scholarly journals Identification and Analysis of Genes Involved in Anaerobic Toluene Metabolism by Strain T1: Putative Role of a Glycine Free Radical

1998 ◽  
Vol 64 (5) ◽  
pp. 1650-1656 ◽  
Author(s):  
Peter W. Coschigano ◽  
Thomas S. Wehrman ◽  
L. Y. Young
Keyword(s):  
Free Radical ◽  
Amino Acid ◽  
Molecular Mass ◽  
Cysteine Residue ◽  
Open Reading Frames ◽  
Site Directed Mutagenesis ◽  
Pyruvate Formate Lyase ◽  
Calculated Molecular Mass ◽  
Acid Protein ◽  
Amino Acid Protein

ABSTRACT The denitrifying strain T1 is able to grow with toluene serving as its sole carbon source. Two mutants which have defects in this toluene utilization pathway have been characterized. A clone has been isolated, and subclones which contain tutD and tutE, two genes in the T1 toluene metabolic pathway, have been generated. ThetutD gene codes for an 864-amino-acid protein with a calculated molecular mass of 97,600 Da. The tutE gene codes for a 375-amino-acid protein with a calculated molecular mass of 41,300 Da. Two additional small open reading frames have been identified, but their role is not known. The TutE protein has homology to pyruvate formate-lyase activating enzymes. The TutD protein has homology to pyruvate formate-lyase enzymes, including a conserved cysteine residue at the active site and a conserved glycine residue that is activated to a free radical in this enzyme. Site-directed mutagenesis of these two conserved amino acids shows that they are also essential for the function of TutD.

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.

scholarly journals Genetic Localization and Molecular Characterization of Two Key Genes (mitAB) Required for Biosynthesis of the Antitumor Antibiotic Mitomycin C

1999 ◽  
Vol 181 (7) ◽  
pp. 2199-2208 ◽  
Author(s):  
Yingqing Mao ◽  
Mustafa Varoglu ◽  
David H. Sherman
Keyword(s):  
Amino Acid ◽  
Mitomycin C ◽  
Site Directed Mutagenesis ◽  
Nucleotide Sequence Analysis ◽  
Resistance Locus ◽  
Antitumor Antibiotic ◽  
Carbamoyl Phosphate ◽  
Acid Protein ◽  
Key Genes ◽  
Amino Acid Protein

ABSTRACT Mitomycin C (MC) is an antitumor antibiotic derived biosynthetically from 3-amino-5-hydroxybenzoic acid (AHBA),d-glucosamine, and carbamoyl phosphate. A gene (mitA) involved in synthesis of AHBA has been identified and found to be linked to the MC resistance locus, mrd, inStreptomyces lavendulae. Nucleotide sequence analysis showed that mitA encodes a 388-amino-acid protein that has 71% identity (80% similarity) with the rifamycin AHBA synthase fromAmycolatopsis mediterranei, as well as with two additional AHBA synthases from related ansamycin antibiotic-producing microorganisms. Gene disruption and site-directed mutagenesis of theS. lavendulae chromosomal copy of mitAcompletely blocked the production of MC. The function ofmitA was confirmed by complementation of an S. lavendulae strain containing a K191A mutation in MitA with AHBA. A second gene (mitB) encoding a 272-amino-acid protein (related to a group of glycosyltransferases) was identified immediately downstream of mitA that upon disruption resulted in abrogation of MC synthesis. This work has localized a cluster of key genes that mediate assembly of the unique mitosane class of natural products.

scholarly journals Ribosomal proteins L34 and S29 of amphioxus Branchiostoma belcheri tsingtauense: cDNAs cloning and gene copy number.

2005 ◽  
Vol 52 (4) ◽  
pp. 857-862 ◽  
Author(s):  
Lina Liu ◽  
Shicui Zhang ◽  
Zhenhui Liu ◽  
Hongyan Li ◽  
Mei Liu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Ribosomal Proteins ◽  
Amino Acid Sequence Identity ◽  
Fruit Fly ◽  
Calculated Molecular Mass ◽  
Sequence Identity ◽  
Acid Protein ◽  
Branchiostoma Belcheri ◽  
Amino Acid Protein

