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Although the universe of protein structures is vast, these innumerable structures can be categorized into a finite number of folds. New functions commonly evolve by elaboration of existing scaffolds, for example, via domain insertions. Thus, understanding structural diversity of a protein fold evolving via domain insertions is a fundamental challenge. The haloalkanoic dehalogenase superfamily serves as an excellent model system wherein a variable cap domain accessorizes the ubiquitous Rossmann-fold core domain. Here, we determine the impact of the cap-domain insertion on the sequence and structure divergence of the core domain. Through quantitative analysis on a unique dataset of 154 core-domain-only and cap-domain-only structures, basic principles of their evolution have been uncovered. The relationship between sequence and structure divergence of the core domain is shown to be monotonic and independent of the corresponding type of domain insert, reflecting the robustness of the Rossmann fold to mutation. However, core domains with the same cap type share greater similarity at the sequence and structure levels, suggesting interplay between the cap and core domains. Notably, results reveal that the variance in structure maps to α-helices flanking the central β-sheet and not to the domain-domain interface. Collectively, these results hint at intramolecular coevolution where the fold diverges differentially in the context of an accessory domain, a feature that might also apply to other multidomain superfamilies.

The crystal structure studies of a second conformational state of Clostridium symbiosum pyruvate phosphate dikinase (PPDK) are reported. The studies confirm the swivel motion of the His domain adjacent to the phosphoenolpyruvate (PEP)-binding domain.

The chemical pathway of the Lactococcus lactis beta-phosphoglucomutase (betaPGM) catalyzed conversion, based on conformational cycling of the enzyme, of beta-D-glucose 1-phosphate (betaGIP) to beta-D-glucose 6-phosphate (G6P) is examined. Kinetic methods demonstrate the intermediacy of beta-D-glucose 1,6-(bis)phosphate (betaG 1,6bisP) in G6P formation and show that the intermediate is reoriented in the active site by dissociation into solvent and then binding in the opposite orientation.

A study is conducted to understand the structure/function relationship in all of the subfamilies, a type IIA subfamily member, NagD from Escherichia coli K-12. The ability to catabolize related secondary metabolites indicates that there is regulation at the generic level.

Contributions made by Pseudomonas aeruginosa (PaADI) core residues Cys406, His278, and Asp166 and the contribution from Asp280 to catalysis of the formation and hydrolysis of the Cys406-alkyluronium intermediate were assessed by kinetic analysis of site-directed mutants. The results suggest that electrostatic interactions play a dominant role in PaADI catalysis.

Study was conducted to define the chemical function, a new member of the isocitrate lyase enzyme family derived from the flowering plant Dianthus caryophyllus. A substrate screen that employed a (2R)-malate core structure was designed on the basis of an analysis of the structural contexts of sequence markers common to the C-C bond lyases of the isocitrate lyase/phosphoenolpyruvate mutase superfamily.

Expression of PSR132 protein from Dianthus caryophyllus was induced in response to ethylene production that was associated with petal senescence and therefore the protein is named petal death protein (PDP). The crystal structure of PDP was determined at 2.7 angstrom resolution, indicating that a dimer-of-dimers oligomeric association.

Asp8 plays an essential role in lactococcus lactis beta-phosphoglucomutase (beta-PGM) catalysis, during which it is phosphorylated. The beta-PGM aspartyl phosphate group is not stable in water, but the discrimination between solvent and substrate is sufficient to avoid hydrolysis.

The BT4131, a representative of the HAD subfamily IIb class of phosphates, is analyzed in terms of active site structure and substrate specificity. The potential application of the active site solvent cage in conjunction with the identity of the residues stationed along the cap domain substrate specificity loops in predicting substrate structure is discovered.

