Publications (13) View all
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Article: Structural and functional studies of truncated hemolysin A from Proteus mirabilis.
Journal of Biological Chemistry 04/2013; 288(14):10092. · 4.77 Impact Factor -
Article: Crystallographic analysis of active site contributions to regiospecificity in the diiron enzyme toluene 4-monooxygenase.
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ABSTRACT: Crystal structures of toluene 4-monooxygenase hydroxylase in complex with reaction products and effector protein reveal active site interactions leading to regiospecificity. Complexes with phenolic products yield an asymmetric μ-phenoxo-bridged diiron center and a shift of diiron ligand E231 into a hydrogen bonding position with conserved T201. In contrast, complexes with inhibitors p-NH(2)-benzoate and p-Br-benzoate showed a μ-1,1 coordination of carboxylate oxygen between the iron atoms and only a partial shift in the position of E231. Among active site residues, F176 trapped the aromatic ring of products against a surface of the active site cavity formed by G103, E104 and A107, while F196 positioned the aromatic ring against this surface via a π-stacking interaction. The proximity of G103 and F176 to the para substituent of the substrate aromatic ring and the structure of G103L T4moHD suggest how changes in regiospecificity arise from mutations at G103. Although effector protein binding produced significant shifts in the positions of residues along the outer portion of the active site (T201, N202, and Q228) and in some iron ligands (E231 and E197), surprisingly minor shifts (<1 Å) were produced in F176, F196, and other interior residues of the active site. Likewise, products bound to the diiron center in either the presence or absence of effector protein did not significantly shift the position of the interior residues, suggesting that positioning of the cognate substrates will not be strongly influenced by effector protein binding. Thus, changes in product distributions in the absence of the effector protein are proposed to arise from differences in rates of chemical steps of the reaction relative to motion of substrates within the active site channel of the uncomplexed, less efficient enzyme, while structural changes in diiron ligand geometry associated with cycling between diferrous and diferric states are discussed for their potential contribution to product release.Biochemistry 02/2012; 51(6):1101-13. · 3.42 Impact Factor -
Chapter: Toluene 4‐Monooxygenase and its Complex with Effector Protein T4moD
09/2010; , ISBN: 9780470028636 -
Article: Crystallographic and catalytic studies of the peroxide-shunt reaction in a diiron hydroxylase.
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ABSTRACT: A diiron hydroxylase reaction typically begins by combination of O2 with a diferrous center to form reactive intermediates capable of hydrocarbon hydroxylation. In this natural cycle, reducing equivalents are provided by specific interactions with electron transfer proteins. The biological process can be bypassed by combining H2O2 with a diferric center, i.e., peroxide-shunt catalysis. Here we show that toluene 4-monooxygenase has a peroxide-shunt reaction that is approximately 600-fold slower than catalysis driven by biological electron transfer. However, the toluene 4-monooxygenase hydroxylase-effector protein complex was stable in the presence of 300 mM H2O2, suggesting overall benign effects of the exogenous oxidant on active site structure and function. The X-ray structure of the toluene 4-monooxygenase hydroxylase-effector protein complex determined from crystals soaked in H2O2 revealed a bound diatomic molecule, assigned to a cis-mu-1,2-peroxo bridge. This peroxo species resides in an active site position adjacent to the hydrogen-bonding substructure established by effector protein binding and faces into the distal cavity where substrate must bind during regiospecific aromatic ring hydroxylation catalysis. These results provide a new structural benchmark for how activated intermediates may be formed and dispatched during diiron hydroxylase catalysis.Biochemistry 09/2009; 48(38):8932-9. · 3.42 Impact Factor -
Article: Structural and functional studies of truncated hemolysin A from Proteus mirabilis.
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ABSTRACT: In this study we analyzed the structure and function of a truncated form of hemolysin A (HpmA265) from Proteus mirabilis using a series of functional and structural studies. Hemolysin A belongs to the two-partner secretion pathway. The two-partner secretion pathway has been identified as the most common protein secretion pathway among Gram-negative bacteria. Currently, the mechanism of action for the two-partner hemolysin members is not fully understood. In this study, hemolysis experiments revealed a unidirectional, cooperative, biphasic activity profile after full-length, inactive hemolysin A was seeded with truncated hemolysin A. We also solved the first x-ray structure of a TpsA hemolysin. The truncated hemolysin A formed a right-handed parallel beta-helix with three adjoining segments of anti-parallel beta-sheet. A CXXC disulfide bond, four buried solvent molecules, and a carboxyamide ladder were all located at the third complete beta-helix coil. Replacement of the CXXC motif led to decreased activity and stability according to hemolysis and CD studies. Furthermore, the crystal structure revealed a sterically compatible, dry dimeric interface formed via anti-parallel beta-sheet interactions between neighboring beta-helix monomers. Laser scanning confocal microscopy further supported the unidirectional interconversion of full-length hemolysin A. From these results, a model has been proposed, where cooperative, beta-strand interactions between HpmA265 and neighboring full-length hemolysin A molecules, facilitated in part by the highly conserved CXXC pattern, account for the template-assisted hemolysis.Journal of Biological Chemistry 07/2009; 284(33):22297-309. · 4.77 Impact Factor
