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PubMed1989

Structures of D-xylose isomerase from Arthrobacter strain B3728 containing the inhibitors xylitol and D-sorbitol at 2.5 A and 2.3 A resolution, respectively.

The structures of D-xylose isomerase from Arthrobacter strain B3728 containing the polyol inhibitors xylitol and D-sorbitol have been solved at 2.5 A and 2.3 A, respectively. The structures have been refined using restrained least-squares refinement methods. The final crystallographic R-factors for the D-sorbitol (xylitol) bound molecules, for 43,615 (32,989) reflections are 15.6 (14.7). The molecule is a tetramer and the asymmetric unit of the crystal contains a dimer, the final model of which, incorporates a total of 6086 unique protein, inhibitor and magnesium atoms together with 535 bound solvent molecules. Each subunit of the enzyme contains two domains: the main domain is a parallel-stranded alpha-beta barrel, which has been reported in 14 other enzymes. The C-terminal domain is a loop structure consisting of five helical segments and is involved in intermolecular contacts between subunits that make up the tetramer. The structures have been analysed with respect to molecular symmetry, intersubunit contacts, inhibitor binding and active site geometry. The refined model shows the two independent subunits to be similar apart from local deviations due to solvent contacts in the solvent-exposed helices. The enzyme is dependent on a divalent cation for catalytic activity. Two metal ions are required per monomer, and the high-affinity magnesium(II) site has been identified from the structural results presented here. The metal ion is complexed, at the high-affinity site, by four carboxylate side-chains of the conserved residues, Glu180, Glu216, Asp244 and Asp292. The inhibitor polyols are bound in the active site in an extended open chain conformation and complete an octahedral co-ordination shell for the magnesium cation via their oxygen atoms O-2 and O-4. The active site lies in a deep pocket near the C-terminal ends of the beta-strands of the barrel domain and includes residues from a second subunit. The tetrameric molecule can be considered to be a dimer of "active" dimers, the active sites being composed of residues from both subunits. The analysis has revealed the presence of several internal salt-bridges stabilizing the tertiary and quaternary structure. One of these, between Asp23 and Arg139, appears to play a key role in stabilizing the active dimer and is conserved in the known sequences of this enzyme.(ABSTRACT TRUNCATED AT 400 WORDS)

Aldose-Ketose Isomerases↗
PubMed1992

Crystal structure analysis, refinement and enzymatic reaction mechanism of N-carbamoylsarcosine amidohydrolase from Arthrobacter sp. at 2.0 A resolution.

N-carbamoylsarcosine amidohydrolase from Arthrobacter sp., a tetramer of polypeptides with 264 amino acid residues each, has been crystallized and its structure solved and refined at 2.0 A resolution, to a crystallographic R-factor of 18.6%. The crystals employed in the analysis contain one tetramer of 116,000 M(r) in the asymmetric unit. The structure determination proceeded by multiple isomorphous replacement, followed by solvent-flattening and density averaging about the local diads within the tetramer. In the final refined model, the root-mean-square deviation from ideality is 0.01 A for bond distances and 2.7 degrees for bond angles. The asymmetric unit consists of 7853 protein atoms, 431 water molecules and four sulfate ions bound into the putative active site clefts in each subunit. One subunit contains a central six-stranded parallel beta-pleated sheet packed by helices on both sides. On one side, two helices face the solvent, while two of the helices on the other side are buried in the tight intersubunit contacts. The catalytic center of the enzyme, tentatively identified by inhibitor binding, is located at the interface between two subunits and involves residues from both. It is suggested that the nucleophilic group involved in hydrolysis of the substrate is the thiol group of Cys117 and a nucleophilic addition-elimination mechanism is proposed.

Amidohydrolases↗
PubMed1985

Redox reactions in hydrocortisone transformation by Arthrobacter globiformis cells.

