Theme C Highlights Theme C Bacterial (primarily S. aureus) proteins for therapeutic intervention
Sar2676, is pantothenate synthetase from MRSA, and an appealing drug target. With 40% identity to the E. coli enzyme, our structure solution proceeded by molecular replacement. The enzyme is upregulated in MRSA252 compared to MSSA476 suggesting it may important in conferring resistance. We have determined the structure to 2.5 Å and now have a complex with ATP bound to 1.8Å. This structure will be used for fragment screening to identify novel inhibitors.
The gene coding for this protein is annotated as a putative phosphofructose kinase enzyme, because its sequence diverges from other bacterial PFK’s. We have determined its structure and surprisingly it is similar to MRSA tagatose kinase (see below). We have confirmed that the enzyme is in fact an efficient phosphofructo kinase. The enzyme is upregulated in MRSA when compared to MSSA.
Structures of Staphylococcus aureus d-tagatose-6-phosphate kinase (LacC) in two crystal forms have been determined. The structures define LacC in apoform, in binary complexes with ADP or the co-factor analogue AMP-PNP, and in a ternary complex with AMP-PNP and D-tagatose-6-phosphate. The tertiary structure of the LacC monomer, which is closely related to other members of the pfkB subfamily of carbohydrate kinases, is composed of a large alpha/beta core domain and a smaller, largely beta "lid." Four extended polypeptide segments connect these two domains. Dimerization occurs via interactions between lid domains, which form a beta-clasp structure. Residues from both subunits contribute to substrate binding. LacC adopts a closed structure required for phosphoryl transfer only when both substrate and co-factor are bound. A reaction mechanism similar to that used by other phosphoryl transferases is proposed and a new motif of amino acid sequence conservation common to the pfkB subfamily of carbohydrate kinases identified.
DNA glycoylase catalyses the removal of methylated adenosyl residues from DNA. We have deleted the gene in knock out experiment and are looking at its phenotype. We have now determined the complex between the protein and 5-methyl adenine.
Pseudomonas aeruginosa encodes a gene that is annotated as a “sialidase” as it contains the bacterial neuraminidase repeats (BNRs) that are a signature of sialidases. Gene knockout showed that the enzyme plays a key role in the initial stages of pulmonary infection by targeting bacterial glycoconjugates and contributing to the formation of biofilm. It has been suggested that the enzyme removes pseudaminic acid, a 9-carbon acidic sugar related to neuraminic acid that can decorate the LPS, pili and flagella in P. aeruginosa and Campylobacter pylori and C. jejuni. We have determined the crystal structure to 1.9Å. The 47kDa protein forms a trimer with trimerisation formed through a novel C-terminal immunoglobulin-like domain. In silico docking studies suggest that this enzyme is indeed a pseudaminidase.
VC1804 and VC1805 are adjacent genes encoding hypothetical proteins within the VPI-2 pathogenicity island of Vibrio cholerae, and are part of a cluster of genes only present in toxigenic strains of the bacterium. Paralogous adjacent genes, VC0508 and VC0509, are also present within the Vibrio seventh pandemic island-II of V. cholerae El Tor and O139 serogroup isolates. Sequence similarity suggests that VC1804 and VC1805 will share a similar protein fold. Homologues of VC1804 and VC1805 exist in several Vibrio species that are pathogenic or symbionts of marine organisms, and orthologues of both genes exist in other -Proteobacteria. The crystal structure of VC1805 from V. cholerae O1 El Tor strain N16961 has been determined to a resolution of 2.1Å by multiple isomorphous replacement. Mapping conserved residues onto the structure reveals two well-defined areas that may represent conserved binding sites on the protein’s negatively-charged surface. Structural comparison with the protein databank revealed VC1805 to be structurally homologous to the human mitochondrial protein p32 that is known to bind to a variety of partners. Gene knockout experiments are in progress in the University of Delaware. PDB 2v1l.
UDP-3-O-acyl-glucosamine N-acyltransferase (LpxD), which transfers 3-hydroxy-arachidic acid from acyl carrier protein to the 2' amine of UDP-3-O-myristoyl glucosamine. Our study reveals LpxD is a homotrimer, each subunit constructed from a novel combination of an N-terminal uridine-binding domain, a core lipid-binding domain, and a C-terminal helical extension. Highly conserved residues dominate nucleotide binding and place the glucosamine moiety at the catalytic center formed by two subunits. Two histidines contribute to a mechanism involving nucleophilic attack by the amine of one substrate on the carbonyl carbon of an acyl carrier protein thioester conjugate. Serendipitously, our study reveals palmitic acid binding near the catalytic center and allows for a description of structure and reactivity at an early stage of LPS assembly.
We have solved apo mevalonate kinase from S. aureus, complementing our structure from Leishmania major that was determined last year. Gram positive bacteria like eukaryotes and trypanosomatids synthesize isoprenoids starting from mevalonate. This structure of the enzyme provides a starting point for fragment soaking to explore feature of the enzyme which may allow its specific inhibition of bacteria and trypanosomatids. There is limited data on the pathway enzymes in trypanosomatids. High-resolution Apo (solved last year) and now most importantly the (R)-mevalonate ((R)-MVA) bound crystal structure of Leishmania major (LmMK) provide, for the first time, information concerning binding of mevalonate to an MK. This represents a rare example of any GHMP kinase family member substrate complex. The mevalonate binds in a deep cavity lined by highly conserved residues and the determinants for discrimination of (R)- over (S)-mevalonate elucidated. The C5 hydroxyl, where phosphorylation occurs, points towards catalytic residues, Lys18 and Asp155. In LmMK, an -helix and the preceding polypeptide adopt a conformation, not seen in related kinase structures, impeding access to the nucleotide triphosphate binding site suggesting that a conformational rearrangement is required to allow ATP binding.

Theme C Highlights