Haloacid dehalogenase (HAD) superfamily mainly consists of structurally homologous enzymes, which have several conserved sequence motifs in their active site. HAD can be divided into three sub-classes, based on the domain organization. Studies and observations showing that the target of magnesium dependent phosphate-1 (MDP-1) is a phosphotyrosine in an unknown protein and not a sugar based substrate are explained.

It is proposed that enzymic phosphoryl-transfer reactions occur by concerted, step-wise, associative (phosphorane-intermediate) or dissociative (metaphosphateintermediate) mechanisms, as dictated by the catalytic scaffold and the reactants. During the evolution of a phosphotransferase family, the mechanism of the phosphoryl-transfer reaction is in constant flux, potentially changing with each adaptation of the catalytic scaffold to a new phosphoryl-donor-acceptor pair. Phosphotransferases of the recently discovered haloacid dehalogenase superfamiiy of enzymes, one of the largest and most ubiquitous of the phosphotransferase families characterized to date, are described in the context of the co-evolution of the catalytic scaffold and mechanism.

The three-dimensional structure of the bacterial enzyme 4-chlorobenzoyl-CoA ligase (CBAL), both unliganded and bound to its substrate 4-chlorobenzoate (4CBA) is reported. Insights into the structure/function relationships of this family of enzymes are provided by analysis of the enzyme active site and overall confirmation in relation to previously determined structures.

In this study, we have shown that recombinant BH1999 from Bacillus halodurans catalyzes the hydrolysis of gentisyl coenzyme A (CoA) (2,5-dihydroxybenzoyl-coenzyme A) at physiological pH with a [k.sup.cat]/[K.sup.m] of 1.6 x [10.sup.6] [M.sup.-1] [S.sup.-1] and the hydrolysis of 3-hydroxybenzoyI-CoA with a [k.sup.cat]/[K.sup.m] of 3.0 x [10.sup.5] [M.sup.-1] [S.sup.-1]. All other acyl-CoA thioesters tested had low or no substrate activity. The BH1999 gene is juxtaposed with a gene cluster that contains genes believed to function in gentisate oxidative degradation. It is hypothesized that BH1999 functions as a gentisyl-CoA thioesterase. GentisyI-CoA thioesterase shares the backbone fold and the use of an active site aspartate residue to mediate catalysis with the 4-hydroxybenzoyl-CoA thioesterase of the hotdog fold enzyme superfamily. A comparative study of these two enzymes showed that they differ greatly in the rate contribution made by the catalytic aspartate, in the pH dependence of catalysis, and in substrate specificity.

Research has been conducted on phosphonoacetaldehyde hydrolase from Bacillus cereus. This hydrolase catalyzes hydrolytic P-C bond phosphonoacetaldehyde cleavage via Schiff base intermediate formed with Lys53 and the results indicate that ligand binding and dissociation occur from the open conformer and catalysis occurs from the closed conformer.

This article discusses three aspects of the Pseudomonas 4-hydroxybenzoyl-CoA thioesterase catalysis, namely, the binding of the CoA nucleotide to the active site, Asp17 contribution, and the polarization of the substrate thioester C=O using spectral, mutagenesis, and kinetic analyses.

Research has been conducted on Mytilus edulis PEP mutase. The crystal structure of this mutase in complex with the substrate-analogue inhibitor has been investigated and the results indicate that the phosphoryl group transfer occurs on PEP Si-face with the configuration retention at phosphorus.

Results reveal that the 2-aminoethylphosphonate transaminase is homologous to members of the aminotransferase family with respect to both structure and catalytic function. Substrate specificity analysis indicate that the enzyme singularly participates in the synthesis and degradation of 2-aminoethylphosphonate.

Research has been conducted on the beta-phosphoglucomutase. The three-dimensional structure of this beta-phosphoglucomutase has been investigated and the results are reported.

Research has been conducted on the 4-chlorobenzoyl-coenzyme A dehalogenase. Results indicate that this dehalogenase catalyzes hydrolytic dehalogenation of 4-chlorobenzoyl-coenzyme A dehalogenase to 4-hydroxybenzoyl-coenzyme A dehalogenase via the active site Asp145 carboxylate using it as the nucleophile.

