scholarly journals Characterization of the Gallate Dioxygenase Gene: Three Distinct Ring Cleavage Dioxygenases Are Involved in Syringate Degradation by Sphingomonas paucimobilis SYK-6

2005 ◽  
Vol 187 (15) ◽  
pp. 5067-5074 ◽  
Author(s):  
Daisuke Kasai ◽  
Eiji Masai ◽  
Keisuke Miyauchi ◽  
Yoshihiro Katayama ◽  
Masao Fukuda
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Terminal Region ◽  
Ring Cleavage ◽  
Sphingomonas Paucimobilis ◽  
Acid Protein ◽  
Demethylation Pathway ◽  
Dioxygenase Gene ◽  
Amino Acid Protein

ABSTRACT Sphingomonas paucimobilis SYK-6 converts vanillate and syringate to protocatechuate (PCA) and 3-O-methylgallate (3MGA) in reactions with the tetrahydrofolate-dependent O-demethylases LigM and DesA, respectively. PCA is further degraded via the PCA 4,5-cleavage pathway, whereas 3MGA is metabolized via three distinct pathways in which PCA 4,5-dioxygenase (LigAB), 3MGA 3,4-dioxygenase (DesZ), and 3MGA O-demethylase (LigM) are involved. In the 3MGA O-demethylation pathway, LigM converts 3MGA to gallate, and the resulting gallate appears to be degraded by a dioxygenase other than LigAB or DesZ. Here, we isolated the gallate dioxygenase gene, desB, which encodes a 418-amino-acid protein with a molecular mass of 46,843 Da. The amino acid sequences of the N-terminal region (residues 1 to 285) and the C-terminal region (residues 286 to 418) of DesB exhibited ca. 40% and 27% identity with the sequences of the PCA 4,5-dioxygenase β and α subunits, respectively. DesB produced in Escherichia coli was purified and was estimated to be a homodimer (86 kDa). DesB specifically attacked gallate to generate 4-oxalomesaconate as the reaction product. The Km for gallate and the V max were determined to be 66.9 ± 9.3 μM and 42.7 ± 2.4 U/mg, respectively. On the basis of the analysis of various SYK-6 mutants lacking the genes involved in syringate degradation, we concluded that (i) all of the three-ring cleavage dioxygenases are involved in syringate catabolism, (ii) the pathway involving LigM and DesB plays an especially important role in the growth of SYK-6 on syringate, and (iii) DesB and LigAB are involved in gallate degradation.

scholarly journals The bglA Gene of Aspergillus kawachii Encodes Both Extracellular and Cell Wall-Bound β-Glucosidases

1999 ◽  
Vol 65 (12) ◽  
pp. 5546-5553 ◽  
Author(s):  
Kazuhiro Iwashita ◽  
Tatsuya Nagahara ◽  
Hitoshi Kimura ◽  
Makoto Takano ◽  
Hitoshi Shimoi ◽  
...  
Keyword(s):  
Cell Wall ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Amino Acid Sequences ◽  
Enzyme Assays ◽  
Partial Amino Acid Sequence ◽  
Reading Frame ◽  
Acid Protein ◽  
Aspergillus Kawachii ◽  
Amino Acid Protein

ABSTRACT We cloned the genomic DNA and cDNA of bglA, which encodes β-glucosidase in Aspergillus kawachii, based on a partial amino acid sequence of purified cell wall-bound β-glucosidase CB-1. The nucleotide sequence of the cloned bglA gene revealed a 2,933-bp open reading frame with six introns that encodes an 860-amino-acid protein. Based on the deduced amino acid sequence, we concluded that the bglA gene encodes cell wall-bound β-glucosidase CB-1. The amino acid sequence exhibited high levels of homology with the amino acid sequences of fungal β-glucosidases classified in subfamily B. We expressed the bglA cDNA inSaccharomyces cerevisiae and detected the recombinant β-glucosidase in the periplasm fraction of the recombinant yeast.A. kawachii can produce two extracellular β-glucosidases (EX-1 and EX-2) in addition to the cell wall-bound β-glucosidase.A. kawachii in which the bglA gene was disrupted produced none of the three β-glucosidases, as determined by enzyme assays and a Western blot analysis. Thus, we concluded that thebglA gene encodes both extracellular and cell wall-bound β-glucosidases in A. kawachii.

