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1.  A Structural and Biochemical Model of Processive Chitin Synthesis* 
The Journal of Biological Chemistry  2014;289(33):23020-23028.
Background: Chitin synthesis is an attractive drug target in a range of organisms but is not understood at the molecular level.
Results: The chitooligosaccharide synthase NodC can be assayed with a novel HTS assay, and the mechanism/fold can be probed by site-directed mutagenesis and topology mapping.
Conclusion: NodC is a model system to probe chitin synthesis.
Significance: This work enables the exploitation of chitin synthesis as a drug target.
Chitin synthases (CHS) produce chitin, an essential component of the fungal cell wall. The molecular mechanism of processive chitin synthesis is not understood, limiting the discovery of new inhibitors of this enzyme class. We identified the bacterial glycosyltransferase NodC as an appropriate model system to study the general structure and reaction mechanism of CHS. A high throughput screening-compatible novel assay demonstrates that a known inhibitor of fungal CHS also inhibit NodC. A structural model of NodC, on the basis of the recently published BcsA cellulose synthase structure, enabled probing of the catalytic mechanism by mutagenesis, demonstrating the essential roles of the DD and QXXRW catalytic motifs. The NodC membrane topology was mapped, validating the structural model. Together, these approaches give insight into the CHS structure and mechanism and provide a platform for the discovery of inhibitors for this antifungal target.
doi:10.1074/jbc.M114.563353
PMCID: PMC4132801  PMID: 24942743
Carbohydrate Biosynthesis; Enzyme Inhibitor; Enzyme Mechanism; Glycosyltransferase; Protein Structure
2.  Structure of a bacterial putative acetyltransferase defines the fold of the human O-GlcNAcase C-terminal domain 
Open Biology  2013;3(10):130021.
The dynamic modification of proteins by O-linked N-acetylglucosamine (O-GlcNAc) is an essential posttranslational modification present in higher eukaryotes. Removal of O-GlcNAc is catalysed by O-GlcNAcase, a multi-domain enzyme that has been reported to be bifunctional, possessing both glycoside hydrolase and histone acetyltransferase (AT) activity. Insights into the mechanism, protein substrate recognition and inhibition of the hydrolase domain of human OGA (hOGA) have been obtained via the use of the structures of bacterial homologues. However, the molecular basis of AT activity of OGA, which has only been reported in vitro, is not presently understood. Here, we describe the crystal structure of a putative acetyltransferase (OgpAT) that we identified in the genome of the marine bacterium Oceanicola granulosus, showing homology to the hOGA C-terminal AT domain (hOGA-AT). The structure of OgpAT in complex with acetyl coenzyme A (AcCoA) reveals that, by homology modelling, hOGA-AT adopts a variant AT fold with a unique loop creating a deep tunnel. The structures, together with mutagenesis and surface plasmon resonance data, reveal that while the bacterial OgpAT binds AcCoA, the hOGA-AT does not, as explained by the lack of key residues normally required to bind AcCoA. Thus, the C-terminal domain of hOGA is a catalytically incompetent ‘pseudo’-AT.
doi:10.1098/rsob.130021
PMCID: PMC3814719  PMID: 24088714
signalling; O-GlcNAc; glycobiology; protein structure
3.  O-GlcNAc transferase invokes nucleotide sugar pyrophosphate participation in catalysis 
Nature chemical biology  2012;8(12):969-974.
Protein O-GlcNAcylation is an essential post-translational modification on hundreds of intracellular proteins in metazoa, catalyzed by O-GlcNAc transferase using unknown mechanisms of transfer and substrate recognition. Through crystallographic snapshots and mechanism-inspired chemical probes, we define how human O-GlcNAc transferase recognizes the sugar donor and acceptor peptide and employs a novel catalytic mechanism of glycosyl transfer, involving the sugar donor α-phosphate as the catalytic base, as well as an essential lysine. This mechanism appears to be a unique evolutionary solution to the spatial constraints imposed by a bulky protein acceptor substrate, and explains the unexpected specificity of a recently reported metabolic O-GlcNAc transferase inhibitor.
doi:10.1038/nchembio.1108
PMCID: PMC3509171  PMID: 23103942
4.  Purification, crystallization and preliminary X-ray diffraction data of UDP-galactopyranose mutase from Aspergillus fumigatus  
The cloning, overexpression, purification, crystallization and preliminary X-ray diffraction data are described for UDP-galactopyranose mutase, an enzyme involved in cell-wall synthesis in A. fumigatus.
Aspergillus fumigatus UDP-galactopyranose mutase (AfUGM) is a potential drug target involved in the synthesis of the cell wall of this fungal pathogen. AfUGM was recombinantly produced in Escherichia coli, purified and crystallized by the sitting-drop method, producing orthorhombic crystals that diffracted to a resolution of 3.25 Å. The crystals contained four molecules per asymmetric unit and belonged to space group P212121, with unit-cell parameters a = 127.72, b = 134.30, c = 173.84 Å. Incorporation of selenomethionine was achieved, but the resulting crystals did not allow solution of the phase problem.
doi:10.1107/S1744309112017915
PMCID: PMC3370916  PMID: 22684076
UDP-galactopyranose mutase; Aspergillus fumigatus
5.  Functional domains of the Xenopus replication licensing factor Cdt1 
Nucleic Acids Research  2005;33(1):316-324.
During late mitosis and early G1, replication origins are licensed for subsequent replication by loading heterohexamers of the mini-chromosome maintenance proteins (Mcm2-7). To prevent re-replication of DNA, the licensing system is down-regulated at other cell cycle stages. A small protein called geminin plays an important role in this down-regulation by binding and inhibiting the Cdt1 component of the licensing system. We examine here the organization of Xenopus Cdt1, delimiting regions of Cdt1 required for licensing and regions required for geminin interaction. The C-terminal 377 residues of Cdt1 are required for licensing and the extreme C-terminus contains a domain that interacts with an Mcm(2,4,6,7) complex. Two regions of Cdt1 interact with geminin: one at the N-terminus, and one in the centre of the protein. Only the central region binds geminin tightly enough to successfully compete with full-length Cdt1 for geminin binding. This interaction requires a predicted coiled-coil domain that is conserved amongst metazoan Cdt1 homologues. Geminin forms a homodimer, with each dimer binding one molecule of Cdt1. Separation of the domains necessary for licensing activity from domains required for a strong interaction with geminin generated a construct, whose licensing activity was partially insensitive to geminin inhibition.
doi:10.1093/nar/gki176
PMCID: PMC546161  PMID: 15653632

Results 1-5 (5)