CRYSTAL STRUCTURE DETERMINATION OF METALLOPROTEINS:PEPTIDE DEFORMYLASE, FIXL HEME DOMAIN, MONOMETHYLAMINE METHYLTRANSFERASE, AND CARBON MONOXIDE DEHYDROGENASE
This research was focused on the structure determination of four metalloproteins by x-ray crystallography. The first target was E. coli peptide deformylase that is responsible for deformylation of the N-terminus of nascent bacterial proteins and represents a potential drug target. We have determined the first crystal structures of formate- and inhibitor-bound deformylase complexes in different metal forms (Fe2+, Co2+ and Zn2+). The different formate-binding modes between the Zn and the other two metallated forms provide a possible explanation for the low activity of Zn enzyme as compared to Fe and Co enzymes. The inhibitor-bound structures reveal that the bound transition-state analog, (S)-2-O-(H-phosphonoxy)-L-caproyl-L-leucyl-p-nitroanilide (PCLNA), adopts an extended conformation and forms an interaction network with the protein. Based on these structures, a mechanism for deformylation is proposed and guidelines for the design of high-affinity deformylase inhibitors are suggested. The second part of this research involved structural studies of the FixL heme domain from Bradyrhizobium japonicum (BjFixLH). FixL proteins are biological oxygen sensors that regulate nitrogen fixation gene expression in Rhizobia. In these proteins, the activity of the C-terminus kinase domain is regulated by the binding of O2 and other strong-field ligands to the N-terminus heme domain. We have determined eight BjFixLH structures including two unliganded and six ligand-bound forms. These structures reveal a novel heme-binding fold that has been conserved in PAS-domain sensor superfamily. Comparison of these structures has also revealed a heme-mediated conformational change that is distinct from that in classic globins. In BjFixLH, binding of O2 to the heme results in the flattening of the heme plane, the rotation of a critical heme-pocket arginine, and the shift of a FG loop. We have proposed that this arginine plays a central role in ligand discrimination of BjFixLH. The third project in this research involved the identification of an in-frame UAG-encoded amino acid in Methanosarcina barkeri monomethylamine methyltransferase (MtmB). We have determined the structures of MtmB in two crystal forms. Both structures suggest that the UAG-encoded residue is distinct from the other 21 natural amino acids. Instead it appears consistent with a lysine in amide-linkage to 4-substituted-pyrroline-5-carboxylate, which is named as L-pyrrolysine. Current data support the idea that pyrrolysine represents the 22nd genetically encoded amino acid. A model for how pyrrolysine participates in activating the methyl group of methylamine for transfer to corrinoid proteins is suggested. The last target of this research was the a2e2 carbon monoxide dehydrogenase (NiCODH) component of acetyl coenzyme-A decarbonylase synthase (ACDS) complex from Methanosarcina barkeri. This component catalyzes the reversible oxidation of CO to CO2 at an unusual nickel-iron sulfur center. Our major effort has been devoted to the purification, crystallization, data collection, and phasing of this protein. So far, we have obtained two crystal forms and collected several MAD and MIR data sets with resolution up to 2.2 Å. Five distinct iron centers have been identified in each ae monomer. The structure determination is in progress.
School:The Ohio State University
School Location:USA - Ohio
Source Type:Master's Thesis
Keywords:crystal structure metalloproteins peptide deformylase bjfixlh monomethylamine methyltransferase carbon monoxide dehydrogenase
Date of Publication:01/01/2002