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Research has been conducted on thiamine biosynthesis in Salmonella enterica serovar Typhimurium. The connections between thiamine biosynthesis and cellular coenzyme A have been investigated.

A paramagnetic probe for the magnesium cation cofactor of H(super)+ -ATPase shows that the cofactor binds to either the alpha or beta subunit with at least one nitrogen ligand. In the hexamer, the cation binds to a second nitrogen with properties similar to a histidine imidazole.

Alpha-melanocyte stimulating hormone can bind to the cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin and thereby regulate tyrosinase activity in epidermal cells. Researchers report the first evidence of such binding using Near Infrared Fourier Transform Raman spectroscopy.

The nucleotide tail attached to the D ring of corrinoid cofactors has little influence on cofactor binding to B(sub)12-dependent enzymes. However, it does play a critical role in the active site of the enzyme.

Results show the crotonyl coenzyme A reductase, encoded by the ccr gene, plays a significant role in the production of butyryl-CoA for monensin A and B biosynthesis by the bacteria grown in yeast extract-malt extract and a complex fermentation media.

The initiation of coenzyme binding mechanism in horse liver alcohol dehydrogenase has been found to effect conformation change in such compounds, as shown in an x-ray crystallography conducted on a sample of proteins. Experimental results suggest that such conformational alterations are made possible by forming the necessary configuration to accomodate all amino acids residues and their coenzyme. The entire process is also characterized by a low-barrier hydrogen bond formed by hydride transfer from a ground zinc alkoxide.

Research was conducted to examine the mechanism of reactivation of diol dehydratase by its reactivating factor through the use of enzyme*cyanocobalamin complex as a model for inactivated holoenzyme. The intermediary formation of apoenzyme in the reactivating factor-mediated cobalamin exchanges and the role of ATP in the reactivation process are described. It is proposed that the reactivating factor is a new type of molecular chaperone that participates in reactivation of the inactivated enzymes.

Research was conducted to investigate the assembly characteristics of the repressor of biotin biosynthesis (BirA) at higher protein concentrations than those previously utilized via equilibrium sedimentation. Results suggest that while apoBirA and the BirA*biotin complex reveal no tendency to aggregate at concentrations up to 100 microns, holoBirA undergoes association to dimer in the micromolar range of protein concentration. The one mechanism by which the adenylate promotes binding of BirA to the biotin operator is by promoting repressor dimerization.

The coenzyme Q10 acts as an electron carrier in the mitochondrial electron transport chain. The effects and safety of Q10 as a dietary supplement are discussed. Relatively low rates of Q10 in myocardial tissue have been reported in different cardiovascular diseases, and positive clinical and haemodynamic effects of oral Q10 have been seen in double-blind trials. No significant adverse effects have been reported from daily supplements of up to 200 mg for 6-12 months and 100mg for up to 6 y.

Protein guidance of reactivity in the vitamin B12 coenzyme-dependent enzymes is characterized by the acceleration of the effective cobalt-carbon bond cleavage rate by approximately 10(to the 12th power)-fold relative to the rate for the free coenzyme in solution. This feature is attributed to substrate-derived radical formation and the specific interactions of the cobalamin moeity of the protein. Techniques of X-band electron spin-echo envelope modulation spectroscopy confirmed these protein-specific effects on cob(II)alamin structure and reactivity.

Alanine-scanning site-directed mutagenesis of ionizable neutral acids that are conserved for all malic enzymes available were used to map the active site topography of the NAD-malic enzyme from Ascaris suum. Each of the conserved ionizable neutral residues were altered to alanine to examine their individual function. Results indicate that it is a possible candidate for the general base that accepts a proton from the malate hydroxyl in the oxidation step.

Resonance Raman (RR) spectroscopy is used to characterize the methylmalonyl-coenzyme A (MM-CoA) mutase, a prototype of adenosylcobalamin- (AdoCbl) dependent isomerase enzymes. The RR spectra suggest that a Co-C tilting is part of the Co-C activation mechanism in MM-CoA mutase. Moreover, the observed RR enhancement patterns are reversed when a bound substrate or product is contained in the enzyme, indicating that the steric forces responsible for the tilting are relieved when the substrate or product is rearranged in the active site.