Hydrocortisone and prednisolone transformation by Arthrobacter globiformis cells in aerobic and anaerobic conditions was studied. 3-Ketosteroid-1-en-dehydrogenase activity was shown to be the major factor regulating the direction of transformation. When it is high (aerobic conditions), the end products of hydrocortisone transformation are prednisolone or its 20 beta-hydroxy derivative. The latter is produced via 1-en-dehydrogenation, which is not a limiting stage of the process. Low 3-ketosteroid-1-en-dehydrogenase activity (in the presence of cyanide) or its complete inhibition (strictly anaerobic conditions) result in the direct reduction of 20-keto group of hydrocortisone.

Aerobiosis↗
PubMed1995

Limonoate dehydrogenase from Arthrobacter globiformis: the native enzyme and its N-terminal sequence.

Bitter limonoids in citrus juice lower the quality and value of commercial juices. Limonoate dehydrogenase converts the precursor of bitter limonin, limonoate A-ring lactone, to nonbitter 17-dehydrolimonoate A-ring lactone. This enzyme was isolated from Arthrobacter globiformis cells by a combination of ammonium sulfate fractionation, Cibacron Blue affinity chromatography and DEAE ion exchange HPLC. Using this protocol a 428-fold purification of the enzyme was obtained. Gel filtration HPLC indicated a M(r) of 118,000 for the native enzyme. SDS-PAGE indicated an individual subunit M(r) of 31,000. N-Terminal sequencing of the protein provided a sequence of the first 16 amino acid residues. Since LDH activity in citrus is very low, cloning the gene for this bacterial enzyme into citrus trees should enhance the natural debittering mechanism in citrus fruit.

Alcohol Oxidoreductases↗
PubMed1993

Transformation of cholic acid by Arthrobacter simplex.

Fermentation of cholic acid with Arthrobacter simplex (IICB 227) under aerobic conditions yielded 3,12-dioxo-23,24-dinorchola-4,6-dienoic acid, 7 alpha-hydroxy-3,12-dioxo-23,24-dinorchol-4-enoic acid, 3 alpha, 7 alpha-dihydroxy-12-oxo-5 beta-cholan-24-oic acid, 3 alpha, 7 alpha-dihydroxy-12-oxo-5 beta-23,24-dinorcholan-22-oic acid, 7 alpha, 12 alpha-dihydroxy-3-oxo-5 beta-cholan-24-oic acid, 7 alpha-12 alpha-dihydroxy-3-oxo-4-cholenoic acid, 7 alpha, 12 alpha-dihydroxy-3-oxo-23,24-dinorchol-4-enoic acid, and methyl-3 alpha-7 alpha, 12 alpha-trihydroxy-5 beta-cholan-24-oate in addition to a new metabolite 2 beta-hydroxy-3,12-dioxo-23,24-dinorchola-4,6-dienoic acid. Each microbial metabolite was characterized by the application of various spectroscopic methods. The availability of some of the metabolites' enabled complete elucidation of their 13C NMR spectra.

Aerobiosis↗
PubMed1990

NADH production from NAD+ with a formate dehydrogenase system involving immobilized cells of a methylotrophic Arthrobacter strain.

A convenient and economical method of NADH production from NAD+ has been established using a formate dehydrogenase system involving immobilized cells of a methanol-utilizing bacterium. Arthrobacter sp, KM62. Four kinds of cell entrapment were studied. An immobilized cell preparation showing a high NADH production activity was obtained by entrapment in a kappa-carrageenan gel lattice. The NADH-producing activity of the immobilized cells was investigated under various conditions. The NADH-producing activity was evoked on the addition of Triton X-100 to the reaction mixture. The conditions for the continuous production of NADH with an immobilized cell column were also investigated. When a reaction mixture containing 10 mumol (6.63 mg) ml-1 NAD+ was passed through the column (1.2 x 20 cm) containing 1.62 g (as dry weight) of immobilized cells, at a space velocity of 0.125 at 35 degrees C, complete conversion was attained.

Arthrobacter↗
PubMed1988

Induction of a light-inducible gene in Arthrobacter sp. by exposure of cells to chelating agents and pH 5.