Heterologous Expression and Characterization of Tyrosine Decarboxylase from Enterococcus faecalis R612Z1 and Enterococcus faecium R615Z1

2014 ◽  
Vol 77 (4) ◽  
pp. 592-598 ◽  
Author(s):  
FANG LIU ◽  
WENJUAN XU ◽  
LIHUI DU ◽  
DAOYING WANG ◽  
YONGZHI ZHU ◽  
...  
Keyword(s):  
Amino Acid ◽  
Heterologous Expression ◽  
Molecular Mass ◽  
Enterococcus Faecalis ◽  
Enterococcus Faecium ◽  
Maximal Activity ◽  
Tyrosine Decarboxylase ◽  
Acid Protein ◽  
Amino Acid Protein

Tyrosine decarboxylase (TDC) is responsible for tyramine production and can catalyze phenylalanine to produce β-phenylethylamine. Enterococcus strains are a group of bacteria predominantly producing tyramine and β-phenylethylamine in water-boiled salted duck. In this study, the heterologous expression and characterization of two TDCs from Enterococcus faecalis R612Z1 (612TDC) and Enterococcus faecium R615Z1 (615TDC) were studied. The recombinant putative proteins of 612TDC and 615TDC were heterologously expressed in Escherichia coli. 612TDC is a 620-amino-acid protein with a molecular mass of 70.0 kDa, whereas 615TDC is a 625-amino-acid protein with a molecular mass of 70.3 kDa. Both 612TDC and 615TDC showed an optimum temperature of 25°C for the tyrosine and phenylalanine substrates. However, 612TDC revealed maximal activity at pH 5.5, whereas 615TDC revealed maximal activity at pH 6.0. Kinetic studies showed that 612TDC and 615TDC exhibited higher specificity for tyrosine than for phenylalanine. The catalysis abilities of both 612TDC and 615TDC for phenylalanine were restrained significantly with the increase in NaCl concentration, but this was not the case for tyrosine. This study revealed that the enzyme properties of the purified recombinant 612TDC and 615TDC were similar, although their amino acid sequences had 84% identity.

Amino acid sequence of penicillopepsin. I. Isolation and characterization of the chymotryptic peptides

1976 ◽  
Vol 54 (10) ◽  
pp. 872-884 ◽  
Author(s):  
Alexander Kurosky ◽  
Theo Hofmann
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Amino Acid Sequences ◽  
Acid Protease ◽  
Terminal Region ◽  
Isolation And Characterization

The amino acid sequences of 48 peptides obtained from a chymotryptic digest of the mould acid protease, penicillopepsin (EC 3.4.23.7), have been determined. These peptides established the sequences of 26 unique fragments of up to 28 residues in length. The 28-residue fragment was identified as the N-terminal region. The C-terminal region is represented by a 13-residue fragment. The amino acids contained in these fragments account for some 85% of the residues of the enzyme.

scholarly journals Molecular Characterization of OXA-20, a Novel Class D β-Lactamase, and Its Integron from Pseudomonas aeruginosa

1998 ◽  
Vol 42 (8) ◽  
pp. 2074-2083 ◽  
Author(s):  
Thierry Naas ◽  
Wladimir Sougakoff ◽  
Anne Casetta ◽  
Patrice Nordmann
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Amino Acid ◽  
Clavulanic Acid ◽  
Antibiotic Resistance Gene ◽  
Amino Acid Identity ◽  
Acid Identity ◽  
Class D ◽  
Acid Protein ◽  
Amino Acid Protein

