scholarly journals Functional Characterization of Alanine Racemase fromSchizosaccharomyces pombe: a Eucaryotic Counterpart to Bacterial Alanine Racemase

2001 ◽  
Vol 183 (7) ◽  
pp. 2226-2233 ◽  
Author(s):  
Takuma Uo ◽  
Tohru Yoshimura ◽  
Naotaka Tanaka ◽  
Kaoru Takegawa ◽  
Nobuyoshi Esaki
Keyword(s):  
Escherichia Coli ◽  
Nitrogen Source ◽  
Functional Characterization ◽  
Open Reading Frame ◽  
Alanine Racemase ◽  
Sole Nitrogen Source ◽  
Recombinant Yeast ◽  
Reading Frame ◽  
Putative Protein

ABSTRACT Schizosaccharomyces pombe has an open reading frame, which we named alr1 +, encoding a putative protein similar to bacterial alanine racemase. We cloned thealr1 + gene in Escherichia coli and purified the gene product (Alr1p), with an M rof 41,590, to homogeneity. Alr1p contains pyridoxal 5′-phosphate as a coenzyme and catalyzes the racemization of alanine with apparentKm and V max values as follows: for l-alanine, 5.0 mM and 670 μmol/min/mg, respectively, and for d-alanine, 2.4 mM and 350 μmol/min/mg, respectively. The enzyme is almost specific to alanine, but l-serine and l-2-aminobutyrate are racemized slowly at rates 3.7 and 0.37% of that ofl-alanine, respectively. S. pombe usesd-alanine as a sole nitrogen source, but deletion of thealr1 + gene resulted in retarded growth on the same medium. This indicates that S. pombe has catabolic pathways for both enantiomers of alanine and that the pathway forl-alanine coupled with racemization plays a major role in the catabolism of d-alanine. Saccharomyces cerevisiae differs markedly from S. pombe: S. cerevisiae uses l-alanine but notd-alanine as a sole nitrogen source. Moreover,d-alanine is toxic to S. cerevisiae. However, heterologous expression of the alr1 + gene enabled S. cerevisiae to grow efficiently ond-alanine as a sole nitrogen source. The recombinant yeast was relieved from the toxicity of d-alanine.

scholarly journals Characterization of the chromosomal aminoglycoside 2'-N-acetyltransferase gene from Mycobacterium fortuitum.

1996 ◽  
Vol 40 (10) ◽  
pp. 2350-2355 ◽  
Author(s):  
J A Aínsa ◽  
C Martin ◽  
B Gicquel ◽  
R Gomez-Lus
Keyword(s):  
Dna Hybridization ◽  
Mycobacterium Smegmatis ◽  
Open Reading Frame ◽  
Mycobacterium Fortuitum ◽  
Aminoglycoside Resistance ◽  
Gene Encoding ◽  
Reading Frame ◽  
Putative Protein ◽  
Providencia Stuartii

A novel gene encoding an aminoglycoside 2'-N-acetyltransferase (AAC) was cloned from Mycobacterium fortuitum. DNA sequencing results identified an open reading frame that we have called aac(2')-Ib encoding a putative protein with a predicted molecular mass of 24,800 Da. The deduced AAC(2')-Ib protein showed homology to the AAC(2')-Ia from Providencia stuartii. This is the second member of a subfamily of AAC(2')-I enzymes to be identified. No homology was found with other acetyltransferases, including all of the AAC(3) and AAC(6') proteins. The aac(2')-Ib gene cloned in a mycobacterial plasmid and introduced in Mycobacterium smegmatis conferred resistance to gentamicin, tobramycin, dibekacin, netilmicin, and 6'-N-ethylnetilmicin. DNA hybridization with an intragenic probe of aac(2')-Ib showed that this gene was present in all 34 strains of M. fortuitum tested. The universal presence of the aac(2')-Ib gene in M. fortuitum was not correlated with any aminoglycoside resistance phenotype, suggesting that this gene may play a role in the secondary metabolism of the bacterium.

Cloning and characterization of theddsAgene encoding decaprenyl diphosphate synthase fromRhodobacter capsulatusB10

2006 ◽  
Vol 52 (12) ◽  
pp. 1141-1147 ◽  
Author(s):  
Xinyi Liu ◽  
Haizhen Wu ◽  
Jiang Ye ◽  
Qinsheng Yuan ◽  
Huizhan Zhang
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Rhodobacter Capsulatus ◽  
Open Reading Frame ◽  
High Similarity ◽  
Reading Frame ◽  
Endogenous Production ◽  
Genome Library

A decaprenyl diphosphate synthase gene (ddsA, GenBank accession No. DQ191802) was cloned from Rhodobacter capsulatus B10 by constructing and screening the genome library. An open reading frame of 1002 bp was revealed from sequence analysis. The deduced polypeptide consisted of 333 amino acids residues with an molecular mass of about 37 kDa. The DdsA protein contained the conserved amino acid sequence (DDXXD) of E-type polyprenyl diphosphate synthase and showed high similarity to others. In contrast, DdsA showed only 39% identity to a solanesyl diphosphate synthase cloned from R. capsulatus SB1003. DdsA was expressed successfully in Escherichia coli. Assaying the enzyme in vivo found it made E.coli synthesize UQ-10 in addition to the endogenous production UQ-8.Key words: ubiquinone, polyprenyl diphosphate synthase, gene expression, Rhodobacter capsulatus.