The solution structure of tGCN5 catalytic domain in complex with coenzyme A has been determined. The structure consists of the amino-terminal domain similar to those of other GCN5-related N-acetyltransferases, and the carboxy-terminal domain. Coenzyme A binds between the two domains, and chemical shift changes on titration with histone H3 peptides suggest a binding site at the domain boundary opposite coenzyme A.

X-ray crystal structure of the enzyme (citrate synthase)-inhibitor (carboxymethyldethia coenzyme) complex shows the presence of an unusually short hydrogen bond between an aspartic acid side chain and the carboxylate group of the inhibitor. Such bond, which is in contrast to the anti hydrogen bond, exerts a substantial effect on the shift tensors for the carboxylate. The nuclear magnetic resonance studies also suggest the presence of a very short hydrogen bond between the Asp carboxylate and the transition state analogue.

The exposure of E91D variant of apo-yeast pyruvate decarboxylase (EC 4.1.1.1) to C2alpha-hydroxybenzylthiamin diphosphate partitioned the putative intermediate on the enzyme between release of the benzaldehyde product and dissociation of the proton at C2alpha to form the enamine-C2alpha-carbanion intermediate. The dramatic stabilization of this zwitterionic enamine intermediate at the active center was sufficient to account for as much as a 10 to the ninth power-fold rate acceleration on the enzyme.

Research was conducted to examine the hydrolytic dealkylation of organocobaloximes related to coenzyme B12. The organocobaloxime comples R-Co(DH)2(L) were prepared and characterized using 1H NMR and UV-vis spectroscopy. The Co-C bond hydrolysis rates were identified as a function of pH and temperature. An assessment and validation of the use of (coII(DH)2(OH2)2) as a radical trap in the kinetic determination of the Co-C bond dissociation enthalpy of coenzyme B12 was performed.

Research was conducted to examine an enzyme system from Nocardioides simplex FJ2-1A that catalyzes a hydride system from NADPH to picrate and 2,4-dinitrophenol (2,4-DNP). The fast and complete carbon removal within four days provides strong evidence that picrate is completely mineralized and used as the sole source of energy and carbon. In contrast, activated sludge or inactivated cells of N. simplex failed to degrade picrate under the same standardized conditions.

A study was conducted to examine the structure of the serotonin N-acetyltransferase (arylalkylamine-N-acetyltransferase[AANAT]). Results show that there is a bisubstrate analog bound to AANAT's enzyme active site, which was synthesized covalently binding the N-acetylated substrate, tryptamine and coenzyme A. It was also observed that analysis of the structure of the complex with bisubstrate analog will be useful in understanding the nature of substrate binding and the mechanism of enzyme action.

N-methyltryptophan oxidase and monomeric sarcosine oxidase catalyze the oxidative demethylation of N-methyl-L-tryptophan and sarcosine, respectively. The two enzymes both contain covalently bound flavin located at the FAD level as indicated in electrospray mass spectrometry data. A covalent flavin attachment site called Cys308 is located in N-methyltryptophan oxidase as reflected by a single conserved cysteine in the enzymes.

A study was conducted on acetyl coenzyme A carboxylase from Escherichia coli. The experiment describes the determination of the solution structures of both the holo and apo forms of the biotinyl domain of E. coli acetyl coenzyme A carboxylase. Both triple resonance nuclear magnetic resonance spectroscopy and an investigation of the existence and strength of the hydrogen bond thought to involve the ureido HN1', which is the site of carboxylation of the biotin ring, were used to determine the solution structures.

A second coenzyme A (CoA) analogue synthon has been isolated through the nonenzymatic synthesis of a racemic phosphopantetheine analogue followed by enzymatic conversion to the corresponding CoA analogue. A thioester was used in replacing the amide bond more distant from the thiol group of the synthon. The novel approach gives greater access to a wider array of CoA analogues modified in the beta-alanylcysteamine moiety. This has been proven in the synthesis of a CoA analogue having an extra methylene group in the beta-alanine moiety.