The complete cDNA and deduced amino-acid sequences of ribosomal proteins L34 (AmphiL34) and S29 (AmphiS29) from the amphioxus Branchiostoma belcheri tsingtauense were identified in this study. The AmphiL34 cDNA is 435 nucleotides in length and encodes a 118 amino-acid protein with calculated molecular mass of 13.6 kDa. It shares 53.6-67.5% amino-acid sequence identity with its eukaryotic counterparts including human, mouse, rat, pig, frog, catfish, fruit fly, mosquito, armyworm, nematode and yeast. The AmphiS29 cDNA comprises 453 nucleotides and codes for a 56 amino-acid protein with a calculated molecular mass of 6.6 kDa. It shows 66.1-78.6% amino-acid sequence identity to eukaryotic S29 proteins from human, mouse, rat, pig, zebrafish, seahorse, fruit fly, nematode, sea hare and yeast. AmphiL34 contains a putative nucleolar localization signal, while AmphiS29 has a zinc finger-like domain. A phylogenetic tree deduced from the conserved sequences of AmphiL34 and AmphiS29 and other known counterparts indicates that the positions of AmphiL34/AmphiS29 are intermediate between the vertebrate and invertebrate L34/S29. Southern blot analysis demonstrates the presence of one copy of the L34 gene and 2-3 copies of the S29 gene in the genome of the amphioxus B. belcheri tsingtauense. This is in sharp contrast to the existence of 7-9 copies of the L34 gene and 14-17 copies of the S29 gene in the rat genome. These date suggest that housekeeping genes like AmphiL34 and AmphiS29 have undergone large-scale duplication in the chordate lineage.

scholarly journals New lnu(C) Gene Conferring Resistance to Lincomycin by Nucleotidylation in Streptococcus agalactiae UCN36

2005 ◽  
Vol 49 (7) ◽  
pp. 2716-2719 ◽  
Author(s):  
Adeline Achard ◽  
Corinne Villers ◽  
Vianney Pichereau ◽  
Roland Leclercq
Keyword(s):  
Amino Acid ◽  
Streptococcus Agalactiae ◽  
Open Reading Frames ◽  
Inverted Repeats ◽  
E Coli ◽  
Acid Protein ◽  
Total Dna ◽  
Plasmid Puc18 ◽  
Reading Frames ◽  
Amino Acid Protein

ABSTRACT Streptococcus agalactiae UCN36 was resistant to lincomycin (MIC = 16 μg/ml) but susceptible to clindamycin (MIC = 0.12 μg/ml) and erythromycin (MIC = 0.06 μg/ml). A 4-kb HindIII fragment was cloned from S. agalactiae UCN36 total DNA on plasmid pUC18 and introduced into Escherichia coli AG100A, where it conferred resistance to lincomycin. The sequence analysis of the fragment showed the presence of a 1,724-bp element delineated by imperfect inverted repeats (22 of 25 bp) and inserted in the operon for capsular synthesis of S. agalactiae UCN36. This element carried two open reading frames (ORF). The deduced amino acid sequence of the upstream ORF displayed similarity with transposases from anaerobes and IS1. The downstream ORF, lnu(C), encoded a 164-amino-acid protein with 26% to 27% identity with the LnuAN2, LnuA, and LnuA′ lincosamide nucleotidyltransferases reported for Bacteroides and Staphylococcus, respectively. Crude lysates of E. coli AG100A containing the cloned lnu(C) gene inactivated lincomycin and clindamycin in the presence of ATP and MgCl2. Mass spectrometry experiments demonstrated that the LnuC enzyme catalyzed adenylylation of lincomycin.

scholarly journals The pcd Gene Encoding Piperideine-6-Carboxylate Dehydrogenase Involved in Biosynthesis of α-Aminoadipic Acid Is Located in the Cephamycin Cluster ofStreptomyces clavuligerus

1998 ◽  
Vol 180 (17) ◽  
pp. 4753-4756 ◽  
Author(s):  
Francisco J. Pérez-Llarena ◽  
Antonio Rodríguez-García ◽  
Francisco J. Enguita ◽  
Juan F. Martín ◽  
Paloma Liras
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Streptomyces Clavuligerus ◽  
Open Reading Frames ◽  
Penicillin Binding Protein ◽  
Gene Encoding ◽  
Acid Protein ◽  
Terminal Amino ◽  
Protein Molecular Weight ◽  
Amino Acid Protein

ABSTRACT Three open reading frames (ORFs) have been located downstream ofcefE in the cephamycin C gene cluster of Streptomyces clavuligerus. ORF13 (pcd) encodes a 496-amino-acid protein (molecular weight [MW], 52,488) with an N-terminal amino acid sequence identical to that of pure piperideine-6-carboxylate dehydrogenase. ORF14 (cmcT) encodes a 523-amino-acid protein (MW, 54,232) analogous to Streptomyces proteins for efflux and resistance to antibiotics. ORF15 (pbp74) encodes a high molecular weight penicillin-binding protein (MW, 74,094).