Transcription of a light-inducible gene in the prokaryote Arthrobacter sp. is induced in the dark when cells are incubated with chelating agents or in medium at pH 5. However, repletion of metal ions such as Ca2+, Mn2+ or Zn2+ or an increase in pH is required for accumulation of the gene product, an Mr 21,000 polypeptide. But such changes in condition restore repression of the gene, and the decay in the rate of synthesis of the polypeptide follows the same time-course as when photodynamically induced cells are transferred to the dark. These results are consistent with regulation of expression of this gene at transcriptional and posttranscriptional steps by mechanisms that involve metal-protein complexes.

Arthrobacter↗
PubMed1990

Cell growth and enzyme synthesis of a mutant of Arthrobacter sp. (DSM 3747) used for the production of L-amino acids from D,L-5-monosubstituted hydantoins.

A microorganism with the ability to form L-tryptophan from D,L-5-(3-indolyl-methyl)hydantoin (D,L-5-IMH) was isolated and identified as Arthrobacter sp. (DSM 3747). After isolation of a mutant with high tryptophan production activity but low tryptophan degradation, cultural conditions were optimized to achieve high amounts of biomass with good specific activities concerning the enzymatic hydantoin-cleaving reactions. The ability of the microorganism to perform these bioconversions was found to be inducible by D,L-5-IMH as well as to be dependent on the presence of Mn2+. The highest specific D,L-5-IMH-cleaving activity of the cells was observed in the exponential phase of growth. The addition of yeast extract to the mineral salts medium was found to be essential for obtaining biomass concentrations of about 25 g l-1 cell dry mass by bioreactor cultivations. In order to obtain a constantly high growth rate, feeding of the C-source was pO2-controlled. The inducer D,L-5-IMH had to be continuously fed to prevent a decline of the L-tryptophan-forming enzyme activities, because it was subjected to degradation with the enzymes induced and higher concentrations of D,L-5-IMH aggravated the growth significantly. The synthesis of the enzymes was also inducible, when inducer and Mn2+ were not added until the late growth phase. Using this process, the consumption of D,L-5-IMH was reduced remarkably. So, under these conditions biomass concentrations of 25 g l-1 cell dry weight with a specific enzymatic activity of 0.20 mmol g-1 h-1 (tryptophan per dry mass per time) could be obtained within 13 h. Using 1 g l-1 of the chemically modified inducer D,L-5-(3-indolylmethyl)-3-N-methylhydantoin, which was not degradable by the microorganisms, a biomass concentration of 28 g l-1 cell dry weight with a specific activity of 0.34 mmol g-1 h-1 (tryptophan per dry mass per time) could be obtained within 28 h.

Amidohydrolases↗
PubMed1990

Production of L-tryptophan from D,L-5-indolylmethylhydantoin by resting cells of a mutant of Arthrobacter species (DSM 3747).

The reaction parameters and the stereospecificity of the enzymatic cleavage of D,L-5-indolylmethylhydantoin in producing L-tryptophan with resting cells of Arthrobacter sp. DSM 3747 were studied. When intact cells were tested, the optimal pH was between 8.5 and 9.0 and the optimal temperature was 50 degrees C. Both, L-N-carbamoylase and hydantoinase could be stabilized over 24 h at 30 and 40 degrees C by the addition of D,L-5-indolylmethylhydantoin. Furthermore, the hydantoinase was stable over 24 h at 50 degrees C by the addition of 0.5 mM Mn2+ ions. The treatment with sodium desoxycholate turned out to be successful in overcoming the poor availability of D,L-5-indolylmethylhydantoin for the cells. The optimal temperature with permeabilized cells decreased to 30 degrees C and therefore ensured a good enzyme stability. While the L-N-carbamoylase proved to be absolutely L-specific, the hydantoinase led to a mixture of enantiomers of N-carbamoyltryptophan. The produced D-N-carbamoyl-tryptophan caused an inhibition of the L-N-carbamoylase. The transformation yield from D,L-5-indolylmethylhydantoin always reached 100%.

Amidohydrolases↗
PubMed1996

Cloning and sequencing of trehalose biosynthesis genes from Arthrobacter sp. Q36.