The steady-state internal redox state in prokaryotes was investigated by growing Escherichia coli MC4100 in anaerobic glucose-limited cultures in the presence of an electron acceptor or fully fermentatively. The steady-state NADH/NAD ratio was found to be dependent on the nature of external electron acceptors. Results also showed that the synthesis and activity of the pyruvate dehydrogenase complex occur under anaerobic respiratory conditions and correlate with the cellular steady-state redox state.

This investigation addressed the hypothesis that stearoyl coenzyme A desaturase (SCD) gene expression would serve as a postnatal marker of adipocyte differentiation in bovine s.c. adipose tissue. Samples of tailhead s.c. adipose tissue were obtained by biopsy from preweaning steer calves 2.5 wk, 5 mo, and 7.5 mo of age and from yearling steers 12 mo of age. Samples also were obtained at slaughter when the steers were 18 mo of age. The steers sampled as yearlings were fed native pasture from weaning until 12 mo of age, and the steers sampled at slaughter were fed a high-concentrate diet from 12 to 18 mo of age. Major peak adipocyte volumes for the 2.5-wk-, 5-mo-, and 7.5-mo-old steers were 14, 270, and 700 pL, respectively (P < .001). The steers did not gain weight during pasture feeding, and at 12 mo of age peak adipocyte volume had decreased (P = .009) to 270 pL. At this time, a second, smaller population of adipocytes had appeared with a peak volume of 115 pL. At slaughter, adjusted fat thickness of the steers was 1.60 [+ or -] .13 cm, the USDA yield grade of the carcasses was 3.51 [+ or -] .31, and peak adipocyte volume had increased (P = .01) to over 2,500 pL. The number of adipocytes per 100 mg of adipose tissue doubled (P = .006) between 2.5 wk and 5 mo of age, concurrent with the nearly 20-fold increase in peak adipocyte volume, indicating that this was a period of apparent adipocyte hyperplasia. Uncoupling protein mRNA was undetectable at all stages of postnatal growth, indicating that differentiating tailhead s.c. adipocytes do not acquire brown adipocyte characteristics postnatally. Lipogenesis expressed on a cellular basis was low in all preweaning samples and increased significantly above preweaning values only in the 18-mo-old steers. Stearoyl coenzyme A desaturase mRNA concentration also was low in all preweaning samples, but it peaked (P = .07) at 12 mo of age. Because the peak in SCD mRNA concentration preceded a significant rise in lipogenesis and lipid filling, we conclude that the level SCD gene expression may be indicative of the extent of terminal differentiation in bovine tailhead s.c. adipose tissue. Key Words: Adipocytes, Differentiation, Acyl-CoA Desaturase, Protein, Genes, Cattle

Research was conducted to analyze the importance of a subset of 26 mutant variants of bovine acyl-coenzyme A binding proteins (ACBP) by investigating their structure, stability, function and folding using single-site mutations. A total of 17 conserved sequence positions and three nonconserved positions were covered by the analysis. Results suggest that conservation of residues in homologous proteins may arise from a summed optimization of an effective folding reaction, a fully active binding site and a stable native protein.

Research was conducted to examine the eubacterial 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) which was purified 3,000-fold from Streptomyces sp. strain CL190. The purification procedure, which proceeded to apparent homogeneity with an overall yield of 2.1%, consisted of (NH4)2SO4 precipitation, hydrophobic interaction, affinity chromatographies and heat treatment and anion exchange. Results indicate a strong evolutionary pressure to maintain amino acid residues at distinct positions.

One of the significant differences in the structures of the active sites of D-amino acid transaminase and alanine racemase is the substitution of a negatively charged Glu in the former enzyme for a positively charged Arg in the latter near the pyridinium nitrogen of the co-enzyme pyridoxal 5'-phosphate ring. Glu-177 of D-amino acid transaminase was substituted with a Lys (E177K) to have a side chain in the titratable range of pH. This made possible the investigation of the effects of the E177K on catalytic efficiency, stereochemical fidelity and circular dichroism properties.

Rhodococcus rhodochrous N75 has been demonstrated to activate 3-methylmuconolactone by the addition of coenzyme A (CoA) before the hydrolysis of the butenolide ring. A lactone-CoA synthase is induced by a growth of R. rhodochrous N75 on p-toluate as the only source of carbon. The enzyme was purified 221 times by ammonium sulfate fractionation. The enzyme was observed to be very specific for its 3-methylmuconolactone substrate and has little or not activity with other monoene and diene lactone analogues.

