Almo SC

References (4)

Title : Biochemical characterization of two haloalkane dehalogenases: DccA from Caulobacter crescentus and DsaA from Saccharomonospora azurea - Carlucci_2016_Protein.Sci_25_877
Author(s) : Carlucci L , Zhou E , Malashkevich VN , Almo SC , Mundorff EC
Ref : Protein Science , 25 :877 , 2016
Abstract : Two putative haloalkane dehalogenases (HLDs) of the HLD-I subfamily, DccA from Caulobacter crescentus and DsaA from Saccharomonospora azurea, have been identified based on sequence comparisons with functionally characterized HLD enzymes. The two genes were synthesized, functionally expressed in E. coli and shown to have activity toward a panel of haloalkane substrates. DsaA has a moderate activity level and a preference for long (greater than 3 carbons) brominated substrates, but little activity toward chlorinated alkanes. DccA shows high activity with both long brominated and chlorinated alkanes. The structure of DccA was determined by X-ray crystallography and was refined to 1.5 A resolution. The enzyme has a large and open binding pocket with two well-defined access tunnels. A structural alignment of HLD-I subfamily members suggests a possible basis for substrate specificity is due to access tunnel size.
ESTHER : Carlucci_2016_Protein.Sci_25_877
PubMedSearch : Carlucci_2016_Protein.Sci_25_877
PubMedID: 26833751
Gene_locus related to this paper: caucr-CC1175

Title : Covalent docking predicts substrates for haloalkanoate dehalogenase superfamily phosphatases - London_2015_Biochemistry_54_528
Author(s) : London N , Farelli JD , Brown SD , Liu C , Huang H , Korczynska M , Al-Obaidi NF , Babbitt PC , Almo SC , Allen KN , Shoichet BK
Ref : Biochemistry , 54 :528 , 2015
Abstract : Enzyme function prediction remains an important open problem. Though structure-based modeling, such as metabolite docking, can identify substrates of some enzymes, it is ill-suited to reactions that progress through a covalent intermediate. Here we investigated the ability of covalent docking to identify substrates that pass through such a covalent intermediate, focusing particularly on the haloalkanoate dehalogenase superfamily. In retrospective assessments, covalent docking recapitulated substrate binding modes of known cocrystal structures and identified experimental substrates from a set of putative phosphorylated metabolites. In comparison, noncovalent docking of high-energy intermediates yielded nonproductive poses. In prospective predictions against seven enzymes, a substrate was identified for five. For one of those cases, a covalent docking prediction, confirmed by empirical screening, and combined with genomic context analysis, suggested the identity of the enzyme that catalyzes the orphan phosphatase reaction in the riboflavin biosynthetic pathway of Bacteroides.
ESTHER : London_2015_Biochemistry_54_528
PubMedSearch : London_2015_Biochemistry_54_528
PubMedID: 25513739

Title : Computational, structural, and kinetic evidence that Vibrio vulnificus FrsA is not a cofactor-independent pyruvate decarboxylase - Kellett_2013_Biochemistry_52_1842
Author(s) : Kellett WF , Brunk E , Desai BJ , Fedorov AA , Almo SC , Gerlt JA , Rothlisberger U , Richards NG
Ref : Biochemistry , 52 :1842 , 2013
Abstract : The fermentation-respiration switch (FrsA) protein in Vibrio vulnificus was recently reported to catalyze the cofactor-independent decarboxylation of pyruvate. We now report quantum mechanical/molecular mechenical calculations that examine the energetics of C-C bond cleavage for a pyruvate molecule bound within the putative active site of FrsA. These calculations suggest that the barrier to C-C bond cleavage in the bound substrate is 28 kcal/mol, which is similar to that estimated for the uncatalyzed decarboxylation of pyruvate in water at 25 degrees C. In agreement with the theoretical predictions, no pyruvate decarboxylase activity was detected for recombinant FrsA protein that could be crystallized and structurally characterized. These results suggest that the functional annotation of FrsA as a cofactor-independent pyruvate decarboxylase is incorrect.
ESTHER : Kellett_2013_Biochemistry_52_1842
PubMedSearch : Kellett_2013_Biochemistry_52_1842
PubMedID: 23452154
Gene_locus related to this paper: vibvy-y856

Title : Structure of diethyl phosphate bound to the binuclear metal center of phosphotriesterase - Kim_2008_Biochemistry_47_9497
Author(s) : Kim J , Tsai PC , Chen SL , Himo F , Almo SC , Raushel FM
Ref : Biochemistry , 47 :9497 , 2008
Abstract : The bacterial phosphotriesterase (PTE) from Pseudomonas diminuta catalyzes the hydrolysis of organophosphate esters at rates close to the diffusion limit. X-ray diffraction studies have shown that a binuclear metal center is positioned in the active site of PTE and that this complex is responsible for the activation of the nucleophilic water from solvent. In this paper, the three-dimensional structure of PTE was determined in the presence of the hydrolysis product, diethyl phosphate (DEP), and a product analogue, cacodylate. In the structure of the PTE-diethyl phosphate complex, the DEP product is found symmetrically bridging the two divalent cations. The DEP displaces the hydroxide from solvent that normally bridges the two divalent cations in structures determined in the presence or absence of substrate analogues. One of the phosphoryl oxygen atoms in the PTE-DEP complex is 2.0 A from the alpha-metal ion, while the other oxygen is 2.2 A from the beta-metal ion. The two metal ions are separated by a distance of 4.0 A. A similar structure is observed in the presence of cacodylate. Analogous complexes have previously been observed for the product complexes of isoaspartyl dipeptidase, d-aminoacylase, and dihydroorotase from the amidohydrolase superfamily of enzymes. The experimentally determined structure of the PTE-diethyl phosphate product complex is inconsistent with a recent proposal based upon quantum mechanical/molecular mechanical simulations which postulated the formation of an asymmetrical product complex bound exclusively to the beta-metal ion with a metal-metal separation of 5.3 A. This structure is also inconsistent with a chemical mechanism for substrate hydrolysis that utilizes the bridging hydroxide as a base to abstract a proton from a water molecule loosely associated with the alpha-metal ion. Density functional theory (DFT) calculations support a reaction mechanism that utilizes the bridging hydroxide as the direct nucleophile in the hydrolysis of organophosphate esters by PTE.
ESTHER : Kim_2008_Biochemistry_47_9497
PubMedSearch : Kim_2008_Biochemistry_47_9497
PubMedID: 18702530