A 5.1-kbp genomic DNA fragment was cloned from trehalose-producing bacterium Arthrobacter sp. strain Q36. Sequence analysis of the DNA fragment revealed two open reading frames of 2325 and 1794 bp, encoding maltooligosyltrehalose synthase (TreY) and maltooligosyltrehalose trehalohydrolase (TreZ), respectively. The 3' end of treY overlaps with the 5' end of treZ by one nucleotide, and it is suggested that treYZ constitutes and operon. The deduced amino acid sequences of both enzymes have several regions common to amylolytic enzymes belonging to an 'alpha-amylase family'.

Amino Acid Sequence↗
PubMed1991

Cloning and expression of the Arthrobacter globiformis KZT1 fcbA gene encoding dehalogenase (4-chlorobenzoate-4-hydroxylase) in Escherichia coli.

The fsbA gene controlling the first step of 4-chlorobenzoic acid (4CBA) metabolism in the Gram-positive soil bacterium Arthrobacter globiformis KZT1 has been cloned and analysed in Escherichia coli. The E. coli minicells analysis showed that a polypeptide(s) with Mr = 58 kDa (and/or Mr = 32 kDa) can be the fcbA product(s). Despite the gene dose amplification and control of the E. coli inducible Plac promoter, the level of functional expression of the fcbA gene in E. coli cells seems comparable only with that in the parental KZT1 strain. Effective 4CBA dechlorination by recombinant cells during growth in the presence of substrate within a range of concentrations 0.1 g/l to 0.7 g/l as well as a sudden reduction in the reaction efficiency at higher substrate concentrations were observed.

Arthrobacter↗
PubMed1991

Genetic control of degradation of chlorinated benzoic acids in Arthrobacter globiformis, Corynebacterium sepedonicum and Pseudomonas cepacia strains.

The strains of Arthrobacter globiformis KZT1, Corynebacterium sepedonicum KZ4 and Pseudomonas cepacia KZ2 capable of early dehalogenation and complete oxidation of 4-chloro-, 2,4-dichloro-and 2-chlorobenzoic acids, respectively, have been analyzed for the origin of the genetic control of degradation. The occurrence and molecular sizes of plasmids in all the strains have been established. Plasmid pBS1501 was shown to control 4-chlorobenzoate dehalogenation in the case of KZT1 strain. The same possibility is proposed for plasmid pBS1502 for dehalogenation of 2,4DCBA by KZ4 strain. The chromosome localization of the genes controlling oxidation of 4-hydroxybenzoate in strain KZT1 is shown. Localization of the whole set of genes responsible for 2CBA degradation in the strain KZ2 chromosome is suggested.

Arthrobacter↗
PubMed1985

An erythromycin-resistance gene from an erythromycin-producing strain of Arthrobacter sp.

A gene (ermA) coding for a presumed erythromycin-resistance (ErR) determinant from an Er-producing Arthrobacter sp. strain (NRRLB3381) was isolated from a gene bank in phage vector lambda 2001 by probing with a Streptomyces ErR gene. Strongly hybridizing fragments were subcloned and the appropriate segments sequenced. The ermA gene is 76 mol% G + C in content and specifies a protein of 340 aa with an Mr of 37454. S1 nuclease mapping and primer extension identified the putative promoter, which resembles the consensus sequence of Escherichia coli promoters particularly in the -10 region. A potential ribosome-binding site (RBS) (AGGAG) was also located. Unexpectedly, the majority of in vivo ermA transcripts detected were only 245 nt long, suggesting that expression of ErR may be regulated post-transcriptionally. Substantial homology is observed between the predicted aa sequences of the ermA-coded protein and the products of three other ErR determinants, from organisms that do not produce Er.

Arthrobacter↗
PubMed1993

Cloning and characterization of a gene coding for the catechol 1,2-dioxygenase of Arthrobacter sp. mA3.

The catA gene, coding for the catechol 1,2-dioxygenase (C12O) of the bacterial strain Arthrobacter sp. mA3, was cloned and expressed in Escherichia coli. One plasmid containing a 6.1-kb EcoRI insert was selected by its ability to degrade catechol and to accumulate cis-cis-muconate. The DNA insert of this plasmid was mapped with restriction enzymes. The catA gene was subcloned on a 1.3-kb PstI-EcoRI fragment by deleting the adjacent restriction fragments. The nucleotide sequence of catA was determined. The C12O is coded for by a gene spanning 849 nucleotides and the deduced M(r) of the protein is 30,560. The polypeptide encoded by the cloned catA gene was expressed in an E. coli minicell system and detected by gel electrophoresis.

