scholarly journals Characterization of a Bordetella pertussisDiaminopimelate (DAP) Biosynthesis Locus Identifies dapC, a Novel Gene Coding for anN-Succinyl-l,l-DAP Aminotransferase

2000 ◽  
Vol 182 (13) ◽  
pp. 3626-3631 ◽  
Author(s):  
Thilo M. Fuchs ◽  
Boris Schneider ◽  
Karin Krumbach ◽  
Lothar Eggeling ◽  
Roy Gross
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Binding Motif ◽  
Aminotransferase Activity ◽  
Phosphate Binding ◽  
E Coli ◽  
Gene Coding ◽  
Sequence Similarities

ABSTRACT The functional complementation of two Escherichia colistrains defective in the succinylase pathway ofmeso-diaminopimelate (meso-DAP) biosynthesis with a Bordetella pertussis gene library resulted in the isolation of a putative dap operon containing three open reading frames (ORFs). In line with the successful complementation of the E. coli dapD and dapE mutants, the deduced amino acid sequences of two ORFs revealed significant sequence similarities with the DapD and DapE proteins of E. coli and many other bacteria which exhibit tetrahydrodipicolinate succinylase and N-succinyl-l,l-DAP desuccinylase activity, respectively. The first ORF within the operon showed significant sequence similarities with transaminases and contains the characteristic pyridoxal-5′-phosphate binding motif. Enzymatic studies revealed that this ORF encodes a protein withN-succinyl-l,l-DAP aminotransferase activity converting N-succinyl-2-amino-6-ketopimelate, the product of the succinylase DapD, to N-succinyl-l,l-DAP, the substrate of the desuccinylase DapE. Therefore, this gene appears to encode the DapC protein of B. pertussis. Apart from the pyridoxal-5′-phosphate binding motif, the DapC protein does not show further amino acid sequence similarities with the only other known enzyme with N-succinyl-l,l-DAP aminotransferase activity, ArgD of E. coli.

scholarly journals Isolation and Characterization of Genes Responsible for Naphthalene Degradation from Thermophilic Naphthalene Degrader, Geobacillus sp. JF8

2019 ◽  
Vol 8 (1) ◽  
pp. 44 ◽  
Author(s):  
Daisuke Miyazawa ◽  
Le Thi Ha Thanh ◽  
Akio Tani ◽  
Masaki Shintani ◽  
Nguyen Hoang Loc ◽  
...  
Keyword(s):  
Amino Acid ◽  
Thermophilic Bacterium ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Pcr Analysis ◽  
Naphthalene Degradation ◽  
Isolation And Characterization ◽  
Dihydrodiol Dehydrogenase ◽  
Reading Frames

Geobacillus sp. JF8 is a thermophilic biphenyl and naphthalene degrader. To identify the naphthalene degradation genes, cis-naphthalene dihydrodiol dehydrogenase was purified from naphthalene-grown cells, and its N-terminal amino acid sequence was determined. Using a DNA probe encoding the N-terminal region of the dehydrogenase, a 10-kb DNA fragment was isolated. Upstream of nahB, a gene for dehydrogenase, there were two open reading frames which were designated as nahAc and nahAd, respectively. The products of nahAc and nahAd were predicted to be alpha and beta subunit of ring-hydroxylating dioxygenases, respectively. Phylogenetic analysis of amino acid sequences of NahB indicated that it did not belong to the cis-dihydrodiol dehydrogenase group that includes those of classical naphthalene degradation pathways. Downstream of nahB, four open reading frames were found, and their products were predicted as meta-cleavage product hydrolase, monooxygenase, dehydrogenase, and gentisate 1,2-dioxygenase, respectively. A reverse transcriptase-PCR analysis showed that transcription of nahAcAd was induced by naphthalene. These findings indicate that we successfully identified genes involved in the upper pathway of naphthalene degradation from a thermophilic bacterium.

scholarly journals Characterization of redox properties of FAD cofactor of Thermotoga maritima thioredoxin reductase