The stereospecific 1H and 13C resonance assignments of hexadienoyl-coenzyme A (CoA) and crotonyl-CoA were investigated and compared with protein-CoA complexes. The two diastereospecific methyl groups at the C2' carbon were assigned via transferred nuclear Overhauser effect experiments. Results showed that the assignments result in a conformational analysis of the uncomplexed form of CoA in solution and that there is a correlation between the conformational tendencies of free CoA and the structure of CoA in protein-CoA complexes.

Research was conducted to examine the coupling of cobalt-carbon bond homolysis and hydrogen atom abstraction in adenosylcobalamin-dependent glutamate mutase. Stopped-flow spectroscopy was used to measure the rate of substrate-induced cleavage of AdoCbl by glutamate mutase using various concentrations of glutamate and methylaspartate. Results provide insight into a mechanism in which Co-C bond homolysis is coupled to substrate hydrogen abstration.

Research was conducted to examine the biochemical controls that regulate the biosynthesis of poly-3-hydroxybutyrate (PHB) in Rhizobium (Cicer) sp. strain CC 1192. Results provide evidence indicating that CC 1192 cells retain the enzymic capacity to synthesize PHB when they differentiate from the free-living state to the bacteroid state. The presence of an NADPH-dependent acetoacetyl coenzyme A reductase was also demonstrated in extracts from free-living and bacteroid cells of CC 1192.

Time-resolved spectroscopic experiments were performed to investigate the reactivity of the carbon-cobalt bond in adensylcobalamin during photolysis of the coenzyme B12(5'-deoxyadenosylcobalamin). Transient spectral data and photoexcitation of the carbon-cobalt bond were performed. Analysis of the results suggested a 76+/-4% recombination of the geminate radical pairs which led to 0.24+/-0.04 yield for the formation of solvent separated radicals. The effective recombination rate was identified at (0.250 ns)-1 + 4ns-1.

The electrochemical behavior of the redox coenzyme pyrroloquinolinequinone (PQQ) was examined in aprotic organic solvents using the trimethyl ester of PQQ and its 1-methylated derivative. Electric skin resonance (ESR) and ultraviolet visibility spectroscopies were performed. Moreover, cyclic voltammetry and ESR were performed to assess the interaction between radical anion species and Ca ion. The findings strongly supported the participation of coenzyme PQQ in enzymatic and nonenzymatic systems.

Mutations occurring in patients with deficiencies in isovaleryl-CoA dehydrogenase (IVD) were investigated through the polymerase chain reaction of IVD genomic and complementary DNA. The molecular defects caused by the amino acid substitutions were studied using in vitro mitochondrial import, prokaryotic expression and kinetic studies of IVD mutants. Results showed that seven precursors of IVD mutant peptides and the IVD Leu13Pro mutant were synthesized and imported into the mitochondria. Moreover, contrasting mitochondrial stability were exhibited by mutant and wild type peptides.

Experiments were performed to identify the biochemistry of the steps involved in the conversion of alpha-ketoglutarate and acetylCoA to alpha-ketosuberate in Methanosarcina thermophila. Trans-homoaconitrate was formed with the condensation of alpha-ketoglutarate and acetylCoA. Two consecutive elongation reactions involving alpha-ketoadipate that was formed from the dehydration and rehydration of cis-homoaconitate resulted to alpha-ketosuberate. A protein product from the Methanococcus gene served as a catalyst in one of the process.

A series of redox titrations of carbon monoxide dehydrogenase from Clostridium thermoaceticum using increasing partial pressures of CO in the absence and presence of CO2 was performed to examine the effects of CO2 on the redox reaction of the enzyme and the CO/acetyl-CoA exchange activity. Results showed that the reduction potentials of the clusters were increased and that the strength of CO binding to the reduced A cluster was increased in the presence of CO2. Moreover, CO2 also stabilized the intermediate EPR-silent state of the C-cluster.