ABSTRACT The Pseudomonas aeruginosa Mus clinical isolate produces OXA-18, a pI 5.5 class D extended-spectrum β-lactamase totally inhibited by clavulanic acid (L. N. Philippon, T. Naas, A.-T. Bouthors, V. Barakett, and P. Nordmann, Antimicrob. Agents Chemother. 41:2188–2195, 1997). A second β-lactamase was cloned, and the recombinant Escherichia coli clone pPL10 expressed a pI 7.4 β-lactamase which conferred high levels of amoxicillin and ticarcillin resistance and which was partially inhibited by clavulanic acid. The 2.5-kb insert from pPL10 was sequenced, and a 266-amino-acid protein (OXA-20) was deduced; this protein has low amino acid identity with most of the class D β-lactamases except OXA-2, OXA-15, and OXA-3 (75% amino acid identity with each). OXA-20 is a restricted-spectrum oxacillinase and is unusually inhibited by clavulanic acid. OXA-20 is a peculiar β-lactamase because its translation initiates with a TTG (leucine) codon, which is rarely used as a translational origin in bacteria. Exploration of the genetic environment of oxa20revealed the presence of the following integron features: (i) a second antibiotic resistance gene, aacA4; (ii) anintI1 gene; and (iii) two 59-base elements, each associated with either oxa20 or aacA4. This integron is peculiar because it lacks the 3′ conserved region, and therefore is not a sul1-associated integron like most of them, and because its 3′ end is located within tnpR, a gene involved in the transposition of Tn5393, a gram-negative transposon.P. aeruginosa Mus produces two novel and unrelated oxacillinases, OXA-18 and OXA-20, both of which are inhibited by clavulanic acid.

Isolation and characterization of the muscle-specific isoform of creatine kinase from the zebrafish, Danio rerio

2001 ◽  
Vol 79 (6) ◽  
pp. 779-782 ◽  
Author(s):  
Gregory Harder ◽  
Ross McGowan
Keyword(s):  
Creatine Kinase ◽  
Amino Acid ◽  
Danio Rerio ◽  
Cdna Sequence ◽  
Reading Frame ◽  
Isolation And Characterization ◽  
Acid Protein ◽  
Amino Acid Levels ◽  
Amino Acid Protein

We have isolated and characterized a cDNA sequence corresponding to the zebrafish muscle-specific isoform of creatine kinase. The sequence is 1552 bases in length and contains an open reading frame capable of producing a 381 amino acid protein. The sequence is very similar to muscle-specific creatine kinases isolated from other species at both the nucleotide and amino acid levels but contains some differences from a previously reported zebrafish clone.Key words: creatine kinase, muscle isoform, zebrafish, Danio rerio.

Characterization of the small endogenous plasmid of Halobacterium strain SB3 and its use in transformation of H. halobium

1989 ◽  
Vol 35 (1) ◽  
pp. 86-91 ◽  
Author(s):  
Neil R. Hackett ◽  
Shiladitya DasSarma
Keyword(s):  
Amino Acid ◽  
Nucleotide Sequence ◽  
Molecular Biology ◽  
Base Pair ◽  
Copy Number ◽  
Halobacterium Halobium ◽  
Acid Protein ◽  
Amino Acid Protein

To study the molecular biology of the halophilic archaebacterium Halobacterium halobium, the introduction of DNA engineered in vitro is desirable. As a first step in developing a cloning vector, the complete 1736 base pair nucleotide sequence of the natural, high copy number, Halobacterium plasmid pHSB1 has been determined. The plasmid was found to show homology to the small plasmids of Halobacterium strains GRB and GN101. Plasmid pHSB1 encodes a 317 amino acid protein of unknown function. The related halophile, H. halobium, could be transformed by pHSB1, demonstrating its utility as the basis of a cloning vector.Key words: archaebacteria, Halobacterium, plasmid.

scholarly journals Novel β-Lactamase Genes from Two Environmental Isolates of Vibrio harveyi

2000 ◽  
Vol 44 (5) ◽  
pp. 1309-1314 ◽  
Author(s):  
Jeanette W. P. Teo ◽  
Antonius Suwanto ◽  
Chit Laa Poh
Keyword(s):  
Amino Acid ◽  
Vibrio Harveyi ◽  
Amino Acid Sequences ◽  
Gene Probe ◽  
Reading Frame ◽  
Class A ◽  
Acid Protein ◽  
Low Levels ◽  
High Level ◽  
Amino Acid Protein

