Cloning and functional expression of a novel GDP-6-deoxy-D-talose synthetase from Actinobacillus actinomycetemcomitans

Minna Maki 1 Nina Jarvinen 1 Jarkko Rabina 1 Hannu Maaheimo 0 Pirkko Mattila 3 Risto Renkonen 1 2 0 VTT Biotechnology and Programme for Structural Biology and Biophysics , P.O. Box 65, 00014 Helsinki, Finland 1 Department of Bacteriology and Immunology, Haartman Institute and Biomedicum , P.O. Box 63, FIN-00014 University of Helsinki , Helsinki, Finland 2 HUCH Laboratory Diagnostics, Helsinki University Central Hospital , P.O. Box 401, FIN-00029 HUCH, Helsinki, Finland 3 MediCel, Haartmaninkatu 8, FIN-00290 Helsinki, Finland Glycobiology vol. 13 no. 4 # Oxford University Press 2003; all rights reserved. - Received on September 30, 2002; revised on November 29, 2002; accepted on December 1, 2002 Actinobacillus actinomycetemcomitans is a Gram-negative coccobacillus that can cause various forms of severe periodontitis and other nonoral infections in human patients. The serotype aspecific polysaccharide antigen of A. actinomycetemcomitans contains solely 6-deoxy-D-talose and its O-2 acetylated modification. This polysaccharide is synthesized from the donor GDP-6-deoxy-D-talose with the relevant talosylation enzyme(s). In the synthesis of GDP-6deoxy-D-talose, GDP-D-mannose is first converted by GDP-mannose-4,6-dehydratase (GMD) to GDP-4-keto6-deoxy-D-mannose and then reduced to GDP-6-deoxy-Dtalose by GDP-6-deoxy-D-talose synthetase (GTS). In this study, we cloned and overexpressed in Escherichia coli the A. actinomycetemcomitans GTS enzyme responsible for the synthesis of GDP-6-deoxy-D-talose. The recombinant A. actinomycetemcomitans GTS enzyme expressed in E. coli converted the GDP-4-keto-6-deoxy-intermediate to a novel GDP-deoxyhexose. The synthesized GDP-deoxyhexose was shown to be GDP-6-deoxy-D-talose by HPLC, MALDITOF MS, and NMR spectroscopy. The functional expression of gts provides another enzymatically defined pathway for the synthesis of GDP-deoxyhexoses, which can be used as donors for the corresponding glycosyltransferases. Introduction Actinobacillus actinomycetemcomitans is a Gram-negative coccobacillus that can cause various forms of severe periodontitis (Buchmann et al., 2000; Doungudomdacha et al., 2000; Slots and Ting, 2000) as well as systemic infections (Kulekci et al., 2001; van Winkelhoff and Slots, 2000). 1To whom correspondence should be addressed; e-mail: A. actinomycetemcomitans strains produce several virulence factors, including a leukotoxin (Haraszthy et al., 2000; Johansson et al., 2000) and iron- and hemin-binding proteins (Graber et al., 1998). The strains are divided into six different serotypes (af ) based on their capsular polysaccharides (Gmur et al., 1993; Kaplan et al., 2001; Saarela et al., 1992, 1993). These polysaccharides constitute the outermost layer of the cell and have thus been suggested to play a role in the virulence of this bacterium (FivesTaylor et al., 2000; Wilson and Henderson, 1995). The serotype aspecific polysaccharide antigen (SPA) of A. actinomycetemcomitans has been characterized as 6deoxy-D-talan, which is composed of a repeating disaccharide a1,3- (6-deoxy-D-talose)-a1,2-(6-deoxy-D-talose), where the O-2 position of a1,3-linked 6-deoxy-D-talose is acetylated (Perry et al., 1996; Shibuya et al., 1991). This glycan is rarely found in bacterial polysaccharides; currently Burkholderia (Pseudomonas) plantarii is the only other bacteria known to synthesize it (Zahringer et al., 1997). Various strains of