Structural analysis of the enzyme-substrate complex reveals that phosphonopyruvate binds in the alpha/beta-barrel's central channel of the Beta-strands C-termini. The structure resembles that of pyruvate kinase but differes in the acid/base catalysis function.

Research discusses the Gibbs free energies of the binding interactions in dehalogenase catalysis involving enzyme-substrate complex and enzyme-transition state complex steps. Data indicate that together, the binding interactions generate about 10(sup)6 kilocalories.

Research has been conducted on the 4-chlorobenzoyl-coenzyme A dehalogenase that catalyzes hydrolytic dehalogenation of 4-CBA-CoA by the Asp145's attack on the C(4) of the substrate benzoyl ring. The effect of the histidine 90 on the the catalysis has been examined and the results are presented.

Research has been conducted on the pyruvate phosphate dikinase which catalyzes a reversible reaction using Mg (super)2+ and NH (sub)4 (super)+ ions as cofactors. The ability of the the connecting linkers to direct the central domain movement between active sites during the catalytic turnover has been investigated and the results are reported.

The crystal structure of Bacillus cereus phosphonoacetaldehyde hydrolase has been determined to gain insight into catalysis of phosphorus bond cleavage and catalytic diversification in the HAD enzyme superfamily. Analysis of the usage of polar amino acid residues at stations by the dehalogenases, phosphatases, phosphonatases and phosphomutases of the HAD superfamily suggests possible ways in which the active site of an ancient enzyme ancestor might have been diversified for catalysis of P-C, C-X, and P-O bond cleavage reactions.

The mechanism of phosphoenolpyruvate mutase (PEP mutase) catalysis is discussed relative to site-directed mutagenesis studies of active site residues. The sequencing, expression, cloning, and mutagenesis of the Mytilus edulis PEP mutase gene in Escherichia coli are covered. A catalytic motif has been proposed. It could serve for both phosphoryl transfer and the C--C cleavage enzymes of the PEP mutase superfamily.

The effects of site selective mutations at the dehalogenase residue Asp 154 on the product complex, 4-hydroxybenzoyl-CoA (4-HBA-CoA), were investigated by Raman spectroscopy. The product complex 4-HBA-CoA was formed by the hydration of 4-chlorobenzoyl-CoA by the enzyme 4-chlorobenzoyl-CoA dehalogenase. The results showed that strong electron pull is exerted on the benzol carbonyl group by hydrogen bonds from the peptide NHs of Gly 114 and Phe 65. In addition, a major rearrangement of the benzol pi-electrons occurs for binding in the active site of WT enzyme.

The genes encoding phosphonatase in 'Bacillus cereus' and 'Salmonella typhimurium' were cloned for high-level expression in Escherichia coli using mutagenesis and sequenced. The kinetic properties and catalytic pathways of the two purified enzymes were compared. Results suggest that the phosphonatase superfamily scaffold is stable both as an independent unit and as a domain within a larger protein structure and that this scaffold has been used in a variety of combinations as a functional and structural module in the evolution of new protein functions.

Two core motifs found in Pseudomonas sp. strain CBS3 4-chlorobenzoate:coenzyme A ligase and conserved among the sequences representing the acyl-adenylate/thioester-forming enzyme family were examined for possible involvement in substrate binding and/or catalysis. Findings showed reduced catalysis of the adenylation of 4-chlorobenzoate in the mutant enzymes. Results were interpreted with regards to the three-dimensional structure of the acyl-adenylate/thioester-forming enzyme family member, firefly luciferase.

The polarizing forces that promote catalysis in 4-Chlorobenzoate-CoA dehologenase was investigated using the Raman spectroscopy method. Results revealed that polarization encourages catalysis by reducing electron density. Furthermore, the p-hydroxy group oxygen makes no significant contribution to the main normal means as reflected in the Raman spectrum.

