Elucidation of the K32 Capsular Polysaccharide Structure and Characterization of the KL32 Gene Cluster of Acinetobacter baumannii LUH5549.

Capsular polysaccharide (CPS), isolated from Acinetobacter baumannii LUH5549 carrying the KL32 capsule biosynthesis gene cluster, was studied by sugar analysis, Smith degradation, and one- and two-dimensional 1H and 13C NMR spectroscopy. The K32 CPS was found to be composed of branched pentasaccharide repeats (K units) containing two residues of ß-D-GalpNAc and one residue of ß-D-GlcpA (ß-D-glucuronic acid) in the main chain and one residue each of ß-D-Glcp and α-D-GlcpNAc in the disaccharide side chain. Consistent with the established CPS structure, the KL32 gene cluster includes genes for a UDP-glucose 6-dehydrogenase (Ugd3) responsible for D-GlcA synthesis and four glycosyltransferases that were assigned to specific linkages. Genes encoding an acetyltransferase and an unknown protein product were not involved in CPS biosynthesis. Whilst the KL32 gene cluster has previously been found in the global clone 2 (GC2) lineage, LUH5549 belongs to the sequence type ST354, thus demonstrating horizontal gene transfer between these lineages.

The Mycobacterium tuberculosis cmaA2 gene encodes a mycolic acid trans-cyclopropane synthetase.

Infection with Mycobacterium tuberculosis remains a major global health emergency. Although detailed understanding of the molecular events of M. tuberculosis pathogenesis is still limited, recent genetic analyses have implicated specific lipids of the cell envelope as important effectors in M. tuberculosis pathogenesis. We have shown that pcaA, a novel member of a family of M. tuberculosis S-adenosyl methionine (SAM)-dependent methyl transferases, is required for alpha-mycolic acid cyclopropanation and lethal chronic persistent M. tuberculosis infection. To examine the apparent redundancy between pcaA and cmaA2, another cyclopropane synthetase of M. tuberculosis thought to be involved in alpha-mycolate synthesis, we have disrupted the cmaA2 gene in virulent M. tuberculosis by specialized transduction. Inactivation of cmaA2 causes accumulation of unsaturated derivatives of both the methoxy- and ketomycolates. Analysis by proton NMR indicates that the mycolic acids of the cmaA2 mutant lack trans-cyclopropane rings but are otherwise intact with respect to cyclopropane and methyl branch content. Thus, cmaA2 is required for the synthesis of the trans cyclopropane rings of both the methoxymycolates and ketomycolates. These results define cmaA2 as a trans-cyclopropane synthetase and expand our knowledge of the substrate specificity of a large family of highly homologous mycolic acid methyl transferases recently shown to be critical to M. tuberculosis pathogenesis.

The 2.0 A structure of malarial purine phosphoribosyltransferase in complex with a transition-state analogue inhibitor.

Malaria is a leading cause of worldwide mortality from infectious disease. Plasmodium falciparum proliferation in human erythrocytes requires purine salvage by hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase). The enzyme is a target for the development of novel antimalarials. Design and synthesis of transition-state analogue inhibitors permitted cocrystallization with the malarial enzyme and refinement of the complex to 2.0 A resolution. Catalytic site contacts in the malarial enzyme are similar to those of human hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) despite distinct substrate specificity. The crystal structure of malarial HGXPRTase with bound inhibitor, pyrophosphate, and two Mg(2+) ions reveals features unique to the transition-state analogue complex. Substrate-assisted catalysis occurs by ribooxocarbenium stabilization from the O5' lone pair and a pyrophosphate oxygen. A dissociative reaction coordinate path is implicated in which the primary reaction coordinate motion is the ribosyl C1' in motion between relatively immobile purine base and (Mg)(2)-pyrophosphate. Several short hydrogen bonds form in the complex of the enzyme and inhibitor. The proton NMR spectrum of the transition-state analogue complex of malarial HGXPRTase contains two downfield signals at 14.3 and 15.3 ppm. Despite the structural similarity to the human enzyme, the NMR spectra of the complexes reveal differences in hydrogen bonding between the transition-state analogue complexes of the human and malarial HG(X)PRTases. The X-ray crystal structures and NMR spectra reveal chemical and structural features that suggest a strategy for the design of malaria-specific transition-state inhibitors.

Motional dynamics of the catalytic loop in OMP synthase.

