Gene cloning and characterization of thiourocanate hydratase from Burkholderia sp. HME13

2019 ◽  
Vol 167 (3) ◽  
pp. 333-341
Author(s):  
Hisashi Muramatsu ◽  
Haruna Miyaoku ◽  
Syuya Kurita ◽  
Hidenori Matsuo ◽  
Takehiro Kashiwagi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Crude Extract ◽  
Maximum Activity ◽  
Luria Bertani ◽  
Sole Nitrogen Source ◽  
Gene Encoding ◽  
Gene Cloning And Characterization

Abstract A novel enzyme, thiourocanate hydratase, which catalyses the conversion of thiourocanic acid to 3-(5-oxo-2-thioxoimidazolidin-4-yl) propionic acid, was isolated from the ergothioneine-utilizing strain, Burkholderia sp. HME13. When the HME13 cells were cultured in medium containing ergothioneine as the sole nitrogen source, thiourocanate-metabolizing activity was detected in the crude extract from the cells. However, activity was not detected in the crude extract from HME13 cells that were cultured in Luria-Bertani medium. The gene encoding thiourocanate hydratase was cloned and expressed in Escherichia coli, and the recombinant enzyme was purified to homogeneity. The enzyme showed maximum activity at pH 7.5 and 55°C and was stable between pH 5.0 and 10.5, and at temperatures up to 45°C. The Km and Vmax values of thiourocanate hydratase towards thiourocanic acid were 30 μM and 7.1 μmol/min/mg, respectively. The enzyme was strongly inhibited by CuCl2 and HgCl2. The amino acid sequence of the enzyme showed 46% identity to urocanase from Pseudomonas putida, but thiourocanate hydratase had no urocanase activity.

scholarly journals Genetic and Biochemical Characterization of a Highly Thermostable α-l-Arabinofuranosidase fromThermobacillus xylanilyticus

2000 ◽  
Vol 66 (4) ◽  
pp. 1734-1736 ◽  
Author(s):  
Takoua Debeche ◽  
Nicola Cummings ◽  
Ian Connerton ◽  
Philippe Debeire ◽  
Michael J. O'Donohue
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Classification System ◽  
Biochemical Characterization ◽  
Glycosyl Hydrolase ◽  
Gene Encoding ◽  
Ph Range ◽  
Sequence Similarities

ABSTRACT The gene encoding an α-l-arabinofuranosidase fromThermobacillus xylanilyticus D3, AbfD3, was isolated. Characterization of the purified recombinant α-l-arabinofuranosidase produced in Escherichia coli revealed that it is highly stable with respect to both temperature (up to 90°C) and pH (stable in the pH range 4 to 12). On the basis of amino acid sequence similarities, this 56,071-Da enzyme could be assigned to family 51 of the glycosyl hydrolase classification system. However, substrate specificity analysis revealed that AbfD3, unlike the majority of F51 members, displays high activity in the presence of polysaccharides.

scholarly journals Expression in Escherichia coli and characterization of a reconstituted recombinant 7Fe ferredoxin from Desulfovibrio africanus

1996 ◽  
Vol 314 (1) ◽  
pp. 63-71 ◽  
Author(s):  
Johanneke L. H. BUSCH ◽  
Jacques L. J. BRETON ◽  
Barry M. BARTLETT ◽  
Richard JAMES ◽  
E. Claude HATCHIKIAN ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Magnetic Circular Dichroism ◽  
Wild Type ◽  
E Coli ◽  
Excess Iron ◽  
Expression In Escherichia Coli ◽  
Type D

