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The crystal structure of the Y140F mutant of ADP-L-glycero-D-manno-heptose 6-epimerase bound to ADP-beta-D-mannose suggests a one base mechanism.

ABSTRACT: Bacteria synthesize a wide array of unusual carbohydrate molecules, which they use in a variety of ways. The carbohydrate L-glycero-D-manno-heptose is an important component of lipopolysaccharide and is synthesized in a complex series of enzymatic steps. One step involves the epimerization at the C6'' position converting ADP-D-glycero-D-manno-heptose into ADP-L-glycero-D-manno-heptose. The enzyme responsible is a member of the short chain dehydrogenase superfamily, known as ADP-L-glycero-D-manno-heptose 6-epimerase (AGME). The structure of the enzyme was known but the arrangement of the catalytic site with respect to the substrate is unclear. We now report the structure of AGME bound to a substrate mimic, ADP-beta-D-mannose, which has the same stereochemical configuration as the substrate. The complex identifies the key residues and allows mechanistic insight into this novel enzyme.

SUBMITTER: Kowatz T

PROVIDER: S-EPMC2974825 | biostudies-literature | 2010 Jul

REPOSITORIES: biostudies-literature

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The crystal structure of the Y140F mutant of ADP-L-glycero-D-manno-heptose 6-epimerase bound to ADP-beta-D-mannose suggests a one base mechanism.

Kowatz Thomas T Morrison James P JP Tanner Martin E ME Naismith James H JH

Protein science : a publication of the Protein Society 20100701 7

Bacteria synthesize a wide array of unusual carbohydrate molecules, which they use in a variety of ways. The carbohydrate L-glycero-D-manno-heptose is an important component of lipopolysaccharide and is synthesized in a complex series of enzymatic steps. One step involves the epimerization at the C6'' position converting ADP-D-glycero-D-manno-heptose into ADP-L-glycero-D-manno-heptose. The enzyme responsible is a member of the short chain dehydrogenase superfamily, known as ADP-L-glycero-D-manno ...[more]

PMID: 20506248

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Project description:Haemophilus influenzae is an important human pathogen. The lipooligosaccharide (LOS) of H. influenzae has been implicated as a virulence determinant. To better understand the assembly of LOS in nontypeable H. influenzae (NtHi), we have cloned and characterized the rfaD and rfaF genes of NtHi 2019, which encode the ADP-L-glycero-D-manno-heptose-6-epimerase and heptosyltransferase II enzymes, respectively. This cloning was accomplished by the complementation of Salmonella typhimurium lipopolysaccharide (LPS) biosynthesis gene mutants. These deep rough mutants are novobiocin susceptible until complemented with the appropriate gene. In this manner, we are able to use novobiocin resistance to select for specific NtHi LOS inner core biosynthesis genes. Such a screening system yielded a plasmid with a 4.8-kb insert. This plasmid was able to complement both rfaD and rfaF mutants of S. typhimurium. The LPS of these complemented strains appeared identical to the wild-type Salmonella LPS. The genes encoding the rfaD and rfaF genes from NtHi 2019 were sequenced and found to be similar to the analogous genes from S. typhimurium and Escherichia coli. The rfaD gene encodes a polypeptide of 35 kDa and the rfaF encodes a protein of 39 kDa, as demonstrated by in vitro transcription-translation studies. Isogenic mutants which demonstrated truncated LOS consistent with inner core biosynthesis mutants were constructed in the NtHi strain 2019. Primer extension analysis demonstrated the presence of a strong promoter upstream of rfaD but suggested only a very weak promoter upstream of rfaF. Complementation studies, however, suggest that the rfaF gene does have an independent promoter. Mass spectrometric analysis shows that the LOS molecules expressed by H. influenzae rfaD and rfaF mutant strains have identical molecular masses. Additional studies verified that in the rfaD mutant strain, D-glycero-D-manno-heptose is added to the LOS molecule in place of the usual L-glycero-D-manno-heptose. Finally, the genetic organizations of the inner core biosynthesis genes of S. typhimurium, E. coli, and several strains of H. influenzae were examined, and substantial differences were uncovered.