In de novo pyrimidine biosynthesis, orotate phosphoribosyltransferase catalyzes the formation of orotidine 5'-monophosphate (OMP) from orotic acid and alpha-D-5-phosphoribosyl-1-pyrophosphate (PRPP). The known three-dimensional structure of the dimeric enzyme from Salmonella typhimurium is similar to that of other Type I phosphoribosyltransferases (nucleotide synthases) with a solvent-exposed active site atop a Rossman-type nucleotide binding fold. The three-dimensional structure of an enzyme-inhibitor complex [Henriksen et al. (1996) Biochemistry 35, 3803-3809] indicates that one of the two identical solvent-exposed loops can descend to cover the active site of the adjacent subunit of the dimeric enzyme. Catalytically essential residues are known to reside on this loop. In the present work, sensitivity toward limited proteolysis by trypsin confirms that the loop is solvent-exposed. Protection by PRPP and, to a lesser extent, by OMP demonstrates the existence of a second, trypsin-inaccessible, loop position. Two-dimensional 1H-15N NMR relaxation experiments on [alpha-15N]histidine-labeled WT OPRTase yielded backbone 15N T1 and T2 relaxation times and 15N[1H] NOE for His-105 (a loop residue) that are characteristic of small peptides. These results document that the surface loop is highly flexible in the unliganded enzyme. Addition of a hydrolytically stable PRPP analogue to the enzyme resulted in a significant reduction of His-105 peak intensity, indicating a dramatic change in the dynamic properties of the loop backbone in the analogue-ligated enzyme. 1H NMR titrations on histidine C2 protons, coupled with 1H and 31P titrations monitoring the C1H and 5-phosphate PRPP resonances, allowed the quantitation of the rates of loop movement during product release, and relate protein motion to enzymatic catalysis. These results suggest that loop opening and PRPP release is a two-step process, whose overall rate is partially rate-limiting in the reverse pyrophosphorolysis reaction.

Solution structure and dynamics of the bioactive retroviral M domain from Rous sarcoma virus.

A biologically active construct of the retroviral M domain from the avian Rous sarcoma virus is defined and its solution structure described. This M domain is fully active in budding and infectivity without myristylation. In spite of a sequence homology level that suggests no relationship among M domains and the family of matrix proteins in mammalian retroviruses, the conserved structural elements of a central core allow an M domain sequence motif to be described for all retroviruses. The surface of the M domain has a highly clustered positive patch comprised of sequentially distant residues. An analysis of the backbone dynamics, incorporating rotational anisotropy, is used to estimate the thermodynamics of proposed domain oligomerization.

The solution structure of the pleckstrin homology domain of human SOS1. A possible structural role for the sequential association of diffuse B cell lymphoma and pleckstrin homology domains.

A large subset of pleckstrin homology (PH) domains are immediately to the C terminus of diffuse B cell lymphoma (Dbl) homology (DbH) domains. Dbl domains are generally considered to be GTPase-exchange factors; many are proto-oncogenes. PH domains appear to function as membrane-recruitment factors, or have specific protein-protein interactions. Since dual domain (DbH/PH) constructs are known to have significant properties in other pathways, it is possible that a defined interdomain relationship is required for DbH/PH function. We determined the solution structure of the human SOS1 PH domain for a construct partially extended into the preceding DbH domain. There are specific structural contacts between the PH and the vestigial DbH domain. This appears to involve structural elements common to this subfamily of PH domains, and to DbH domains. The human SOS1 PH domain binds to inositol 1,4,5-triphosphate with a approximately 60 mu M affinity. Using chemical shift titration, the binding site is identified to be essentially identical to that observed crystallographically for the inositol 1,4,5-triphosphate complex with the PH domain of phospholipase Cdelta. This site may serve as an interdomain regulator of DbH or other domains' functions. While the overall fold of the human SOS1 PH domain is similar to other PH domains, the size and position of the intrastrand loops and the presence of an N-terminal alpha-helix of the vestigial DbH domain suggest that the subfamily of PH domains associated with DbH domains may be a well defined structural group in which the PH domain is a membrane recruiter and modulator.

Identification of the binding site for acidic phospholipids on the pH domain of dynamin: implications for stimulation of GTPase activity.

It has recently been suggested that pleckstrin homology (PH) domains bind specifically to phospholipids, with phosphatidylinositol-4,5-bisphosphate (PtdIns(4,5)P2) being most strongly bound. This observation suggests that PH domains may be responsible for membrane association of proteins in which they occur. Further, this membrane association may be regulated by enzymes that modify lipid head groups to which PH domains may bind. We have studied the binding of phospholipids to the PH domain of human dynamin, a 100 kDa GTPase that is involved in the initial stages of endocytosis. We describe a rapid method for screening PH domain/ligand interactions that gives precise binding constants. We confirm that PtdIns(4,5)P2 can bind to dynamin PH domain, although not in an aggregated state. Using NMR spectroscopy, we have mapped a specific site on the surface of dynamin PH domain of which binding of gIns(1,4,5)P3 (the head-group skeleton of PtdIns(4,5)P2) occurs. The relative affinity of acidic phospholipids for dynamin PH domain correlates with their ability to activate the GTPase of dynamin. We propose, therefore, that the interaction of these phospholipids with dynamin is likely to occur via the PH domain. Given the fact that PH domains are often found in proteins associated with GTPase activity, or in guanine nucleotide exchange factors, we suggest that one role of PH domains may be to couple phosphatidylinositol signalling to GTP hydrolysis.