Desulfovibrio africanus ferredoxin III is a monomeric protein (molecular mass of 6585 Da) that contains one [3Fe-4S]1+/0 and one [4Fe-4S]2+/1+ cluster when isolated aerobically. The amino acid sequence consists of 61 amino acids, including seven cysteine residues that are all involved in co-ordination to the clusters. In order to isolate larger quantities of D. africanus ferredoxin III, we have overexpressed it in Escherichia coli by constructing a synthetic gene based on the amino acid sequence of the native protein. The recombinant ferredoxin was expressed in E. coli as an apoprotein. We have reconstituted the holoprotein by incubating the apoprotein with excess iron and sulphide in the presence of a reducing agent. The reconstituted recombinant ferredoxin appeared to have a lower stability than that of wild-type D. africanus ferredoxin III. We have shown by low-temperature magnetic circular dichroism and EPR spectroscopy that the recombinant ferredoxin contains a [3Fe-4S]1+/0 and a [4Fe-4S]2+/1+ cluster similar to those found in native D. africanus ferredoxin III. These results indicate that the two clusters have been correctly inserted into the recombinant ferredoxin.

scholarly journals A Novel Archaeal Alanine Dehydrogenase Homologous to Ornithine Cyclodeaminase and μ-Crystallin

2004 ◽  
Vol 186 (22) ◽  
pp. 7680-7689 ◽  
Author(s):  
Imke Schröder ◽  
Alexander Vadas ◽  
Eric Johnson ◽  
Sierin Lim ◽  
Harold G. Monbouquette
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Maximum Activity ◽  
The Novel ◽  
Amino Acid Sequence Homology ◽  
Gene Encoding ◽  
Alanine Dehydrogenase ◽  
Significant Amino Acid Sequence ◽  
Significant Amino Acid ◽  
Ornithine Cyclodeaminase

ABSTRACT A novel alanine dehydrogenase (AlaDH) showing no significant amino acid sequence homology with previously known bacterial AlaDHs was purified to homogeneity from the soluble fraction of the hyperthermophilic archaeon Archaeoglobus fulgidus. AlaDH catalyzed the reversible, NAD+-dependent deamination of l-alanine to pyruvate and NH4 +. NADP(H) did not serve as a coenzyme. The enzyme is a homodimer of 35 kDa per subunit. The K m values for l-alanine, NAD+, pyruvate, NADH, and NH4 + were estimated at 0.71, 0.60, 0.16, 0.02, and 17.3 mM, respectively. The A. fulgidus enzyme exhibited its highest activity at about 82°C (203 U/mg for reductive amination of pyruvate) yet still retained 30% of its maximum activity at 25°C. The thermostability of A. fulgidus AlaDH was increased by more than 10-fold by 1.5 M KCl to a half-life of 55 h at 90°C. At 25°C in the presence of this salt solution, the enzyme was ∼100% stable for more than 3 months. Closely related A. fulgidus AlaDH homologues were found in other archaea. On the basis of its amino acid sequence, A. fulgidus AlaDH is a member of the ornithine cyclodeaminase-μ-crystallin family of enzymes. Similar to the μ-crystallins, A. fulgidus AlaDH did not exhibit any ornithine cyclodeaminase activity. The recombinant human μ-crystallin was assayed for AlaDH activity, but no activity was detected. The novel A. fulgidus gene encoding AlaDH, AF1665, is designated ala.

scholarly journals The cloning and expression of the aroL gene from Escherichia coli K12. Purification and complete amino acid sequence of shikimate kinase II, the aroL-gene product

1986 ◽  
Vol 237 (2) ◽  
pp. 427-437 ◽  
Author(s):  
G Millar ◽  
A Lewendon ◽  
M G Hunter ◽  
J R Coggins
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Transcriptional Start Site ◽  
Cloning And Expression ◽  
Potential Operator ◽  
Gene Encoding ◽  
Shikimate Kinase ◽  
Complete Amino Acid Sequence ◽  
Escherichia Coli K12

The aroL gene encoding the enzyme shikimate kinase II was cloned from Escherichia coli K12. Construction of over-expressing strains permitted for the first time the purification to homogeneity of a monofunctional shikimate kinase. The complete amino acid sequence of shikimate kinase II was determined by a combined nucleotide and direct amino acid sequencing strategy. E. coli shikimate kinase II is a monomeric enzyme containing 173 amino acid residues with a calculated Mr 18,937. The amino acid sequence contains a region homologous with other kinases and ATP-requiring enzymes. Evidence is presented suggesting that the transcriptional start site of the aroL gene is located within a potential operator site.

scholarly journals Molecular cloning and characterization of the structural gene for the major iron-regulated protein expressed by Neisseria gonorrhoeae.