Amino Acid Sequence↗
PubMed1994

The penicillin amidase of Arthrobacter viscosus (ATCC 15294).

The nucleotide (nt) sequence of the gene encoding penicillin G amidase (PA) of Arthrobacter viscosus strain ATCC 15,294 was determined. The sequence contained an open reading frame of 2406 nt with a G+C content of 37%. The deduced amino-acid sequence shows significant homology with other so far identified beta-lactam amidases of Gram- bacteria.

Amino Acid Sequence↗
PubMed1994

Secretion of a temporally controlled cell-associated protein in Arthrobacter aureus: production of the protein and inactivation of its structural gene.

Arthrobacter aureus secretes 2 proteins of 22 and 100 kDa (P22 and P100, respectively). P22 is a protease, while P100, of unknown function, is associated with the cells before being released into the medium. An immunologically related P100 protein was also found in the culture supernatant of A. ureafasciens. Despite the fact that production of P22 and P100 proteins is induced by growth in the presence of bovine serum albumin, their synthesis is not always induced coordinately. Inactivation of the P100 chromosomal structural gene had no effect on growth characteristics of the mutant under inducing conditions.

Arthrobacter↗
PubMed2004

Cloning, sequence analysis, and purification of choline oxidase from Arthrobacter globiformis: a bacterial enzyme involved in osmotic stress tolerance.

Choline oxidase catalyzes the four-electron oxidation of choline to glycine betaine, one of a limited number of compounds that accumulate to high levels in the cytoplasm of cells to prevent dehydration and plasmolysis in adverse hyperosmotic environments. In the present study, the highly GC rich codA gene encoding for choline oxidase was cloned from genomic DNA of Arthrobacter globiformis strain ATCC 8010 and expressed to high yields in Escherichia coli strain Rosetta(DE3)pLysS. The resulting enzyme was purified to high levels in a single chromatographic step using DEAE-Sepharose, as shown by SDS-PAGE analysis. Denaturation and mass spectroscopic analyses showed that the covalent linkage between the FAD cofactor and the protein is preserved in recombinant choline oxidase, consistent with protein flavinylation being a self-catalytic process. The enzyme was shown to be a homodimer of 120,000 Da by size-exclusion chromatography and to be active with both choline and betaine aldehyde as substrate. Sequencing analysis indicated that the nucleotide sequence of codA originally reported in GenBank contains seven flaws, resulting in a translated protein with a significantly altered amino acid sequence between position 298 and 410.

Adaptation, Physiological↗
PubMed2006

N-ethylmaleimide-resistant acyl-coenzyme A oxidase from Arthrobacter ureafaciens NBRC 12140: molecular cloning, gene expression and characterization of the recombinant enzyme.

N-ethylmaleimide (NEM)-resistant acyl-coenzyme A oxidase (ACO) has been desired for the determination of free fatty acids (FFAs). In order to meet this demand, we prepared recombinant ACO from Arthrobacter ureafaciens NBRC 12140. The coding region of the gene was 2109, encoding a protein of 703 amino acids with a predicted molecular mass of 76.5 kDa. The heterologous expression level in Escherichia coli was 520-fold higher than that in the native strain. The purified enzyme retained more than 60% activity after incubation in the presence of 10 mM NEM at 37 degrees C for 4 h, while other commercially available ACOs showed only less than 10% activities after the same NEM treatment. We presume that this is due to the presence of only three cysteines in ACO from A. ureafaciens. Site-directed mutagenesis studies and close scrutiny of the three-dimensional structures of other related ACOs suggested that these cysteines were buried in the protein and unreactive to NEM. The recombinant enzyme was used for the colorimetric determination of free fatty acid, which gave a linear calibration.

Acyl-CoA Oxidase↗