The synthesis and characterization of a new structural analogue of coenzyme B12, 5'-deoxyadenosylcobalamin, AdoCbl, is conducted using X-ray crystallography, NMR and NMR-restrained molecular modeling. The objective was to determine if the configuration of the N-glycosidic linkage in the Ado ligand caused the conformation adopted by the ligand in AdoCbl. Results suggest that the southern conformation was the most stable and the predominant, if not the only, conformation adopted in solution.

The main photochemistry of methylcobalamin was examined through the ultrafast transient absorption spectroscopy. It was estimated that 27% of the initially excited methylcobalamine experienced a bond homolysis on a subpicosecond time scale while the remaining 73% developed a metastable photoproduct having a spectrum resembling the cob(III)alamin compounds. Findings indicated that methylcobalamin was not subjected to a clean photohomolysis of the carbon cobalt bond.

A general method to synthesize an acyl coenzyme A (CoA) analogue synthon with a reversed thioester orientation is described. A triortho ester was utilized as a protected thioester in the aminolysis reaction to prepare the analogue. The acyl coenzyme analogue formed an unnatural acyl-enzyme where the CoA moiety was covalently linked to an active site cysteine. The reversed succinate thioester together with the hydroxamate and the hydrazide analogues were synthesized to show the role of the acyl CoA as an analogue synthon of CoA.

A study was conducted to examine the aminoacylation of coenzyme A and pantetheine by aminoacyl-tRNA synthetases. Enzyme-bound radiolabeled aminoacyl adenylates were determined and incubated with thiols in the same mixture. Results indicated that Ile-pantetheine adduct was sensitive to NaOH and hydroxylamine. Findings also suggested that the thioester-dependent mechanism of peptide synthesis may have followed the RNA-dependent mechanism.

Tryptophanase has an absorption maximum at 338 nm at high pH and 422 nm at low pH. The 422-nm absorption species has been considered to be the catalytically competent ketoenamine form of the Schiff base of pyridoxal 5[prime]-phosphate with a lysine residue. The 338-nm absorption band showed an intense fluorescence band at 390 nm and not around 500 nm, indicating that the 338-nm absorption species is the substituted aldamine rather than an enolimine form of the Schiff base which has been suggested previously. To explore the mechanism of the enzyme that can exert its catalytic ability at high pH where most of its coenzyme exists as the catalytically incompetent aldamine structure, the reaction of tryptophanase with 3-indolepropionate, a substrate analogue that stops the reaction at the step of the Michaelis complex, was studied at various pH values and analogue concentrations. Kinetic analysis was done based on a scheme involving eight forms of the enzyme, i.e., the liganded and unliganded forms of the ketoenamine, the substituted aldamine structures, and their protonated and deprotonated forms. Kinetic parameters were obtained for each interconversion step. The results showed that the binding of 3-indolepropionate to tryptophanase shifts the equilibrium from the substituted aldamine to the ketoenamine structure over the entire pH region studied. This implies that in the reaction of tryptophanase with tryptophan at high pH, where the enzyme shows maximum activity, the binding of the substrate to the enzyme converts the inactive aldamine form of the coenzyme to the active ketoenamine form. Mechanisms for the activation process, in which a nucleophile is expelled from the aldamine either by steric hindrance of the nucleophile with the ligand or by the negative charge of the ligand [Alpha]-carboxylate group that stabilizes the aldimine structure, were discussed.

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 ribonucleotide reductases from three ancient eubacteria, the hyperthermophilic Thermotoga maritima (TM), the radioresistant Deinococcus radiodurans (DR), and the thermophilic photosynthetic Chloroflexus aurantiacus, were found to be coenzyme-[B.sub.12] (class II) enzymes, similar to the earlier described reductases from the archaebacteria Thermoplasma acidophila and Pyrococcus furiosus. Reduction of CDP by the purified TM and DR enzymes requires adenosylcobalamin and DTT. dATP is a positive allosteric effector, but stimulation of the TM enzyme only occurs close to the temperature optimum of 80-90 [degrees] C. The TM and DR genes were cloned by PCR from peptide sequence information. The TM gene was sequenced completely and expressed in Escherichia coli. The deduced amino acid sequences of the two eubacterial enzymes are homologous to those of the archaebacteria. They can also be aligned to the sequence of the large protein of the aerobic E. coli ribonucleotide reductase that belongs to a different class (class I), which is not dependent on [B.sup.12]. Structure determinations of the E. coli reductase complexed with substrate and allosteric effectors earlier demonstrated a 10-stranded [Beta]/[Alpha]-barrel in the active site. From the conservation of substrate- and effector-binding residues we propose that the [B.sub.12]-dependent class II enzymes contain a similar barrel.

