Crystal structures of two putative phosphoheptose isomerases. Proteins

Biology Department, Brookhaven National Laboratory, Upton, New York 11973, USA.
Proteins Structure Function and Bioinformatics (Impact Factor: 2.63). 06/2006; 63(4):1092-6. DOI: 10.1002/prot.20908
Source: PubMed
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Available from: Christopher D Lima, May 09, 2015
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    • "Structural superimposition of HobA with two SIS domain-containing proteins. Ribbon diagram of the HobA structure (light orange) superimposed with the phosphoheptose isomerase from Pseudomonas aeruginosa (pdb code 1X92) coloured in blue (left) and from Campylobacter jejuni (pdb code 1TK9; Seetharaman et al., 2006 "
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    ABSTRACT: In prokaryotes, DNA replication is initiated by the binding of DnaA to the oriC region of the chromosome to load the primosome machinery and start a new replication round. Several proteins control these events in Escherichia coli to ensure that replication is precisely timed during the cell cycle. Here, we report the crystal structure of HobA (HP1230) at 1.7 A, a recently discovered protein that specifically interacts with DnaA protein from Helicobacter pylori (HpDnaA). We found that the closest structural homologue of HobA is a sugar isomerase (SIS) domain containing protein, the phosphoheptose isomerase from Pseudomonas aeruginosa. Remarkably, SIS proteins share strong sequence homology with DiaA from E. coli; yet, HobA and DiaA share no sequence homology. Thus, by solving the structure of HobA, we unexpectedly discovered that HobA is a H. pylori structural homologue of DiaA. By comparing the structure of HobA to a homology model of DiaA, we identified conserved, surface-accessible residues that could be involved in protein-protein interaction. Finally, we show that HobA specifically interacts with the N-terminal part of HpDnaA. The structural homology between DiaA and HobA strongly supports their involvement in the replication process and these proteins could define a new structural family of replication regulators in bacteria.
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    ABSTRACT: The barrier imposed by lipopolysaccharide (LPS) in the outer membrane of Gram-negative bacteria presents a significant challenge in treatment of these organisms with otherwise effective hydrophobic antibiotics. The absence of l-glycero-d-manno-heptose in the LPS molecule is associated with a dramatically increased bacterial susceptibility to hydrophobic antibiotics and thus enzymes in the ADP-heptose biosynthesis pathway are of significant interest. GmhA catalyzes the isomerization of d-sedoheptulose 7-phosphate into d-glycero-d-manno-heptose 7-phosphate, the first committed step in the formation of ADP-heptose. Here we report structures of GmhA from Escherichia coli and Pseudomonas aeruginosa in apo, substrate, and product-bound forms, which together suggest that GmhA adopts two distinct conformations during isomerization through reorganization of quaternary structure. Biochemical characterization of GmhA mutants, combined with in vivo analysis of LPS biosynthesis and novobiocin susceptibility, identifies key catalytic residues. We postulate GmhA acts through an enediol-intermediate isomerase mechanism.
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    ABSTRACT: MurNAc etherases cleave the unique D-lactyl ether bond of the bacterial cell wall sugar N-acetylmuramic acid (MurNAc). Members of this newly discovered family of enzymes are widely distributed among bacteria and are required to utilize peptidoglycan fragments obtained either from the environment or from the endogenous cell wall (i.e., recycling). MurNAc etherases are strictly dependent on the substrate MurNAc possessing a free reducing end and a phosphoryl group at C6. They carry a single conserved sugar phosphate isomerase/sugar phosphate-binding (SIS) domain to which MurNAc 6-phosphate is bound. Two subunits form an enzymatically active homodimer that structurally resembles the isomerase module of the double-SIS domain protein GlmS, the glucosamine 6-phosphate synthase. Structural comparison provides insights into the two-step lyase-type reaction mechanism of MurNAc etherases: beta-elimination of the D-lactic acid substituent proceeds through a 2,3-unsaturated sugar intermediate to which water is subsequently added.
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