The mutants D145A, H90Q, W137F, W89F, W89Y, F64L, F82L and G114A of 4-chlorobenzoyl-coenzyme A (4-CBA-CoA) were prepared to investigate the effect of amino acid substitution on catalysis and on perturbation of the UV-visible spectral characteristics of the substrate benzoyl ring. Substitution of the two catalytic residues D145 and H90 inhibited catalysis but not ligand binding or the induction of the red ring binding or polarization. The F64L, F82L, W89F and W137F mutants retained substantial catalytic activity and the capability to induce the red shift.

The intermediacy of 4-chlorobenzoyl-adenosine 5'-phosphate diester (4-CBA-AMP) in the reaction of 4-chlorobenzoate:coenzyme A ligase as the first step for degrading 4-CBA in Pseudomonas sp. strain CBS3, was studied. Findings indicated that the 4-CBA-AMP intermediate formed attained a maximum of 25% of the initial 4-CBA within 20 ms and then decreases with the formation of the 4-CBA-CoA. The rates of adenylation and thioesterification partial reactions were almost the same. Substitution of CoA with the slow substrate pantetheine did not substantially affect the rate of adenylation step but decreased the rate of the thioesterification step to 2/sec.

Conserved aspartate, histidine and tryptophan residues which are important for 4-chlorobenzoyl-coenzyme A (4-CBA-CoA) catalysis, are identified using chemical modification and site-directed mutagenesis methods. Results suggest that aspartate 145 acts as the active site nucleophile, that tryptophan 137 functions as a hydrogen bond donor to the aspartate 145 C=O, and that histidine 90 helps in the deprotonation of the bound water molecule.

Photoaffinity labeling, site-directed mutagenesis and structural analysis of pyruvate phosphate dikinase from Clostridium symbiosum reveals that the nucleotide binding site is present in the N-terminal. X-ray crystal structure studies indicates that the enzyme folds into N-terminal, central and C-terminal regions. ATP binds in a crevice formed by subdomains one and three in a manner similar to ADP binding in D-alanine-D-alanine-ligase.

The three-dimensional structure of 4-chlorobenzoyl-CoA dehalogenase from Pseudomonas sp. strain CBS-3 is studied. Crystallographic analysis showed that the enzyme is a trimer and each subunit folds into two distinct motifs. The bigger, N-terminal domain has 10 strands of beta-pleated sheet that form two distinct layers which are almost perpendicular to each other. Alpha-helices are found on the sides of the layers of beta-sheet. The C-terminal domain is made of three amphiphilic alpha-helices. The two domains of the subunit are linked by a cation.

The phosphoenolpyruvate phosphomutase (PEP mutase) catalysis of phosphorus-carbon bond formation of phosphoenolpyruvate (PEP) to phosphonopyruvate (P-pyr) was studied in terms of kinetics and mechanism. The catalysis was measured by a designed spectrophotometric P-pyr assay. Interestingly, the PEP mutase does not promote self phosphorylation with oxalyl phosphate which indicates nonrecognition of the phosphate as a substrate. Likewise, the PEP mutase-catalyzed reaction of PEP does not form a phospho-enzyme intermediate.

A spectroscopic study of the benzoyl coenzyme A (CoA) attached to the active site of 4-chlorobenzoyl coenzyme A (4-CBA-CoA) dehalogenase reveals that there is a rearrangement of the pi electrons around the benzoyl ring when the substrate ligand binds to the enzyme. This suggests that the reaction of 4-CBA-CoA dehalogenase is catalyzed in an electrophilic manner. The reaction involves the formation of Meisenheimer complex by the attack of an active site carboxylate at C(4) of the benzoyl ring. The enzyme catalyzes the hydrolytic dehalogenation of 4-CBA-CoA to hydroxybenzoyl coenzyme A.