1990 ◽  
Vol 171 (5) ◽  
pp. 1535-1546 ◽  
Author(s):  
S A Berish ◽  
T A Mietzner ◽  
L W Mayer ◽  
C A Genco ◽  
B P Holloway ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Neisseria Gonorrhoeae ◽  
Biochemical Characterization ◽  
Iron Acquisition ◽  
Regulatory Region ◽  
Single Copy ◽  
Oligonucleotide Probes ◽  
Gene Encoding

This report describes the cloning and sequencing of the major iron-regulated protein (termed Fbp) of Neisseria gonorrhoeae strain F62. Attempts to identify recombinants expressing the Fbp using specific antibody proved unsuccessful. Therefore, an alternative cloning strategy using oligonucleotide probes derived from NH2-terminal and tryptic fragments of this protein was used to identify short fragments of the gene. Using this methodology, the gene encoding the precursor of Fbp was cloned on three separate overlapping fragments and sequenced, and the amino acid sequence was deduced. These data were unambiguously confirmed by the known NH2-terminal amino acid sequence and were supported by the sequences from tryptic fragments that lie outside of this region. Using oligonucleotide probes, we were unable to obtain clones encoding the potential regulatory region of this protein. Therefore, the technique of inverse polymerase chain reaction was used to amplify a fragment containing an additional 200 bp. This fragment was cloned and sequenced and found to contain a consensus ribosome binding site and potential -10 and -35 sequences. Hybridization analysis of genomic DNA from gonococcal strain F62 indicated that only a single copy of the Fbp gene exists per genome. These results complement the biochemical characterization of the Fbp expressed by gonococci and further suggest that it has a role in iron-acquisition.

scholarly journals Expression of the Methanobacterium thermoautotrophicum hpt Gene, Encoding Hypoxanthine (Guanine) Phosphoribosyltransferase, in Escherichia coli

1999 ◽  
Vol 181 (6) ◽  
pp. 1958-1962 ◽  
Author(s):  
Jørgen Sauer ◽  
Per Nygaard
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Methanobacterium Thermoautotrophicum ◽  
Recombinant Enzyme ◽  
Functional Complementation ◽  
Gene Encoding ◽  
Apparent Homogeneity ◽  
Hypoxanthine Guanine Phosphoribosyltransferase

ABSTRACT The hpt gene from the archaeon Methanobacterium thermoautotrophicum, encoding hypoxanthine (guanine) phosphoribosyltransferase, was cloned by functional complementation into Escherichia coli. The hpt-encoded amino acid sequence is most similar to adenine phosphoribosyltransferases, but the encoded enzyme has activity only with hypoxanthine and guanine. The synthesis of the recombinant enzyme is apparently limited by the presence of the rare arginine codons AGA and AGG and the rare isoleucine AUA codon on the hpt gene. The recombinant enzyme was purified to apparent homogeneity.

Characterization of two plasmid-encoded cefotaximases found in clinical Escherichia coli isolates: CTX-M-65 and a novel enzyme, CTX-M-87

2009 ◽  
Vol 58 (6) ◽  
pp. 811-815 ◽  
Author(s):  
Jun Yin ◽  
Jun Cheng ◽  
Zhen Sun ◽  
Ying Ye ◽  
Yu-Feng Gao ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Catalytic Activity ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Hydrolytic Activity ◽  
E Coli ◽  
High Affinity ◽  
Extended Spectrum ◽  
M 14

