Glycobiology Advance Access originally published online on January 3, 2007
Glycobiology 2007 17(4):433-443; doi:10.1093/glycob/cwl084
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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
3 Zentrum für NanoBiotechnologie
4 Department für Chemie, Universität für Bodenkultur Wien, A-1190 Wien, Austria
1 To whom correspondence should be addressed; Tel: +43-1-47654-2202; Fax: +43-1-4789112; e-mail: paul.messner{at}boku.ac.at
Received on November 13, 2006; revised on December 22, 2006; accepted on December 24, 2006
The glycan chain of the S-layer protein of Geobacillus tepidamans GS5-97T 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 
-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--D-fucose. We identified the fcd gene in G. tepidamans GS5-97T 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 1H and 13C nuclear magnetic resonance spectroscopy. This is the first characterization of the dTDP--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 GS5-97T. 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.
Key words: surface layer glycoprotein / fcd / dTDP-4-dehydro-6-deoxyglucose reductase / glycosylation gene cluster / Geobacillus tepidamans
2 Present address: Sonn & Partner, Patent attorneys, Riemergasse 14, A-1010 Wien, Austria