scholarly journals Purification, Characterization, and Molecular Analysis of Thermostable Cellulases CelA and CelB fromThermotoga neapolitana

1998 ◽  
Vol 64 (12) ◽  
pp. 4774-4781 ◽  
Author(s):  
Jin-Duck Bok ◽  
Dinesh A. Yernool ◽  
Douglas E. Eveleigh
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Genomic Library ◽  
Cellulose Hydrolysis ◽  
Thermotoga Neapolitana ◽  
Glycosyl Hydrolases ◽  
Transglycosylation Activity ◽  
Acid Protein ◽  
High Level ◽  
Amino Acid Protein

ABSTRACT Two thermostable endocellulases, CelA and CelB, were purified fromThermotoga neapolitana. CelA (molecular mass, 29 kDa; pI 4.6) is optimally active at pH 6.0 at 95°C, while CelB (molecular mass, 30 kDa; pI 4.1) has a broader optimal pH range (pH 6.0 to 6.6) at 106°C. Both enzymes are characterized by a high level of activity (high V max value and low apparentKm value) with carboxymethyl cellulose; the specific activities of CelA and CelB are 1,219 and 1,536 U/mg, respectively. With p-nitrophenyl cellobioside theV max values of CelA and CelB are 69.2 and 18.4 U/mg, respectively, while the Km values are 0.97 and 0.3 mM, respectively. The major end products of cellulose hydrolysis, glucose and cellobiose, competitively inhibit CelA, and CelB. The Ki values for CelA are 0.44 M for glucose and 2.5 mM for cellobiose; the Ki values for CelB are 0.2 M for glucose and 1.16 mM for cellobiose. CelB preferentially cleaves larger cellooligomers, producing cellobiose as the end product; it also exhibits significant transglycosylation activity. This enzyme is highly thermostable and has half-lives of 130 min at 106°C and 26 min at 110°C. A single clone encoding thecelA and celB genes was identified by screening a T. neapolitana genomic library in Escherichia coli. The celA gene encodes a 257-amino-acid protein, while celB encodes a 274-amino-acid protein. Both proteins belong to family 12 of the glycosyl hydrolases, and the two proteins are 60% similar to each other. Northern blots of T. neapolitana mRNA revealed that celA andcelB are monocistronic messages, and both genes are inducible by cellobiose and are repressed by glucose.

scholarly journals Alkylation damage repair protein O6-alkylguanine-DNA alkyltransferase from the hyperthermophiles Aquifex aeolicus and Archaeoglobus fulgidus

2003 ◽  
Vol 375 (2) ◽  
pp. 449-455 ◽  
Author(s):  
Sreenivas KANUGULA ◽  
Anthony E. PEGG
Keyword(s):  
Amino Acid ◽  
Molecular Mass ◽  
Protein Denaturation ◽  
Alkylating Agents ◽  
Archaeoglobus Fulgidus ◽  
Aquifex Aeolicus ◽  
Acid Protein ◽  
Repair Protein ◽  
Dna Alkyltransferase ◽  
Amino Acid Protein

AGT (O6-alkylguanine DNA alkyltransferase) is an important DNA-repair protein that protects cells from killing and mutagenesis by alkylating agents. The AGT genes from two extremely thermophilic organisms, the bacterium Aquifex aeolicus and the archaeon Archaeoglobus fulgidus were PCR-derived and cloned into an expression vector. The nucleotide sequence of the Aq. aeolicus AGT encodes a 201-amino-acid protein with a molecular mass of 23000 Da and Ar. fulgidus AGT codes for a 147-amino-acid protein with a molecular mass of 16718 Da. The Aq. aeolicus and Ar. fulgidus AGTs were expressed at high levels in Escherichia coli fused to an N-terminal polyhistidine tag that allowed single-step isolation and purification by metal-affinity chromatography. Both AGTs formed inclusion bodies and were not soluble under native purification conditions. Therefore AGT isolation was performed under protein-denaturation conditions in the presence of 8.0 M urea. Soluble AGT was obtained by refolding the AGT in the presence of calf thymus DNA. Both AGTs were active in repairing O6-methylguanine and, at a lower rate, O4-methylthymine in DNA. They exhibited thermostability and optimum activity at high temperature. The thermostable AGTs, particularly that from Aq. aeolicus, were readily inactivated by the low-molecular-mass inhibitor O6-benzylguanine, which is currently in clinical trials to enhance cancer chemotherapy.

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 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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