A. actinomycetemcomitans also harbor an isomer of 6-deoxy-D-talose, that is, 6-deoxy-L-talose (Nakano et al., 2000) or other deoxyhexoses, such as L-rhamnose and/or D-fucose on their capsular polysaccharides (Shibuya et al., 1991). An epimer of 6-deoxy-D-talose, D-rhamnose is a deoxyhexose sugar, which differs from 6-deoxy-D-talose only in the orientation of the OH group at the C-4 position (Figure 1). Rhamnose is widely found in bacteria and plants but not in mammals. Of the two isomers, L- and D-rhamnose, the former is more common (Sadovskaya et al., 2000). L-fucose is another deoxyhexose and the only representative of this glycan family also found in eukaryotic cells. Some fucosylated glycoproteins have been shown to be crucial both in bacterial adherence to host cells (Herron et al., 2000; Karlsson, 2000) and leukocyte trafficking in mammals (Lowe, 2001; Satomaa et al., 2002). The synthetic pathways for closely related deoxyhexoses Lfucose, D-rhamnose, 6-deoxy-D-talose begin from GDP-Dmannose, which is first converted to the labile intermediate product GDP-4-keto-6-deoxy-D-mannose (Figure 1). This intermediate product is reduced to various GDPdeoxyhexoses (in the case of GDP-L-fucose, epimerization occurs prior to reduction). The enzymes GDP-4-keto-6deoxy-D-mannose-3,5 epimerase/4-reductase (GMER) and GDP-4-keto-6-deoxy-D-mannose-4-reductase (RMD) synthesizing two of these sugar nucleotides, GDP-L-fucose and GDP-D-rhamnose, respectively, have already been described in detail (Butler and Elling, 1999; Jarvinen et al., 2001; Kneidinger et al., 2001; Mattila et al., 2000; Maki et al., 2002). The aim of this study was to seek a reductase responsible for converting the labile intermediate product Fig. 1. (A) Biosynthetic pathways and (B) structures of the deoxyhexoses reduced from the common intermediate product GDP-4-keto-6-deoxy-Dmannose. GDP-D-mannose is first converted to the intermediate product GDP-4-keto-6-deoxy-D-mannose, which is reduced alternatively to various deoxyhexoses (in the case of GDP-L-fucose the 3,5 epimerization occurs prior reduction). The enzymes involved in GDP-L-fucose, GDP-D-rhamnose, and GDP-6-deoxy-D-talose pathways have been characterized. GDP-4-keto-6-deoxy-D-mannose to GDP-6-deoxy-Dtalose. We searched for an amino acid sequence similar to the closely related reductases responsible for the synthesis of other GDP-deoxyhexoses. From the gene cluster associated with the serotype aSPA of A. actinomycetemcomitans (Suzuki et al., 2000), we identified a putative GDP-6deoxy-D-talose synthetase (gts) gene, which we cloned and overexpressed in Escherichia coli. GDP-4-keto-intermediate, produced by the recombinant Helicobacter pylori GDP-mannose-4,6-dehydratase (GMD) enzyme, was used as a substrate for the recombinant GDP-6-deoxy-D-talose synthetase (GTS) enzyme. The reaction product of the GTS enzyme was analyzed with high-pressure liquid chromatography (HPLC), matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), and nuclear magnetic resonance (NMR) spectroscopy. The results indicated that the GTS enzyme represents a novel reductase responsible for converting GDP-4-keto-6-deoxyD-mannose to GDP-6-deoxy-D-talose. Results Sequence analysis A gene cluster responsible for the biosynthesis of SPA has been sequenced from serotype a A. actinomycetemcomitans (the EMBL/GenBank/DDBJ accession number 9309318) (Suzuki et al., 2000). From this gene cluster we identified a putative GDP-6-deoxy-D-talose synthetase (gts) gene. The corresponding gene product conta (...truncated)