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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.

Enzymes provide enormous rate enhancements, unmatched by any other type of catalyst. The stabilization of high-energy states along the reaction coordinate is the crux of the catalytic power of enzymes. We report the atomic-resolution structure of a high-energy reaction intermediate stabilized in the active site of an enzyme. Crystallization of phosphorylated β-phosphoglucomutase in the presence of the Mg(II) cofactor and either of the substrates glucose 1-phosphate or glucose 6-phosphate produced crystals of the enzyme-Mg(II)-glucose 1,6-(bis)phosphate complex, which diffracted x-rays to 1.2 and 1.4 angstroms, respectively. The structure reveals a stabilized pentacovalent phosphorane formed in the phosphoryl transfer from the C(1)O of glucose 1,6-(bis)phosphate to the nucleophilic Asp8 carboxylate., The unique ability of enzymes to synchronize multiple interactions at specific sites with reactants, intermediates, and products defines the reaction pathway and is the source of the enormous rate acceleration [...]

CHLOROAROMATICS RANK AMONG the most prevalent and enviromentally persistent class of synthetic chemicals. Because standard methodologies have proved ineffective in removing these pollutants from the environment, other strategies are evolving. [...]

The features of the oxidative cleavage reactions of diastereomers of dimeric lignin model compounds are examined, the cation radicals of these substances are generated by using SET-sensitized photochemical and Ce(IV) and lignin peroxidase promoted oxidative processes and the C-C bond cleavage reactions are determined. The results have shown that the C1-C2 bond dissociation energies of the [beta]-1 lignin model compounds are lower than those of the [beta]-O-4 models, which have provided clear evidence for the source of the rate differences.

The X-ray crystallographic structural characterization of Escherichia coli GmhB in the apo form, in a complex with [Mg.sup.2+] and orthophosphate, and in a complex with [Mg.sup.2+] and D-glycero-D-manno-heptose 1[beta],7-bisphosphate along with structure of Bordetella bronchiseptica GmhB bound to [Mg.sup.2+] and orthophosphate are reported. GmhB and the histidinol-phosphate phosphatase domain which used the same design of three substrate recognition loops inserted into the cap domain achieved unique substrate specificity through selective residue usage and hence have novel biochemical function.

The comparative analysis results for substrate recognition in selected D-glycero-D-manno-heptose-1,7-bisphosphate phosphatase (GmhB) which is a member of the histidinol-phosphate phosphatase (HisB) subfamily of the haloalkanoic acid dehalogenase (HAD) enzyme superfamily are reported. The study results concluded that the evolution of the ancestor to GmhB in the HisB subfamily provided for specialization toward two distinct biochemical functions.

Single turnover kinetic experiments were performed to examine the impact of D402P mutation on the ability of the enzyme 4-chlorobenzoate:CoA ligase (CBL) to catalyze the complete reaction to transfer the carboxylate to the pantetheine group of either coenzyme A or an acyl-carrier protein. The significant impact of the alanine replacement of Asp402 on the thioester-forming reaction provide evidence for the contribution of Asp402 carboxylate group, through ion pair formation with N-terminal domain residue Arg400, in the transition to the thioester-forming conformer.

Several studies are conducted to explain the substrate binding and stereoselectivity phenomena observed in the Giardia lamblia fructose-1,6-bisphosphate aldolase (FBPA), a member of the II zinc-dependent aldolase family. The polarization of the substrate for protein transfer in the FBPA compound is shown to be highly dependent on the zinc mobility within it.

Several theoretical, as well as experimental studies are conducted to determine the various structural factors that help in the distinction between substrate and solvent in [beta]-phosphoglucomutase catalysis ([beta]G1P). The binding of the complex to the core domain is shown to be extremely useful for inducing cap closure during the catalysis process.

The study results of an extensive human thioesterase superfamily member 2 (hTHEM2) substrate screens, the structural characterization of hTHEM2 complexed with the inert substrate analogue undecan-2-one-CoA and the kinetic analysis of active site mutants is presented. The results obtained for the structure-function analysis of hTHEM2 offer insight into the physiological role of hTHEM2, which involves catalysis of the hydrolysis of cytosolic medium-to-long-chain acyl-CoA thioesters.

In vitro kinetic analysis has shown two potential routes to EntB misacylation and has provided evidence for two mechanisms by which the hot dog-fold thioesterase EntH can potentially prevent or reverse EntB misacylation. The studies have shown that EntH has prevented EntD-catalyzed mischarging of EntB through hydrolysis of endogenous acyl- or aroyl-CaAs and that EntH has released the holo-EntB from misacylated holo-EntB.

The study report on the steady-state kinetic and transient state kinetic analysis of wild-type 4-chlorobenzoate:CoA ligase (CBL) and a series of site-directed CBL active site mutants is presented. The results obtained provide insight into the contribution of the active site residues in substrate/cofactor binding and catalysis and the role of domain alternation in catalysis.

