A Structural Study of GDP-4-Keto-6-Deoxy-d-Mannose-3-Dehydratase:  Caught in the Act of Geminal Diamine Formation^,

Abstract

Di-and tri-deoxysugars are an important class of carbohydrates synthesized by certain plants, fungi, and bacteria. Colitose, for example, is a 3,6-dideoxysugar found in the O-antigens of Gram-negative bacteria such as Escherichia coli, Salmonella enterica, Yersinia pseudotuberculosis, and Vibrio cholerae, among others. These types of dideoxysugars are thought to serve as antigenic determinants and to play key roles in bacterial defense and survival. Four enzymes are required for the biochemical synthesis of colitose starting from mannose-1-phosphate. The focus of this investigation, GDP-4-keto-6-deoxy-D-mannose-3-dehydratase (ColD), catalyzes the third step in the pathway, namely the PLP-dependent removal of the C3′-hydroxyl group from GDP-4-keto-6-deoxymannose. Whereas most PLP-dependent enzymes contain an active site lysine, ColD utilizes a histidine as its catalytic acid/base. The ping-pong mechanism of the enzyme first involves the conversion of PLP to PMP followed by the dehydration step. Here we present the three-dimensional structure of a site-directed mutant form of ColD whereby the active site histidine has been replaced with a lysine. The electron density reveals that the geminal diamine, a tetrahedral intermediate in the formation of PMP from PLP, has been trapped within the active site region. Functional assays further demonstrate that this mutant form of ColD cannot catalyze the dehydration reaction.Di-and tri-deoxysugars are an intriguing class of carbohydrates that are synthesized by plants, fungi, and bacteria (1,2). The biosynthetic pathways for the production of these unusual sugars typically begin with simple monosaccharides such as glucose-1-phosphate or fructose-6-phosphate as starting materials. Via an impressive array of reactions including dehydrations, reductions, amino transfers, and methylations, the pathways result in an astonishing array of sugars with differing three-dimensional shapes and hydrophilicities. Many of these unusual sugars have been isolated from the O-antigens of a variety of Gram-negative bacteria, where they contribute to the structural variations observed in bacterial cell walls (3). It has been suggested that these sugars play a role in the virulence of a given pathogen (4). Other deoxysugars have been found in polyketide antibiotics including erythromycin, azithromycin, and clarithromycin, where they are known to enhance the biological activities of the respective drugs (5).The focus of this investigation is an enzyme involved in the biosynthesis of colitose, a 3,6-dideoxysugar found in the O-antigen of Gram-negative bacteria such as Escherichia coli (6), Yersinia pseudotuberculosis(7) , Salmonella enterica (8), Vibrio cholerae (9), and in marine bacteria such as Pseudoalteromonas tetraodonis (9) carrageenovora (10). The biosynthesis of the O-antigen requires nucleotide-linked sugars, which are transferred to the oligosaccharide chain by an array of glycosyltransferases. In the case of colitose, the sugar is linked to GDP 1 .As indicated in Scheme 1, four...

Mechanisms and structures of vitamin B6-dependent enzymes involved in deoxy sugar biosynthesis

“…Like E 1 and CDP-D-ascarylose, attempts to co-crystallize ColD and its substrate consistently failed [90,91]. It was thought that perhaps this was due to the instability of the substrate during the crystallization process, so the enzyme was crystallized in the presence of GDP-D-perosamine ( 58 ), which differs from the natural substrate in that it features an amino group instead of a ketone at the C4-position [92].…”

Two Site-Directed Mutations Are Required for the Conversion of a Sugar Dehydratase into an Aminotransferase

“…The ColD gene used for this study had already been mutagenized to H188K and inserted into the pET28t plasmid (12, 14). The S187N/H188K mutant protein was expressed and purified in a manner identical to that previously described for the wild-type enzyme (9).…”

The structural biology of enzymes involved in natural product glycosylation

“…To date, NDP-hexose-4,6-dehydratases have been found to utilize the s hort-chain d ehydrogenase/ r eductase (SDR) structural fold and include: dTDP-glucose-4,6-dehydratases (EC 4.2.1.46) RmlB from Salmonella enterica and Streptococcus suis and DesIV from Streptomyces venezuelae 55, 86, 87 ; CDP-glucose-4,6-dehydratase (EC 4.2.1.45) from Yersinia pseudotuberculosis and Salmonella typhi 88, 89 ; GDP-mannose-4,6-dehydratase (EC 4.2.1.47) from Escherichia coli , Pseudomonas aeruginosa and Aquifex aeolicus VF5 90–92 ; and UDP-GlcNAc-4,6 dehydratase (EC 4.2.1.115) FlaA1 from Helicobacter pylori 93 . Dehydrases which subsequently act upon NDP-4-keto-6-deoxyhexoses to facilitate pyridoxamine 5′-phosphate (PMP)-mediated dehydration/deoxygenation adjacent to the C4 carbonyl adopt an aspartate aminotransferase (AAT) fold and include: the GDP-4-keto-6-deoxy-D-mannose-3-dehydratase ColD from Escherichia coli 94 ; and the CDP-6-deoxy-L-threo-D-glycero-4-hexulose 3-dehydrase E1 from Yersinia pseudotuberculosis 53 .…”

“…Dehydratases of SDR family do not exhibit an obligate multimeric preference and have been found as dimeric 55, 86, 87, 90, 94, 95 , tetrameric 88, 91 , and hexameric enzymes 93 . The monomer-monomer association in these multimers occurs principally through hydrophobic interactions via a four-helix bundle (Figures 4A & 4B) and each monomer exhibits an α/β structure that can be divided into two domains.…”

“…While these catalysts adopt an aspartate aminotransferase (AAT) fold and, like typical aminotransferases, use PMP as a cofactor, the conserved transaminase lysine which typically forms an imine with PMP is replaced in these enzymes with a conserved histidine [H188 in ColD and H220 in E1] 53, 94 . Although, they have same structural fold (Figure 6A), a key feature that distinguishes E1 from ColD is the presence of a [2Fe-2S] cluster critical to the final reductive deoxygenation step, the ligands for which are cysteine and a histidine (C251-X 1 -C253-X 7 -C261-X 17 -H278) 100 .…”