Project description:D-Glycero-d-manno-heptose-1,7-bisphosphate phosphatase (GmhB) is a member of the histidinol-phosphate phosphatase (HisB) subfamily of the haloalkanoic acid dehalogenase (HAD) enzyme superfamily. GmhB supports two divergent biochemical pathways in bacteria: the d-glycero-d-manno-heptose-1alpha-GDP pathway (in S-layer glycoprotein biosynthesis) and the l-glycero-d-manno-heptose-1beta-ADP pathway (in lipid A biosynthesis). Herein, we report the comparative analysis of substrate recognition in selected GmhB orthologs. The substrate specificity of the l-glycero-d-manno-heptose-1beta-ADP pathway GmhB from Escherichia coli K-12 was evaluated using hexose and heptose bisphosphates, histidinol phosphate, and common organophosphate metabolites. Only d-glycero-d-manno-heptose 1beta,7-bisphosphate (k(cat)/K(m) = 7 x 10(6) M(-1) s(-1)) and d-glycero-d-manno-heptose 1alpha,7-bisphosphate (k(cat)/K(m) = 7 x 10(4) M(-1) s(-1)) displayed physiologically significant substrate activity. (31)P NMR analysis demonstrated that E. coli GmhB selectively removes the C(7) phosphate. Steady-state kinetic inhibition studies showed that d-glycero-d-manno-heptose 1beta-phosphate (K(is) = 60 microM, and K(ii) = 150 microM) and histidinol phosphate (K(is) = 1 mM, and K(ii) = 6 mM), while not hydrolyzed, do in fact bind to E. coli GmhB, which leads to the conclusion that nonproductive binding contributes to substrate discrimination. High catalytic efficiency and a narrow substrate range are characteristic of a well-evolved metabolic enzyme, and as such, E. coli GmhB is set apart from most HAD phosphatases (which are typically inefficient and promiscuous). The specialization of the biochemical function of GmhB was examined by measuring the kinetic constants for hydrolysis of the alpha- and beta-anomers of d-glycero-d-manno-heptose 1beta,7-bisphosphate catalyzed by the GmhB orthologs of the l-glycero-d-manno-heptose 1beta-ADP pathways operative in Bordetella bronchiseptica and Mesorhizobium loti and by the GmhB of the d-glycero-d-manno-heptose 1alpha-GDP pathway operative in Bacteroides thetaiotaomicron. The results show that although each of these representatives possesses physiologically significant catalytic activity toward both anomers, each displays substantial anomeric specificity. Like E. coli GmhB, B. bronchiseptica GmhB and M. loti GmhB prefer the beta-anomer, whereas B. thetaiotaomicron GmhB is selective for the alpha-anomer. By determining the anomeric configuration of the physiological substrate (d-glycero-d-manno-heptose 1,7-bisphosphate) for each of the four GmhB orthologs, we discovered that the anomeric specificity of GmhB correlates with that of the pathway kinase. The conclusion drawn from this finding is that the evolution of the ancestor to GmhB in the HisB subfamily provided for specialization toward two distinct biochemical functions.

Project description:ADP-activated β-D-manno-heptoses (ADP-β-D-manno-heptoses) are precursors for the biosynthesis of the inner core of lipopolysaccharide in Gram-negative bacteria. Recently, ADP-D-glycero-β-D-manno-heptose (ADP-D,D-manno-heptose) and its C-6'' epimer, ADP-L-glycero-β-D-manno-heptose (ADP-L,D-manno-heptose), were identified as potent pathogen-associated molecular patterns (PAMPs) that can trigger robust innate immune responses. Although the production of ADP-D,D-manno-heptose has been studied in several different pathogenic Gram-negative bacteria, current knowledge of ADP-β-D-manno-heptose biosynthesis in Vibrio strains remains limited. Here, we characterized the biosynthetic enzymes of ADP-D,D-manno-heptose and the epimerase that converts it to ADP-L,D-manno-heptose from Vibrio cholerae (the causative agent of pandemic cholera) and Vibrio parahaemolyticus (non-cholera pathogen causing vibriosis with clinical manifestations of gastroenteritis and wound infections) in comparison with their isozymes from Escherichia coli. Moreover, we discovered that β-D-mannose 1-phosphate, but not α-D-mannose 1-phosphate, could be activated to its ADP form by the nucleotidyltransferase domains of bifunctional kinase/nucleotidyltransferases HldEVC (from V. cholerae) and HldEVP (from V. parahaemolyticus). Kinetic analyses of the nucleotidyltransferase domains of HldEVC and HldEVP together with the E. coli-derived HldEEC were thus carried out using β-D-mannose 1-phosphate as a mimic sugar substrate. Overall, our works suggest that V. cholerae and V. parahaemolyticus are capable of synthesizing ADP-β-D-manno-heptoses and lay a foundation for further physiological function explorations on manno-heptose metabolism in Vibrio strains. KEY POINTS: • Vibrio strains adopt the same biosynthetic pathway as E. coli in synthesizing ADP-β-D-manno-heptoses. • HldEs from two Vibrio strains and E. coli could activate β-D-mannose 1-phosphate to ADP-β-D-mannose. • Comparable nucleotidyltransfer efficiencies were observed in the kinetic studies of HldEs.

Dataset Information

The crystal structure of the Y140F mutant of ADP-L-glycero-D-manno-heptose 6-epimerase bound to ADP-beta-D-mannose suggests a one base mechanism.

Publications

The crystal structure of the Y140F mutant of ADP-L-glycero-D-manno-heptose 6-epimerase bound to ADP-beta-D-mannose suggests a one base mechanism.

Similar Datasets

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