Crystal structures of intermediates in the dehalogenation of haloalkanoates by L-2-haloacid dehalogenase.
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The L-2-haloacid dehalogenase from the 1,2-dichloroethane-degrading bacterium Xanthobacter autotrophicus GJ10 catalyzes the hydrolytic dehalogenation of small L-2-haloalkanoates to their corresponding D-2-hydroxyalkanoates, with inversion of the configuration at the C(2) atom. The structure of the apoenzyme at pH 8 was refined at 1.5-A resolution. By lowering the pH, the catalytic activity of the enzyme was considerably reduced, allowing the crystal structure determination of the complexes with L-2-monochloropropionate and monochloroacetate at 1.7 and 2.1 A resolution, respectively. Both complexes showed unambiguous electron density extending from the nucleophile Asp(8) to the C(2) atom of the dechlorinated substrates corresponding to a covalent enzyme-ester reaction intermediate. The halide ion that is cleaved off is found in line with the Asp(8) Odelta1-C(2) bond in a halide-stabilizing cradle made up of Arg(39), Asn(115), and Phe(175). In both complexes, the Asp(8) Odelta2 carbonyl oxygen atom interacts with Thr(12), Ser(171), and Asn(173), which possibly constitute the oxyanion hole in the hydrolysis of the ester bond. The carboxyl moiety of the substrate is held in position by interactions with Ser(114), Lys(147), and main chain NH groups. The L-2-monochloropropionate CH(3) group is located in a small pocket formed by side chain atoms of Lys(147), Asn(173), Phe(175), and Asp(176). The size and position of the pocket explain the stereospecificity and the limited substrate specificity of the enzyme. These crystallographic results demonstrate that the reaction of the enzyme proceeds via the formation of a covalent enzyme-ester intermediate at the nucleophile Asp(8). (+info)
Analysis of the reaction mechanism and substrate specificity of haloalkane dehalogenases by sequential and structural comparisons.
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Haloalkane dehalogenases catalyse environmentally important dehalogenation reactions. These microbial enzymes represent objects of interest for protein engineering studies, attempting to improve their catalytic efficiency or broaden their substrate specificity towards environmental pollutants. This paper presents the results of a comparative study of haloalkane dehalogenases originating from different organisms. Protein sequences and the models of tertiary structures of haloalkane dehalogenases were compared to investigate the protein fold, reaction mechanism and substrate specificity of these enzymes. Haloalkane dehalogenases contain the structural motifs of alpha/beta-hydrolases and epoxidases within their sequences. They contain a catalytic triad with two different topological arrangements. The presence of a structurally conserved oxyanion hole suggests the two-step reaction mechanism previously described for haloalkane dehalogenase from Xanthobacter autotrophicus GJ10. The differences in substrate specificity of haloalkane dehalogenases originating from different species might be related to the size and geometry of an active site and its entrance and the efficiency of the transition state and halide ion stabilization by active site residues. Structurally conserved motifs identified within the sequences can be used for the design of specific primers for the experimental screening of haloalkane dehalogenases. Those amino acids which were predicted to be functionally important represent possible targets for future site-directed mutagenesis experiments. (+info)
Heterologous expression of bacterial Epoxyalkane:Coenzyme M transferase and inducible coenzyme M biosynthesis in Xanthobacter strain Py2 and Rhodococcus rhodochrous B276.
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Coenzyme M (CoM) (2-mercaptoethanesulfonic acid) biosynthesis is shown to be coordinately regulated with the expression of the enzymes of alkene and epoxide metabolism in the propylene-oxidizing bacteria Xanthobacter strain Py2 and Rhodococcus rhodochrous strain B276. These results provide the first evidence for the involvement of CoM in propylene metabolism by R. rhodochrous and demonstrate for the first time the inducible nature of eubacterial CoM biosynthesis. (+info)
Effects of the Calvin cycle on nicotinamide adenine dinucleotide concentrations and redox balances of Xanthobacter flavus.
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The levels of reduced and oxidized nicotinamide adenine dinucleotides were determined in Xanthobacter flavus during a transition from heterotrophic to autotrophic growth. Excess reducing equivalents are rapidly dissipated following induction of the Calvin cycle, indicating that the Calvin cycle serves as a sink for excess reducing equivalents. The physiological data support the conclusion previously derived from molecular studies in that expression of the Calvin cycle genes is controlled by the intracellular concentration of NADPH. (+info)
The importance of reactant positioning in enzyme catalysis: a hybrid quantum mechanics/molecular mechanics study of a haloalkane dehalogenase.
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Hybrid quantum mechanics/molecular mechanics calculations using Austin Model 1 system-specific parameters were performed to study the S(N)2 displacement reaction of chloride from 1,2-dichloroethane (DCE) by nucleophilic attack of the carboxylate of acetate in the gas phase and by Asp-124 in the active site of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10. The activation barrier for nucleophilic attack of acetate on DCE depends greatly on the reactants having a geometry resembling that in the enzyme or an optimized gas-phase structure. It was found in the gas-phase calculations that the activation barrier is 9 kcal/mol lower when dihedral constraints are used to restrict the carboxylate nucleophile geometry to that in the enzyme relative to the geometries for the reactants without dihedral constraints. The calculated quantum mechanics/molecular mechanics activation barriers for the enzymatic reaction are 16.2 and 19.4 kcal/mol when the geometry of the reactants is in a near attack conformer from molecular dynamics and in a conformer similar to the crystal structure (DCE is gauche), respectively. This haloalkane dehalogenase lowers the activation barrier for dehalogenation of DCE by 2-4 kcal/mol relative to the single point energies of the enzyme's quantum mechanics atoms in the gas phase. S(N)2 displacements of this sort in water are infinitely slower than in the gas phase. The modest lowering of the activation barrier by the enzyme relative to the reaction in the gas phase is consistent with mutation experiments. (+info)
Evidence of substantial carbon isotope fractionation among substrate, inorganic carbon, and biomass during aerobic mineralization of 1, 2-dichloroethane by Xanthobacter autotrophicus.