A novel product analogue, 4-chlorophenacyl-CoA was used to trap the enzyme 4-chlorobenzoate:CoA ligase (CBL) in the thioester-forming conformation and characterize the structure of CBL in the original adenylate-forming conformation, bound to the adenylate intermediate. The findings from complete characterization of CBL enzyme provide insight into the use of the domain alteration strategy by these enzymes.

The pH rate profiles are measured for several L-arginine deiminase (ADIs) from divergent organisms to define the limits of the pH range within which the His-Cys catalytic pair can operate. The results have shown that catalytic turnover commences from the active configuration of the ADI complex consisting of the Cys thiolate and His imidazolium ion.

The biochemical characterization and cystallographic analysis of HI0827 protein isolated from Haemophilus influenza Rd KW20 was carried out to examine the structural variations within the acyl-CoA thioesterase family. The findings provided insight into a new structural class of hexameric hot dog fold enzyme, acyl-coenzyme A (acyl-CoA) thioesterases.

A study was conducted to examine the biochemical and biological function of the hot dog fold thioesterase YciA (EcYciA) from Escherichia coli and its close sequence homologue HI0827 from Haemophilus influenzae (HiYciA). The results provide insight into significant role of YciA for efficient CoA-regulated hydrolysis of cellular acyl-CoA thioesters in a wide range of bacteria.

The isolation, structure and chemical mechanism for catalysis of the Pseudomonas aeruginosa, arginine deiminase (ADI) (L-arginine) which involves initial formation and subsequent hydrolysis of a Cys-alkylthiouronium ion intermediate is described. The findings from the studies would help to design the arginine analogs such as L-canavanine that migaht react with the ADIs to form inactive covalent adduct during catalytic turnover.

The crystal structure of PA4872 from Pseudomonas aeruginosa in complex with oxalate and [Mg.sup.2+] is studied. The results have shown that PA4872 is an oxaloacetate decarboxylase, which is used primarily by pseudomonas, and that the enzyme is used by differential bacterial species to maintain pyruvate and bicarbonate cellular levels in many pathways.

The article explains the phenomenon behind the rational redesign of 4-chlorobenzoate binding site of the 4-chlorobenzoate:CoA (coenzyme A) ligase (CBL). The ligases are found to act as extremely effective catalytic agents.

The structure and properties of recombinant phosphonopyruvate (P-pyr) hydrolase (PPH) from variovorax sp. Pal2 expressed in E. coli is reported. Structure alignment of PPH with other superfamily members revealed two pairs of invariant or conservatively replaced residues that anchor the flexible gating loop.

The mechanism by which the central domain moves between the two catalytic sites located on the N-terminal and C-terminal domains was examined by expressing the domain as an independent protein and measuring its structure, stability and ability to catalyst the ATP/phosphate partial reaction in conjunction. The results were interpreted as evidence that the flexible linkers to the N-terminal domain for the productive domain-domain docking, although solvent-compatible, must tether the central domain, required for efficient catalysis.

A theory-guided mutation experiments that identifies strong electrostatic contributions of a remote residue, namely, Glu232 located on the adjacent subunit, to 4-chlorobenzoyl-CoA dehalogenase catalysis is described. The combination of the experimental and theoretical evidence is used to hypothesize that the Gly232(B) residue contributes to catalysis by providing an electrostatic force that acts on the Asp145 nucleophile.

Study is conducted to show that L-canavanine is a slow substrate inhibitor of pseudomonas aeruginosa arginine deiminase that works by covalent alteration of the active position Cys406 residue in the form of thiouronium salt. Strategies for the design of arginine deiminase (ADI) inhibitors that rely on the mechanism-based covalent modification of the active position Cys406 are feasible.

Experimental evidence that supports the assignment of the beta-phosphoglucomutase-beta-D-glucose-6-phosphate-1-phosphorane structure was demonstrated. The difference between this complex and beta-phosphoglucomutase-beta-D-glucose-6-phosphate-1-MgF3 structure is that the phosphorane complex contains two phosphorus atoms per one enzyme molecule, while the MgF3 complex would contain one phosphorus atom per one enzyme molecule.

The conversion of 4-chlorobenzoyl-CoA to 4-hydroxybenzoyl-CoA catalyzed by 4-chlorobenzoylCoA dehalogenase is investigated with the help of combined QM/MM approaches. The calculated potential of mean force at the PM3/CHARMM level supports the proposed nucleophilic aromatic substitution mechanism.

The results from transient-state kinetic experiments which show that arginine deiminase catalysis proceeds through a covalent adduct formed by activesite Cys406 displacement of ammonia from the guanidium carbon of L-arginine are presented.

A study which will show that the number of carboxylate residues on loop 4 in the positions on the loop are determinants and reliable sequence markers, for metal ion activation among the 2-haloalkanoic acid dehalogenase (HAD) family members, is presented. The results show that a majority of the HAD members use a metal cofactor and Asp186 is important to cofactor binding while Asp190 enhances cofactor binding.