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Fluoroacetate dehalogenase catalyzes the hydrolytic defluorination of fluoroacetate to produce glycolate. The enzyme is unique in that it catalyzes the cleavage of a carbon-fluorine bond of an aliphatic compound: the bond energy of the carbon-fluorine bond is among the highest found in natural products. The enzyme also acts on chloroacetate, although much less efficiently. We here determined the X-ray crystal structure of the enzyme from Burkholderia sp. strain FA1 as the first experimentally determined three-dimensional structure of fluoroacetate dehalogenase. The enzyme belongs to the [alpha]/[beta] hydrolase superfamily and exists as a homodimer. Each subunit consists of core and cap domains. The catalytic triad, Asp104-His271-Asp128, of which Asp104 serves as the catalytic nucleophile, was found in the core domain at the domain interface. The active site was composed of Phe34, Asp104, Arg105, Arg108, Asp128, His271, and Phe272 of the core domain and Tyr147, His149, Trp150, and Tyr212 of the cap domain. An electron density peak corresponding to a chloride ion was found in the vicinity of the [N.sup.[epsilon]1] atom of Trp150 and the [N.sup.[epsilon]2] atom of His149, suggesting that these are the halide ion acceptors. Site-directed replacement of each of the active-site residues, except for Trp150, by Ala caused the total loss of the activity toward fluoroacetate and chloroacetate, whereas the replacement of Trp150 caused the loss of the activity only toward fluoroacetate. An interaction between Trp150 and the fluorine atom is probably an absolute requirement for the reduction of the activation energy for the cleavage of the carbon-fluorine bond.

Shewanella livingstonensis Ac10, a psychrotrophic gram-negative bacterium isolated from Antarctic seawater, produces eicosapentaenoic acid (EPA) as a component of phospholipids at low temperatures. EPA constitutes about 5% of the total fatty acids of cells grown at 4[degrees]C. We found that five genes, termed orf2, orf5, orf6, orf7, and orf8, are specifically required for the synthesis of EPA by targeted disruption of the respective genes. The mutants lacking EPA showed significant growth retardation at 4[degrees]C but not at 18[degrees]C. Supplementation of a synthetic phosphatidylethanolamine that contained EPA at the sn-2 position complemented the growth defect. The EPA-less mutant became filamentous, and multiple nucleoids were observed in a single cell at 4[degrees]C, indicating that the mutant has a defect in cell division. Electron microscopy of the cells by high-pressure freezing and freeze-substitution revealed abnormal intracellular membranes in the EPA-less mutant at 4[degrees]C. We also found that the amounts of several membrane proteins were affected by the depletion of EPA. While polyunsaturated fatty acids are often considered to increase the fluidity of the hydrophobic membrane core, diffusion of a small hydrophobic molecule, pyrene, in the cell membranes and large unilamellar vesicles prepared from the lipid extracts was very similar between the EPA-less mutant and the parental strain. These results suggest that EPA in S. livingstonensis Ac10 is not required for bulk bilayer fluidity but plays a beneficial role in membrane organization and cell division at low temperatures, possibly through specific interaction between EPA and proteins involved in these cellular processes.

We identified a gene cluster that is involved in the [gamma]-resorcylate (2,6-dihydroxybenzoate) catabolism of the aerobic bacterium Rhizobium sp. strain MTP-10005. The cluster consists of the graRDAFCBEK genes, and graA, graB, graC, and graD were heterologously expressed in Escherichia coli. Enzymological studies showed that graD, graA, graC, and graB encode the reductase (GraD) and oxygenase (GraA) components of a resorcinol hydroxylase (EC 1.14.13.x), a maleylacetate reductase (GraC) (EC 1.3.1.32), and a hydroxyquinol 1,2-dioxygenase (GraB) (EC 1.13.11.37). Bioinformatic analyses suggested that graE, graR, and graK encode a protein with an unknown function (GraE), a MarR-type transcriptional regulator (GraR), and a benzoate transporter (GraK). Quantitative reverse transcription-PCR of graF, which encodes [gamma]-resorcylate decarboxylase, revealed that the maximum relative mRNA expression level ([5.93 [+ or -] 0.82] x [10.sup.-4]) of graF was detected in the total RNA of the cells after one hour of cultivation when [gamma]-resorcylate was used as the sole carbon source. Reverse transcription-PCR of graDAFCBE showed that these genes are transcribed as a single mRNA and that the transcription of the gene cluster is induced by [gamma]-resorcylate. These results suggested that the graDAFCBE genes are responsible as an operon for the growth of Rhizobium sp. strain MTP-10005 on [gamma]-resorcylate and are probably regulated by GraR at the transcriptional level. This is the first report of the [gamma]-resorcylate catabolic pathway in an aerobic bacterium.

