Probing the catalytic mechanism of GDP-4-keto-6-deoxy-d-mannose epimerase/reductase by kinetic and crystallographic characterization of site-specific mutants

Background: Modified heptoses are important for bacterial virulence. Results: We characterized novel L-gluco-heptose synthesis enzymes from Campylobacter jejuni and compared their specificity with D-altro-heptose synthesis enzymes. Conclusion: Significant differences of activities and specificities were observed despite high sequence similarities. Significance: The versatility of the enzymes can be exploited to synthesize novel carbohydrates for technological applications and to develop therapeutic inhibitors.

Section: Benefits Of the Surrogate Gdp-6-deoxy-4-keto-d-lyxo-heptose supporting

confidence: 77%

Comparison of Predicted Epimerases and Reductases of the Campylobacter jejuni d-altro- and l-gluco-Heptose Synthesis Pathways

McCallum¹,

Shaw

Creuzenet³

2013

“…In particular, the tyrosine residue acts as a catalytic base that allows the transfer of a hydrogen atom from the 4Ј-hydroxyl of the sugar ring to an acceptor nucleotide cofactor such as NAD ϩ or NADP ϩ (15,33,(37)(38)(39). In agreement with the widely accepted "SYK dogma," alteration of the tyrosine residue in the catalytic triad has resulted in inactive mutants as seen in the UDP-Gal C4 epimerase GalE S124A/Y149F and Y149F mutants (34,35) or in other SDR enzymes (17,40). At most, 0.25% of wild-type catalytic activity was reported using a Y/C dehydrogenase mutant (41).…”

mentioning

confidence: 71%

Structure-Function Studies of Two Novel UDP-GlcNAc C6 Dehydratases/C4 Reductases

Creuzenet¹,

Urbanic²,

Lam³

2002

Two subfamilies of UDP-GlcNAc C6 dehydratases were recently identified. FlaA1, a short soluble protein that exhibits a typical SYK catalytic triad, characterizes one of these subfamilies, and WbpM, a large membrane protein that harbors an altered SMK triad that was not predicted to sustain activity, represents the other subfamily. This study focuses on investigating the structure and function of these C6 dehydratases and the role of the altered triad as well as additional amino acid residues involved in catalysis. The significant activity retained by the FlaA1 Y141M triad mutant and the low activity of the WbpM M438Y mutant indicated that the methionine residue was involved in catalysis. A Glu 589 residue, which is conserved only within the large homologues, was shown to be essential for activity in WbpM. Introduction of this residue in FlaA1 enhanced the activity of the corresponding V266E mutant. Hence, this glutamate residue might be responsible for the retention of catalytic efficiency in the large homologues despite alteration of their catalytic triad. Mutations of residues specific for the short homologues (Asp 70 , Asp 149 -Lys 150 , Cys 103 ) abolished the activity of FlaA1. Among them, C103M prevented dimerization but did not significantly affect the secondary structure. The fact that we could identify subfamily-specific residues that are essential for catalysis suggested an independent evolution for each subfamily of C6 dehydratases. Finally, the loss of activity of the FlaA1 G20A mutant provided evidence that a cofactor is involved in catalysis, and kinetic study of the FlaA1 H86A mutant revealed that this conserved histidine is involved in substrate binding. None of the mutations investigated altered the substrate, product, and function specificity of these enzymes.FlaA1 and WbpM are two novel bifunctional UDP-GlcNAc C6 dehydratases/C4 reductases that were recently characterized at the biochemical level (1, 2). Both enzymes catalyze the stereo-specific conversion of UDP-GlcNAc to Qui2NAc 1 via the formation of a 4-keto, 6-deoxy intermediate. They are both highly specific for their substrate, UDP-GlcNAc, and exhibit no activity with closely related substrates, such as UDP-Glc, UDPGal, or UDP-GalNAc, or with substrates of other known C6 dehydratases such as GDP-mannose or dTDP-glucose. This is consistent with the fact that, despite their dehydratase activity, FlaA1 and WbpM exhibit very limited sequence similarity with other known C6 dehydratases, such as GDP-D-mannose (3-6) and dTDP-and CDP-D-glucose dehydratases (7-14). In contrast, they are homologous to C4 epimerases such as GalE from Escherichia coli (45% homology) (15) and WbpP from P. aeruginosa (49% homology) (16). The molecular basis for their unexpected functional specificity is not understood to date. Hence, FlaA1 and WbpM are representative members of a larger family of bifunctional dehydratases/reductases that is characterized by the presence of five conserved domains organized in the same pattern (1). One of these motifs corresponds to th...

“…The boxed string of identical residues near the C terminus may participate in substrate specificity. It differs greatly from a stretch of residues that are conserved among fucose synthetases at the same place in alignments of the polypeptide sequences (38). preparations is reversion.…”

Section: 5mentioning

confidence: 91%

Genetic Locus and Structural Characterization of the Biochemical Defect in the O-Antigenic Polysaccharide of the Symbiotically Deficient Rhizobium etli Mutant, CE166

Forsberg

Noel

Box

et al. 2003

The O-antigen polysaccharide (OPS) of Rhizobium etli CE3 lipopolysaccharide (LPS) is linked to the core oligosaccharide via an N-acetylquinovosaminosyl (QuiNAc) residue. A mutant of CE3, CE166, produces LPS with reduced amounts of OPS, and a suppressed mutant, CE166 alpha, produces LPS with nearly normal OPS levels. Both mutants are deficient in QuiNAc production. Characterization of OPS from CE166 and CE166 alpha showed that QuiNAc was replaced by its 4-keto derivative, 2-acetamido-2,6-dideoxyhexosyl-4-ulose. The identity of this residue was determined by NMR and mass spectrometry, and by gas chromatography-mass spectrometry analysis of its 2-acetamido-4-deutero-2,6-dideoxyhexosyl derivatives produced by reduction of the 4-keto group using borodeuteride. Mass spectrometric and methylation analyses showed that the 2-acetamido-2,6-dideoxyhexosyl-4-ulosyl residue was 3-linked and attached to the core-region external Kdo III residue of the LPS, the same position as that of QuiNAc in the CE3 LPS. DNA sequencing revealed that the transposon insertion in strain CE166 was located in an open reading frame whose predicted translation product, LpsQ, falls within a large family of predicted open reading frames, which includes biochemically characterized members that are sugar epimerases and/or reductases. A hypothesis to be tested in future work is that lpsQ encodes UDP-2-acetamido-2,6-dideoxyhexosyl-4-ulose reductase, the second step in the synthesis of UDP-QuiNAc from UDP-GlcNAc.