Chemija ◽  
2020 ◽  
Vol 31 (3) ◽  
Author(s):  
Benjaminas Valiauga ◽  
Nicolas Rouhier ◽  
Jean-Pierre Jacquot ◽  
Narimantas Čėnas
Keyword(s):  
Amino Acid ◽  
Redox Potential ◽  
Thermotoga Maritima ◽  
Thioredoxin Reductase ◽  
Amino Acid Sequences ◽  
Redox Properties ◽  
Redox Couple ◽  
E Coli ◽  
Standard Redox Potential

The fluorescence properties of FAD of Thermotoga maritima thioredoxin reductase (TmTR), taken together with the amino acid sequences and structures of similar TRs, are consistent with the interdomain rotation in the catalysis of TmTR. The standard redox potential of FAD of TmTR, –0.230 V, determined by the reactions with 3-acetylpyridine adenine dinucleotide (APAD+/APADH) redox couple, is close to that of E. coli TR. During the reduction of duroquinone with TmTR, the transient formation of neutral FAD semiquinone, and, possibly, FADH2–NAD+ complex was observed. This shows that in spite of obligatory twoelectron (hydride)-transfer between NADH and physiological disulfide oxidants, the FAD cofactor of TmTR may exist under a stable semiquinone form.

Isolation and characterization of two Saccharomyces cerevisiae genes encoding homologs of the bacterial HexA and MutS mismatch repair proteins.

Genetics ◽  
1992 ◽  
Vol 132 (4) ◽  
pp. 963-973 ◽  
Author(s):  
R A Reenan ◽  
R D Kolodner
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Amino Acid ◽  
Mismatch Repair ◽  
Dna Sequences ◽  
Amino Acid Sequences ◽  
Binding Motif ◽  
E Coli ◽  
Conserved Regions ◽  
Isolation And Characterization ◽  
Degenerate Oligonucleotide

Abstract Homologs of the Escherichia coli (mutL, S and uvrD) and Streptococcus pneumoniae (hexA, B) genes involved in mismatch repair are known in several distantly related organisms. Degenerate oligonucleotide primers based on conserved regions of E. coli MutS protein and its homologs from Salmonella typhimurium, S. pneumoniae and human were used in the polymerase chain reaction (PCR) to amplify and clone mutS/hexA homologs from Saccharomyces cerevisiae. Two DNA sequences were amplified whose deduced amino acid sequences both shared a high degree of homology with MutS. These sequences were then used to clone the full-length genes from a yeast genomic library. Sequence analysis of the two MSH genes (MSH = mutS homolog), MSH1 and MSH2, revealed open reading frames of 2877 bp and 2898 bp. The deduced amino acid sequences predict polypeptides of 109.3 kD and 109.1 kD, respectively. The overall amino acid sequence identity with the E. coli MutS protein is 28.6% for MSH1 and 25.2% for MSH2. Features previously found to be shared by MutS homologs, such as the nucleotide binding site and the helix-turn-helix DNA binding motif as well as other highly conserved regions whose function remain unknown, were also found in the two yeast homologs. Evidence presented in this and a companion study suggest that MSH1 is involved in repair of mitochondrial DNA and that MSH2 is involved in nuclear DNA repair.

LlaFI, a Type III Restriction and Modification System in Lactococcus lactis

1999 ◽  
Vol 65 (2) ◽  
pp. 686-693 ◽  
Author(s):  
Ping Su ◽  
Heejeong Im ◽  
Hsiaoling Hsieh ◽  
Simon Kang’A ◽  
Noel W. Dunn
Keyword(s):  
Amino Acid ◽  
Lactococcus Lactis ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Homology Search ◽  
Binding Motif ◽  
Significant Similarity ◽  
Modification System ◽  
Type Iii ◽  
Central Regions