ABSTRACT Two ampicillin-resistant (Ampr) isolates ofVibrio harveyi, W3B and HB3, were obtained from the coastal waters of the Indonesian island of Java. Strain W3B was isolated from marine water near a shrimp farm in North Java while HB3 was from pristine seawater in South Java. In this study, novel β-lactamase genes from W3B (bla VHW-1) and HB3 (bla VHH-1) were cloned and their nucleotide sequences were determined. An open reading frame (ORF) of 870 bp encoding a deduced protein of 290 amino acids (VHW-1) was revealed for the bla gene of strain W3B while an ORF of 849 bp encoding a 283-amino-acid protein (VHH-1) was deduced forbla VHH-1. At the DNA level, genes for VHW-1 and VHH-1 have a 97% homology, while at the protein level they have a 91% homology of amino acid sequences. Neither gene sequence showed homology to any other β-lactamases in the databases. The deduced proteins were found to be class A β-lactamases bearing low levels of homology (<50%) to other β-lactamases of the same class. The highest level of identity was obtained with β-lactamases from Pseudomonas aeruginosa, i.e., PSE-1, PSE-4, and CARB-3, and Vibrio cholerae CARB-6. Our study showed that both strains W3B and HB3 possess an endogenous plasmid of approximately 60 kb in size. However, Southern hybridization analysis employingbla VHW-1 as a gene probe demonstrated that thebla gene was not located in the plasmid. A total of nine ampicillin-resistant V. harveyi strains, including W3B and HB3, were examined by pulsed-field gel electrophoresis ofNotI-digested genomic DNA. Despite a high level of intrastrain genetic diversity, thebla VHW-1 probe hybridized only to an 80- or 160-kb NotI genomic fragment in different isolates.

Molecular cloning and characterization of a novel liver-specific transport protein

1994 ◽  
Vol 107 (4) ◽  
pp. 1065-1072 ◽  
Author(s):  
G.D. Simonson ◽  
A.C. Vincent ◽  
K.J. Roberg ◽  
Y. Huang ◽  
V. Iwanij
Keyword(s):  
Amino Acid ◽  
Transmembrane Domain ◽  
Fold Increase ◽  
Liver Development ◽  
Cdna Expression Library ◽  
Expression Library ◽  
Transporter Family ◽  
Acid Protein ◽  
Amino Acid Protein

Monoclonal antibodies that specifically recognize a membrane component located on the sinusoidal domain of the hepatocyte have been used to screen a rat liver cDNA expression library and a clone encoding a novel transporter (NLT) protein has been identified. Analysis of the deduced 535 amino acid protein sequence indicates that it is unique, but shares the twelve-transmembrane domain hydrophathicity profile as well as the presence of transporter-specific amino acid motifs with bacterial and mammalian transporters. Since overall homology of NLT to known transporter genes is low (20-25% identity) it may represent a new subgroup within the transporter family of proteins. The NLT was characterized further with respect to its tissue distribution and its expression during liver development. A 2.1 kb transcript has been found in liver and at lower levels in kidney, but not in several other tissues tested. Studies on the developing liver indicate that NLT transcripts are present at a very low level from 19 through 21 gestation days with a 4- to 5-fold increase within two weeks after birth. Overall, we have cloned a novel transporter that is preferentially expressed in liver, is located on the sinusoidal domain of the plasma membrane and represents a marker for the late stage of liver development.

scholarly journals LASS3 (longevity assurance homologue 3) is a mainly testis-specific (dihydro)ceramide synthase with relatively broad substrate specificity

2006 ◽  
Vol 398 (3) ◽  
pp. 531-538 ◽  
Author(s):  
Yukiko Mizutani ◽  
Akio Kihara ◽  
Yasuyuki Igarashi
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Family Members ◽  
Fatty Acyl ◽  
Ceramide Synthase ◽  
Broad Substrate Specificity ◽  
Acid Protein ◽  
Amino Acid Protein

The LASS (longevity assurance homologue) family members are highly conserved from yeasts to mammals. Five mouse and human LASS family members, namely LASS1, LASS2, LASS4, LASS5 and LASS6, have been identified and characterized. In the present study we cloned two transcriptional variants of hitherto-uncharacterized mouse LASS3 cDNA, which encode a 384-amino-acid protein (LASS3) and a 419-amino-acid protein (LASS3-long). In vivo, [3H]dihydrosphingosine labelling and electrospray-ionization MS revealed that overproduction of either LASS3 isoform results in increases in several ceramide species, with some preference toward those having middle- to long-chain-fatty acyl-CoAs. A similar substrate preference was observed in an in vitro (dihydro)ceramide synthase assay. These results indicate that LASS3 possesses (dihydro)ceramide synthesis activity with relatively broad substrate specificity. We also found that, except for a weak display in skin, LASS3 mRNA expression is limited almost solely to testis, implying that LASS3 plays an important role in this gland.

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