Research describes cloning, expression, and characterization of the alr1(sp)+ gene and its encoded product alanine racemase ALR1p of Schizosaccharomyces pombe. Data indicate that alanine racemase mediates D-alanine catabolism in S. pombe and enables growth of S. cerevisiae on D-alanine as a sole nitrogen following heterologous expression of the gene.

The cloning and expression in Escherichia coli of the genes encoding NAD+-dependent alanine dehydrogenases (AlaDHs) (EC1.4.1.1) from the Antarctic bacterial organisms Shewanella sp strain Ac10 (SheAlaDH) and Carnobacterium sp strain St2 (CarAlaDH) are described. The highest level of sequence similarity to the AlaDH from the gram-negative bacterium Vibrio pproteolyticus was observed in SheAlaDH. Results indicate that the thermal instability of SheAlaDH and CarAlaDH may be caused by the relatively low numbers of salt bridges in the proteins.

Research was conducted to examine the characteristics of a cold-active recombinant subtilisinlike protease from the psychrotrophic bacterium Shewanella strain Ac10. The amino acid sequence deduced from the 2,442-bp nucleotide sequence showed that the protein was 814 amino acids long with an estimated molecular weight of 85,113. Purification was performed from the culture supernatant of Escherichia coli recombinant cells using affinity chromatography with a bacitracin-Sepharose column.

A nitroalkane-oxidizing enzyme from Neurospora crassa is shown to be a 2-nitropropane dioxygenase after purification to homogeneity. It is the first 2-nitropropane dioxygenase purified to homogeneity from organisms other than yeast. It is also the first flavin mononucleotide-dependent 2-nitropropane dioxygenase. Results of its characterization dispute previous suggestions that the nitroalkane-oxidizing enzyme from N. crassa is an oxidase and that the oxygen atom of the product is derived from water and not from molecular oxygen in the reaction.

The cloning and sequencing of the lipase gene (lipP) from a Pseudomonas strain and the purification and characterization of the recombinant enzyme LipP were studied and reported. The amino acid sequence from the nucleotide sequence of the gene corresponded to a protein of 308 amino acid residues with a molecular weight of 33,714. The LipP enzyme was found to be stable between pH 6 and 9 while ts was unstable with temperatures higher than 45 degrees C. Various organic solvents such as dimethyl sulfoxide and methanol at concentrations of 0 to 30% activated the enzyme.

A research was conducted to study an enzymatic synthesis technique of L and D amino acids from alpha-keto acids with Escherichia coli cells which express heterologous genes. An Applied Biosystems model 370A DNA sequencer and a dye-labelled terminator sequencing kit were utilized to perform DNA sequencing while cloned cells were washed with 0.85% NaCl. Results showed the technique to be suitable for the synthesis of unnatural L and D amino acids with bulky side chains.

DL-2-haloacid dehydrogenase from Pseudomonas sp. strain 113 (DL-DEX) is a hydrolic dehalogenase catalyst which acts on the chiral of the acid substrate and uses both enantiomers as equal substrates. DL-DEX gene encoder isolation and sequencing were done to determine the structural make-up but no similarity between the DL-DEX and L-DEX was detected. Each enantiomer of 2-chloropropionate competitively inhibits the enzymatic dehalogenation of each other, suggesting that DL-DEX has a single common catalytic site for both enantiomers.

A 15-kb region of Pseudomonas putida chromosomal DNA containing the mde operon and an upstream regulatory gene (mdeR) was cloned and sequenced. The mde operon has two structural genes involved in L-methionine degradative metabolism. A rho-independent terminator was found just downstream of mdeB, and open reading frames corresponding to other components of alpha-keto acid dehydrogenase complex were not present. Findings indicated that MdeB plays a significant role in the metabolism of alpha-ketobutyrate produced by MdeA from L-methionine.

The thermostable ornithine 5-aminotransferase from the thermophile Bacillus sp. YM-2 shows stereospecific abstraction of the pro-S hydrogen from C-5 of L- and D-ornithine. It exhibits similar behavior during the transfer of the pro-S hydrogen to the C-4' on the si face of the planar pi-system of the cofactor moiety in the transamination process. The stereospecificity for the transfer does not rely on the enantiomeric state of ornithine. The geometrical relationship between the catalytic action of the enzyme and the bound cofactor is detailed.