Kinetics and substrate binding were utilized to investigate the substrate-coenzyme binding in a reconstructed protein containing simplified subunit structure and kinetic properties. The reconstructed configuration of the subunit of glutamate mutase resulted in simplified kinetic properties. Results have shown the importance of steric compression of the enzyme to stabilize the coenzyme.

Research was conducted to test whether acyltransferase (AT) and beta-ketoacyl synthase (KS) domains from Mycobacterium bovis influence selectivity for methylmalonyl-CoA. Extracts from the cultures expressing AT and KS were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis while a routine assay was carried out to determine the nature of the products. Results showed that the production of mycocerosic acids is catalyzed by a multifunctional FAS-like enzyme called mycocerosic acid synthase.

A study was conducted on the role of propionyl coenzyme A (CoA) carboxylase in the development of Myxococcus xanthus. TnV mutagenesis was employed to isolate a developmental cell morphological mutant (dcm-1 mutant) of M xanthus. The TnV insertions were observed in a Beta subunit of the M xanthus proprionyl CoA carboxylase gene. The findings indicate that propionyl-CoA carboxylase regulates the long-chain fatty acid synthesis in M xanthus through the concentration of malonyl-CoA.

Studies have shown that Acetobacterium woodii, Acetohalobium arabaticum, Clostridium formicoaceticum and Sporomusa silvacetica contain carbonic anhydrase (CA). A woodii exhibited the highest CA specific activity at approximately 14 U mg of protein-1 in extracts of either glucose- or H2-CO2-cultivated cells. CA activity of A woodii was inhibited by intracellular acetate concentrations by 50-85%, suggesting that intracellular acetate may influence in situ CA activity.

The ab initio structures of 2,7,9-tricarboxypyrroloquinoline quinone (PQQ), semiquinone (PQQH), and dihydroquinone (PQQH2) have been determined and compared with ab initio structures of the (PQQ) [Ca.sup.2+], (PQQH)[Ca.sup.2+], and (PQQH2) [Ca.sup.2+] complexes as well as the x-ray structure of (PQQ)[Ca.sup.2+] bound at the active site of the methanol dehydrogenase (MDH) of methyltropic bacteria. Plausible mechanisms for the MDH oxidation of methanol involving the (PQQ)[Ca.sup.2+] complex are explored via ab initio computations and discussed. Considering the reaction of methanol with PQQ in the absence of [Ca.sup.2+], nucleophilic addition of methanol to the PQQ C-5 carbonyl followed by a retro-ene elimination is deemed unlikely due to large energy barrier. A much more favorable disposition of the methanol C-5 adduct to provide formaldehyde involves proton ionization of the intermediate followed by elimination of methoxide concerted with hydride transfer to the oxygen of the C-4 carbonyl. Much the same transition state is reached if one searches for the transition state beginning with Asp-303-[Mathematical Expression Omitted] general-base removal of the methanol proton of the (PQQ)[Ca.sup.2+] O(H)[CH.sub.3] complex concerted with hydride transfer to the oxygen at C-4. For such a mechanism the role of the [Ca.sup.2+] moiety would be to (i) contribute to the formation of the ES complex (ii) provide a modest decrease in the [pK.sub.a] of methanol substrate,; and (iii) polarize the oxygen at C-5.

The role of the conserved histidine-aspartate pair in the catalysis and coenzyme binding by AdoCbl-dependent glutamate mutase was studied using a site-directed mutagenesis. Results show that glutamate mutase mutants were essential in the catalysis and binding of coenzyme. This indicates that the reactivity of AdoCbl is consistent with the varying modes of cobalt bond cleavage in the two coenzymes modulated by glutamate mutase histidine-aspartate pair.