Structural Biology Research On SAM-dependent O-methyltransferase From Leptospira Interrogans And Human MitoNEET

Partâ… Crystal structure of SAM-dependent O-methyltransferase from pathogenic bacterium Leptospira interrogansThe S-adenosylmethionine(SAM)-dependent O-methyltransferase from Leptospira interrogans(LiOMT)expressed by gene LA0415 belongs to the Methyltransf₃ family(Pfam PF01596).In this family all of the five bacterial homologues with known function are reported as SAM-dependent O-methylstransferases involved in antibiotic production.The crystal structure of LiOMT in complex with S-adenosylhomocysteine reported here is the first bacterial protein structure in this family.The LiOMT structure shows a conserved SAM-binding region and a probable metal-dependent catalytic site.The molecules of LiOMT generate homodimers by N-terminal swapping,which assists the pre-organization of the substrate-binding site. Based on the sequence and structural analysis,it is implied by the catalytic and substrate-binding site that the substrate of LiOMT is a phenolic derivative,which probably has a large ring-shaped moiety.Partâ…¡Crystallographic studies of human mitoNEETMitoNEET was identified as an outer mitochondrial membrane protein that can bind the anti-diabetes drug pioglitazone.The crystal structure of the cytoplasmic mitoNEET(residue 33-108)is determined in this study.The structure presents a novel protein fold and contains a[2Fe-2S]cluster-binding domain.The[2Fe-2S] cluster is coordinated to the protein by Cys72,Cys74,Cys83 and His87 residues.This coordination is also novel compared to the traditional[2Fe-2S]cluster coordinated by four cysteines or two cysteines and two histidines.The cytoplasmic mitoNEET forms homodimers in solution and in crystal.The dimerization is mainly mediated by hydrophobic interactions as well as hydrogen bonds coordinated by two water molecules binding at the interface.His87 residue,which plays an important role in the coordination of the[2Fe-2S]cluster,is exposed to the solvent on the dimer surface.It is proposed that mitoNEET dimer may interact with other proteins via the surface residues in close proximity to the[2Fe-2S]cluster.Partâ…¢Priliminary crystallographic study on E.coli diacylglyceroi kinase Diacylglycerol kinase(DAGK)from E.coli is a small(13 kDa)enzyme which catalyzes the phosphorylation the lipid diacylglycerol by MgATP to form another lipid(phosphatidic acid)and MgADP.It forms homotrimer in the membrane.Every monomer has three transmembrane segments,and the active site is likely shared between subunits.DAGK is representative of an entire class of microbial enzymes that share no similarity with other known kinases but catalyze the complicated phosphate transfer as other kinases.To determine the crystal structure of E.coli DAGK could be facilitated to study molecular mechanism of these distinct enzymes. We have over-expressed and purified the DAGK from E.coli.The crystal we have got now can diffract to 20(?).