus DSM 10155

Abstract

ABSTRACT The glycan chains of the surface layer (S-layer) glycoprotein from the gram-positive, thermophilic bacterium Aneurinibacillus (formerly Bacillus ) thermoaerophilus strain DSM 10155 are composed of l -rhamnose- and d - glycero - d - manno -heptose-containing disaccharide repeating units which are linked to the S-layer polypeptide via core structures that have variable lengths and novel O-glycosidic linkages. In this work we investigated the enzymes involved in the biosynthesis of thymidine diphospho- l -rhamnose (dTDP- l -rhamnose) and their specific properties. Comparable to lipopolysaccharide O-antigen biosynthesis in gram-negative bacteria, dTDP- l -rhamnose is synthesized in a four-step reaction sequence from dTTP and glucose 1-phosphate by the enzymes glucose-1-phosphate thymidylyltransferase (RmlA), dTDP- d -glucose 4,6-dehydratase (RmlB), dTDP-4-dehydrorhamnose 3,5-epimerase (RmlC), and dTDP-4-dehydrorhamnose reductase (RmlD). The rhamnose biosynthesis operon from A. thermoaerophilus DSM 10155 was sequenced, and the genes were overexpressed in Escherichia coli . Compared to purified enterobacterial Rml enzymes, the enzymes from the gram-positive strain show remarkably increased thermostability, a property which is particularly interesting for high-throughput screening and enzymatic synthesis. The closely related strain A. thermoaerophilus L420-91 T produces d -rhamnose- and 3-acetamido-3,6-dideoxy- d -galactose-containing S-layer glycan chains. Comparison of the enzyme activity patterns in A. thermoaerophilus strains DSM 10155 and L420-91 T for l -rhamnose and d -rhamnose biosynthesis indicated that the enzymes are differentially expressed during S-layer glycan biosynthesis and that A. thermoaerophilus L420-91 T is not able to synthesize dTDP- l -rhamnose. These findings confirm that in each strain the enzymes act specifically on S-layer glycoprotein glycan formation.