Cloning and functional characterization of a monoterpene synthase gene from Eleutherococcus trifoliatus

Holzforschung ◽  
2015 ◽  
Vol 69 (2) ◽  
pp. 163-171 ◽  
Author(s):  
Kuan-Feng Huang ◽  
Yi-Ru Lee ◽  
Yen-Hsueh Tseng ◽  
Sheng-Yang Wang ◽  
Fang-Hua Chu
Keyword(s):  
Functional Characterization ◽  
Open Reading Frame ◽  
Terpene Synthase ◽  
Gas Chromatography Mass Spectrometry ◽  
Degenerate Primers ◽  
Reading Frame ◽  
Monoterpene Synthase ◽  
Young Leaves ◽  
Rapid Amplification

AbstractEleutherococcus trifoliatusalso known as the three-leavedEleutherococcus, a member of the Araliaceae (ginseng) family, is commonly used in traditional Chinese medicine. Recently, many studies have demonstrated the bioactivities of the secondary metabolites inE. trifoliatus. In this study, a monoterpene synthase fromE. trifoliatushas been characterized. A pair of degenerate primers was designed and a fragment with conserved region of terpene synthase (TPS) was obtained. After 5′- and 3′-rapid amplification of cDNA ends (RACE), the full-length cDNA was obtained. The gene designatedEtLIMcontains an open reading frame of 1752 bp with a predicated molecular mass of 67.3 kDa. It was expressed in young leaves, stems, and drupes. The product ofEtLIMhas been identified by gas chromatography/mass spectrometry (GC/MS) as limonene.

scholarly journals Molecular Characterization of a Glucokinase with Broad Hexose Specificity from Bacillus sphaericus Strain C3-41

2007 ◽  
Vol 73 (11) ◽  
pp. 3581-3586 ◽  
Author(s):  
Bei Han ◽  
Haizhou Liu ◽  
Xiaomin Hu ◽  
Yajun Cai ◽  
Dasheng Zheng ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Genetic Improvement ◽  
Biochemical Analysis ◽  
Additional Data ◽  
Bacillus Sphaericus ◽  
Phosphoglucose Isomerase ◽  
Open Reading Frame ◽  
Reading Frame ◽  
Encoding Gene

ABSTRACT Bacillus sphaericus cannot metabolize sugar since it lacks several of the enzymes necessary for glycolysis. Our results confirmed the presence of a glucokinase-encoding gene, glcK, and a phosphofructokinase-encoding gene, pfk, on the bacterial chromosome and expression of glucokinase during vegetative growth of B. sphaericus strains. However, no phosphoglucose isomerase gene (pgi) or phosphoglucose isomerase enzyme activity was detected in these strains. Furthermore, one glcK open reading frame was cloned from B. sphaericus strain C3-41 and then expressed in Escherichia coli. Biochemical analysis revealed that this gene encoded a protein with a molecular mass of 33 kDa and that the purified recombinant glucokinase had K m values of 0.52 and 0.31 mM for ATP and glucose, respectively. It has been proved that this ATP-dependent glucokinase can also phosphorylate fructose and mannose, and sequence alignment of the glcK gene indicated that it belongs to the ROK protein family. It is postulated that the absence of the phosphoglucose isomerase-encoding gene pgi in B. sphaericus might be one of the reasons for the inability of this bacterium to metabolize carbohydrates. Our findings provide additional data that further elucidate the specific metabolic pathway and could be used for genetic improvement of B. sphaericus.

scholarly journals Antigenic Characterization of ORF2 and ORF3 Proteins of Hepatitis E Virus (HEV)

Viruses ◽  
2021 ◽  
Vol 13 (7) ◽  
pp. 1385
Author(s):  
Giulia Pezzoni ◽  
Lidia Stercoli ◽  
Eleonora Pegoiani ◽  
Emiliana Brocchi
Keyword(s):  
Hepatitis E Virus ◽  
Hepatitis E ◽  
Recombinant Antigen ◽  
Open Reading Frame ◽  
Reading Frame ◽  
E Coli ◽  
Antigenic Characterization ◽  
Antigenic Properties ◽  
Open Reading Frame 2

To evaluate the antigenic properties of Hepatitis E Virus (HEV) Open Reading Frame 2 and 3 (ORF2 and ORF3) codified proteins, we expressed different portions of ORF2 and the entire ORF3 in E. coli, a truncated ORF2, was also expressed in baculovirus. A panel of 37 monoclonal antibodies (MAbs) was raised against ORF2 (1–660 amino acids) and MAbs were mapped and characterized using the ORF2 expressed portions. Selected HEV positive and negative swine sera were used to evaluate ORF2 and ORF3 antigens’ immunogenicity. The MAbs were clustered in six groups identifying six antigenic regions along the ORF2. Only MAbs binding to the sixth ORF2 antigenic region (394–608 aa) were found to compete with HEV positive sera and efficiently catch the recombinant antigen expressed in baculovirus. The ORF2 portion from 394–608 aa demonstrated to include most immunogenic epitopes with 85% of HEV positive swine sera reacting against the region from 461–544 aa. Only 5% of the selected HEV sera reacted against the ORF3 antigen.

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