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

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.Crystalline surface layers (S-layers) represent the outermost cell envelope component of many archaea and bacteria (for reviews see references 41, 45, and 46). Distinct functions of S-layers have been reported only in a few cases (47). For pathogenic bacteria variation of surface antigens is important for evading the immune response of the host. Campylobacter fetus achieves antigenic variation by expression of different S-layer proteins from at least eight homologs of the structural S-layer gene (6). Variations in the expression of S-layer proteins are not limited to pathogenic species and have also been described for nonpathogenic bacteria, such as Bacillus sphaericus (3), Geobacillus (formerly Bacillus) stearothermophilus (24), and Lactobacillus acidophilus (2). These organisms seem to adapt to altered environmental conditions by changing the S-layer protein. Modification of the surface properties is also achieved by posttranslational modification of the S-layer proteins, particularly by glycosylation (for reviews see references 15, 31, 32, 34, 42, and 51). Few analyses have been carried out so far to elucidate the biosynthesis of bacterial S-layer glycoprotein glycans (12,44). The studies that have been performed confirmed that there are differences in activation of mono-and oligosaccharides for eukaryotic glycoprotein biosynthesis (20) and even for the formation of other bacterial glycoconjugates. Most of the nucleotide-activated monosaccharides used for biosynthesis of Slayer glycoproteins are identical to those used for biosynthesis of other glycoconjugates. However, in Thermoanaerobacter thermosaccharolyticum strain E207-71 N-acetylmannosamine is activated not as the common UDP-N-acetylmannosamine but as GDP-N-acetylmannosamine (44). These monosaccharide precursors are not used for the formation of lipid-activated oligosaccharides but as precursors for the synthesis of nucleotide-activated oligosaccharides.In our attempts to elucidate the formation of specific sugar components of bacterial S-layer glycans, we focused on the biosynthesis of L-rhamnose. This sugar has often been found in S-layer glycoproteins (for reviews see references 31, 32, 34, and 42), and its biosynthetic pathway in gram-negative organisms has been known since the 1960s (8,21,36). The reaction steps and enzymes and the corresponding gene loci are as follows (10,11,14,16,38): (i) dTTP ϩ D-Glc-1-P 3 dTDP-D-Glc ϩ PP i (glucose-1-phosphate thymidylyltransferase; EC 2.7.7.24; * Corresponding author. Mailing address: Zentrum für Ultrastrukturforschung,

Biochemical Characterization of dTDP- d -Qui4N and dTDP- d -Qui4NAc Biosynthetic Pathways in Shigella dysenteriae Type 7 and Escherichia coli O7

“…An early study reported purification of a SAT from E. coli strain B for the conversion of TDP-D-Glc4O to TDP-D-Qui4N; however, no structural verification of the enzyme product was carried out, and a putative gene was not proposed (21). RmlA and RmlB catalyzing the first two steps of the pathways have been functionally identified in several other bacterial species (11,30). In this study, we confirmed the functions of RmlA and RmlB from S. dyenteriae type 7 and utilized those two enzymes to produce the substrate (dTDP-D-Glc4O) for the assay of D7 and VioA O7 catalyze the same transamination reaction.…”

“…Glucose-1-phosphate thymidyltransferase (RmlA) catalyzes the conversion of glucose-1-phosphate to dTDP-D-glucose (dTDP-D-Glc), which is then converted to dTDP-4-keto-6-deoxy-D-glucose (dTDP-D-Glc4O) by dTDP-Dglucose 4,6-dehydratase (RmlB). Both reaction steps have been biochemically verified in a number of bacterial strains (11,30). VioA, a putative sugar aminotransferase (SAT) of the DegT/ DnrJ/EryC1/StrS family, was proposed to catalyze the conversion of dTDP-D-Glc4O to dTDP-D-Qui4N.…”

Functional Characterization and Substrate Specificity of Spinosyn Rhamnosyltransferase by in Vitro Reconstitution of Spinosyn Biosynthetic Enzymes

“…In S. spinosa, the 4,6-dehydratase activity of Gdh was shown here to be much lower (137-and 270-fold) than those of RmlB (Salmonella enterica) and RffG (E. coli), respectively, in catalytic efficiency (35,36). This is an interesting observation presumably associated with the fact that, unlike Gdh, RmlB and RffG mainly meditate the primary metabolic pathway for principle survival of the microorganisms.…”

Functional Characterization of the Initiation Enzyme of S-Layer Glycoprotein Glycan Biosynthesis in Geobacillus stearothermophilus NRS 2004/3a

“…The slg gene clusters contain components for glycan precursor biosynthesis and S-layer glycan assembly and export. These findings, together with the characterization of nucleotide diphosphate-activated sugar precursors (13,17,28) and lipid-activated intermediates (14), indicate that S-layer glycan and O-polysaccharide biosyntheses share common pathways.…”

“…The core unit consists of two or three L-rhamnose residues [32)-␣-L-Rhap-(133)-␣-L-Rhap-(133)-␣-L-Rhap- (13], which are O-glycosidically linked via a ␤-D-galactose residue to threonine 590 , threonine 620 , and serine 794 of the mature S-layer protein SgsE (39,43). The glycan chain has, on average, 15 identical L-rhamnose trisaccharide repeating units with the structure 3[2)-␣-LRhap-(133)-␤-L-Rhap-(132)-␣-L-Rhap-(13.…”