Three clinical strains of Escherichia coli (p168, p517 and p667) were collected in 2006 from three hospitals in Anhui Province (China). PCR and DNA sequencing revealed that E. coli p168 carried a novel extended-spectrum β-lactamase (ESBL), which was designated CTX-M-87. The extended-spectrum β-lactamase which was carried by E. coli p517 and E. coli p667 was previously named CTX-M-65. The deduced amino acid sequence of CTX-M-87, with pI 9.1, differed from that of CTX-M-14 by the substitutions Ala77→Val and Pro167→Leu. Like CTX-M-14, CTX-M-87 had a more potent hydrolytic activity against cefotaxime than against ceftazidime and had high affinity for cefuroxime and cefotaxime. These data show that mutations at position 167 in CTX-M do not always affect catalytic activity and substrate preference.

scholarly journals Cloning and Characterization of vuuA, a Gene Encoding the Vibrio vulnificus Ferric Vulnibactin Receptor

2000 ◽  
Vol 68 (2) ◽  
pp. 526-534 ◽  
Author(s):  
Athena C. D. Webster ◽  
Christine M. Litwin
Keyword(s):  
Transcriptional Regulation ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Outer Membrane ◽  
Dna Sequence ◽  
Vibrio Vulnificus ◽  
Membrane Receptors ◽  
Gene Encoding ◽  
Infant Mouse

ABSTRACT The ability of Vibrio vulnificus to acquire iron from the host has been shown to correlate with virulence. Many iron transport genes are regulated by iron, and in V. vulnificus, transcriptional regulation by iron depends on thefur gene. The N-terminal amino acid sequence of a 72-kDa iron-regulated outer membrane protein purified from a V. vulnificus fur mutant had 53% homology with the first 15 amino acids of the mature protein of the Vibrio choleraevibriobactin receptor, ViuA. In this report, we describe the cloning, DNA sequence, mutagenesis, and analysis of transcriptional regulation of the structural gene for VuuA, the vulnibactin receptor of V. vulnificus. Analysis of the DNA sequence of the vuuApromoter region demonstrated a sequence identical to the upstream Fur box of V. cholerae viuA. Northern blot analysis showed that the transcript was strongly regulated by iron. The amino acid sequence of VuuA was 74% identical to the sequence of V. choleraeViuA and was homologous to those of several TonB-dependent outer membrane receptors. An internal deletion of the V. vulnificus vuuA gene resulted in the loss of expression of the 72-kDa protein and the loss of the ability to use transferrin or vulnibactin as a source of iron. This mutant showed reduced virulence in an infant mouse model. Introduction of a plasmid containing the completeviuA coding sequence and 342 bp of upstream DNA into the mutant restored ferric vulnibactin and ferric transferrin utilization to the mutant.

scholarly journals Penicillin-Binding Protein 1 ofStaphylococcus aureus Is Essential for Growth

1998 ◽  
Vol 180 (10) ◽  
pp. 2759-2765 ◽  
Author(s):  
Akihito Wada ◽  
Haruo Watanabe
Keyword(s):  
Escherichia Coli ◽  
Staphylococcus Aureus ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Binding Protein ◽  
Ofstaphylococcus Aureus ◽  
Penicillin Binding Protein ◽  
Gene Encoding ◽  
Chromosomal Copy

ABSTRACT pbpA, a gene encoding penicillin-binding protein (PBP) 1 of Staphylococcus aureus, was cloned in anEscherichia coli MC1061 transformant which grew on a plate containing 512 μg of vancomycin per ml. This gene encodes a 744-amino-acid sequence which conserves three motifs of PBPs, SXXK, SXN, and KTG. The chromosomal copy of pbpA could be disrupted only when RN4220, a methicillin-sensitive S. aureus strain, had additional copies of pbpA in its episome. Furthermore, these episomal copies of pbpA could not be eliminated by an incompatible plasmid when the chromosomal copy of pbpA was disrupted beforehand. Based on these observations, we concluded that pbpA is essential for the growth of methicillin-sensitive S. aureus.

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