The dTDP-4-dehydro-6-deoxyglucose reductase encoding fcd gene is part of the surface layer glycoprotein glycosylation gene cluster of Geobacillus tepidamans GS5-97T

Abstract

The glycan chain of the S-layer protein of Geobacillus tepidamans GS5-97(T) consists of disaccharide repeating units composed of L-rhamnose and D-fucose, the latter being a rare constituent of prokaryotic glycoconjugates. Although biosynthesis of nucleotide-activated L-rhamnose is well established, D-fucose biosynthesis is less investigated. The conversion of alpha-D-glucose-1-phosphate into thymidine diphosphate (dTDP)-4-dehydro-6-deoxyglucose by the sequential action of RmlA (glucose-1-phosphate thymidylyltransferase) and RmlB (dTDP-glucose-4,6-dehydratase) is shared between the dTDP-D-fucose and the dTDP-L-rhamnose biosynthesis pathway. This key intermediate is processed by the dTDP-4-dehydro-6-deoxyglucose reductase Fcd to form dTDP-alpha-D-fucose. We identified the fcd gene in G. tepidamans GS5-97(T) by chromosome walking and performed functional characterization of the recombinant 308-amino acid enzyme. The in vitro activity of the enzymatic cascade (RmlB and Fcd) was monitored by high-performance liquid chromatography and the reaction product was confirmed by (1)H and (13)C nuclear magnetic resonance spectroscopy. This is the first characterization of the dTDP-alpha-D-fucopyranose biosynthesis pathway in a Gram-positive organism. fcd was identified as 1 of 20 open reading frames contained in a 17471-bp S-layer glycosylation (slg) gene cluster on the chromosome of G. tepidamans GS5-97(T). The sgtA structural gene is located immediately upstream of the slg gene cluster with an intergenic region of 247 nucleotides. By comparison of the SgtA amino acid sequence with the known glycosylation pattern of the S-layer protein SgsE of Geobacillus stearothermophilus NRS 2004/3a, two out of the proposed three glycosylation sites on SgtA could be identified by electrospray ionization quadrupole-time-of-flight mass spectrometry to be at positions Ser-792 and Thr-583.

Fig. 1

Schematic drawing of the glycan structure of the S-layer glycoprotein glycan of G. tepidamans GS5-97^T.

Fig. 2

Nucleotide sugar anabolism pathways for dTDP-α-d-fucose and dTDP-β-l-rhamnose.

Fig. 3

Alignment of Fcd (G. tepidamans GS5-97^T) and Fcf1 (E. coli O52). Grey boxes indicate motifs of the SDR enzyme family, bold letters indicate the catalytic triad.

Fig. 4

Monitoring of dTDP-α-d-fucose synthesis. (A) HPLC profiles of nucleotides and nucleotide-activated monosaccharides. (a) Standard mixture (1, dTTP; 2, dTDP; 3, dTDP-d-glucose; and 4, dTMP); (b) no enzyme added to dTDP-d-glucose; and (c) conversion of dTDP-d-glucose with RmlB and Fcd (5, newly formed reaction product dTDP-d-fucose). (B) HPAEC profiles of monosaccharides after TFA hydrolysis. (a) Standard mixture (1, fucose; 2, 2-deoxy-galactose; 3, galactosamine; 4, glucosamine; 5, galactose; and 6, glucose); (b) Product after conversion of dTDP-d-glucose with RmlB, Fcd and TFA hydrolysis.

Fig. 5

300 MHz ¹H NMR spectrum of dTDP-α-d-fucose.

Fig. 6

Comparison of the slg gene clusters of G. tepidamans GS5-97^T (A) and G. stearothermophilus NRS 2004/3a (B). Similarity: black arrows: more than 80%, grey arrows: 50–80%, white arrows: no homology.

Fig. 7

Sequence alignment of the sequences in vicinity of the glycosylation sites of SgtA and SgsE, identified glycosylation sites are indicated by black boxes, putative glycosylation sites by grey boxes.