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Carbon isotope fractionation during aerobic mineralization of 1, 2-dichloroethane (1,2-DCA) by Xanthobacter autotrophicus GJ10 was investigated. A strong enrichment of (13)C in residual 1,2-DCA was observed, with a mean fractionation factor alpha +/- standard deviation of 0.968 +/- 0.0013 to 0.973 +/- 0.0015. In addition, a large carbon isotope fractionation between biomass and inorganic carbon occurred. A mechanistic model that links the fractionation factor alpha to the rate constants of the first catabolic enzyme was developed. Based on the model, it was concluded that the strong enrichment of (13)C in 1,2-DCA arises because the first irreversible step of the initial enzymatic transformation of 1,2-DCA consists of an S(N)2 nucleophilic substitution. S(N)2 reactions are accompanied by a large kinetic isotope effect. The substantial carbon isotope fractionation between biomass and inorganic carbon could be explained by the kinetic isotope effect associated with the initial 1,2-DCA transformation and by the metabolic pathway of 1,2-DCA degradation. Carbon isotope fractionation during 1,2-DCA mineralization leads to 1,2-DCA, inorganic carbon, and biomass with characteristic carbon isotope compositions, which may be used to trace the process in contaminated environments. (+info)
Evidence that a linear megaplasmid encodes enzymes of aliphatic alkene and epoxide metabolism and coenzyme M (2-mercaptoethanesulfonate) biosynthesis in Xanthobacter strain Py2.
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The bacterial metabolism of propylene proceeds by epoxidation to epoxypropane followed by a sequence of three reactions resulting in epoxide ring opening and carboxylation to form acetoacetate. Coenzyme M (2-mercaptoethanesulfonic acid) (CoM) plays a central role in epoxide carboxylation by serving as the nucleophile for epoxide ring opening and the carrier of the C(3) unit that is ultimately carboxylated to acetoacetate, releasing CoM. In the present work, a 320-kb linear megaplasmid has been identified in the gram-negative bacterium Xanthobacter strain Py2, which contains the genes encoding the key enzymes of propylene oxidation and epoxide carboxylation. Repeated subculturing of Xanthobacter strain Py2 under nonselective conditions, i.e., with glucose or acetate as the carbon source in the absence of propylene, resulted in the loss of the propylene-positive phenotype. The propylene-negative phenotype correlated with the loss of the 320-kb linear megaplasmid, loss of induction and expression of alkene monooxgenase and epoxide carboxylation enzyme activities, and the loss of CoM biosynthetic capability. Sequence analysis of a hypothetical protein (XecG), encoded by a gene located downstream of the genes for the four enzymes of epoxide carboxylation, revealed a high degree of sequence identity with proteins of as-yet unassigned functions in the methanogenic archaea Methanobacterium thermoautotrophicum and Methanococcus jannaschii and in Bacillus subtilis. The M. jannaschii homolog of XecG, MJ0255, is located next to a gene, MJ0256, that has been shown to encode a key enzyme of CoM biosynthesis (M. Graupner, H. Xu, and R. H. White, J. Bacteriol. 182: 4862-4867, 2000). We propose that the propylene-positive phenotype of Xanthobacter strain Py2 is dependent on the selective maintenance of a linear megaplasmid containing the genes for the key enzymes of alkene oxidation, epoxide carboxylation, and CoM biosynthesis. (+info)
Persistently conserved positions in structurally similar, sequence dissimilar proteins: roles in preserving protein fold and function.
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Many protein pairs that share the same fold do not have any detectable sequence similarity, providing a valuable source of information for studying sequence-structure relationship. In this study, we use a stringent data set of structurally similar, sequence-dissimilar protein pairs to characterize residues that may play a role in the determination of protein structure and/or function. For each protein in the database, we identify amino-acid positions that show residue conservation within both close and distant family members. These positions are termed "persistently conserved". We then proceed to determine the "mutually" persistently conserved (MPC) positions: those structurally aligned positions in a protein pair that are persistently conserved in both pair mates. Because of their intra- and interfamily conservation, these positions are good candidates for determining protein fold and function. We find that 45% of the persistently conserved positions are mutually conserved. A significant fraction of them are located in critical positions for secondary structure determination, they are mostly buried, and many of them form spatial clusters within their protein structures. A substitution matrix based on the subset of MPC positions shows two distinct characteristics: (i) it is different from other available matrices, even those that are derived from structural alignments; (ii) its relative entropy is high, emphasizing the special residue restrictions imposed on these positions. Such a substitution matrix should be valuable for protein design experiments. (+info)