ABSTRACT We describe a type III restriction and modification (R/M) system,LlaFI, in Lactococcus lactis. LlaFI is encoded by a 12-kb native plasmid, pND801, harbored in L. lactisLL42-1. Sequencing revealed two adjacent open reading frames (ORFs). One ORF encodes a 680-amino-acid polypeptide, and this ORF is followed by a second ORF which encodes an 873-amino-acid polypeptide. The two ORFs appear to be organized in an operon. A homology search revealed that the two ORFs exhibited significant similarity to type III restriction (Res) and modification (Mod) subunits. The complete amino acid sequence of the Mod subunit of LlaFI was aligned with the amino acid sequences of four previously described type III methyltransferases. Both the N-terminal regions and the C-terminal regions of the Mod proteins are conserved, while the central regions are more variable. An S-adenosyl methionine (Ado-Met) binding motif (present in all adenine methyltransferases) was found in the N-terminal region of the Mod protein. The seven conserved helicase motifs found in the previously described type III R/M systems were found at the same relative positions in the LlaFI Res sequence.LlaFI has cofactor requirements for activity that are characteristic of the previously described type III enzymes. ATP and Mg2+ are required for endonucleolytic activity; however, the activity is not strictly dependent on the presence of Ado-Met but is stimulated by it. To our knowledge, this is the first type III R/M system that has been characterized not just in lactic acid bacteria but also in gram-positive bacteria.

scholarly journals Molecular Cloning and Characterization of the Gene Coding for the Aerobic Azoreductase from Xenophilus azovorans KF46F

2002 ◽  
Vol 68 (8) ◽  
pp. 3948-3955 ◽  
Author(s):  
Silke Blümel ◽  
Hans-Joachim Knackmuss ◽  
Andreas Stolz
Keyword(s):  
Amino Acid ◽  
Azo Dyes ◽  
Sole Source ◽  
Amino Acid Sequences ◽  
Resting Cells ◽  
E Coli ◽  
Amino Terminal ◽  
Cell Extracts ◽  
Gene Coding

ABSTRACT The gene coding for an aerobic azoreductase was cloned from Xenophilus azovorans KF46F (formerly Pseudomonas sp. strain KF46F), which was previously shown to grow with the carboxylated azo compound 1-(4′-carboxyphenylazo)-2-naphthol (carboxy-Orange II) as the sole source of carbon and energy. The deduced amino acid sequence encoded a protein with a molecular weight of 30,278 and showed no significant homology to amino acid sequences currently deposited at the relevant data bases. A presumed NAD(P)H-binding site was identified in the amino-terminal region of the azoreductase. The enzyme was heterologously expressed in Escherichia coli and the azoreductase activities of resting cells and cell extracts were compared. The results suggested that whole cells of the recombinant E. coli strains were unable to take up sulfonated azo dyes and therefore did not show in vivo azoreductase activity. The turnover of several industrially relevant azo dyes by cell extracts from the recombinant E. coli strain was demonstrated.

scholarly journals Cloning, Overexpression, and Mutagenesis of theSporobolomyces salmonicolor AKU4429 Gene Encoding a New Aldehyde Reductase, Which Catalyzes the Stereoselective Reduction of Ethyl 4-Chloro-3-Oxobutanoate to Ethyl (S)-4-Chloro-3-Hydroxybutanoate

1999 ◽  
Vol 65 (12) ◽  
pp. 5207-5211 ◽  
Author(s):  
Keiko Kita ◽  
Takanobu Fukura ◽  
Koh-Ichi Nakase ◽  
Kenji Okamoto ◽  
Hideshi Yanase ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Substrate Inhibition ◽  
Amino Acid Sequences ◽  
Yeast Cells ◽  
Binding Motif ◽  
Aldehyde Reductase ◽  
Amino Acid Residues ◽  
Gene Encoding ◽  
E Coli

ABSTRACT We cloned and sequenced the gene encoding an NADPH-dependent aldehyde reductase (ARII) in Sporobolomyces salmonicolorAKU4429, which reduces ethyl 4-chloro-3-oxobutanoate (4-COBE) to ethyl (S)-4-chloro-3-hydroxybutanoate. The ARII gene is 1,032 bp long, is interrupted by four introns, and encodes a 37,315-Da polypeptide. The deduced amino acid sequence exhibited significant levels of similarity to the amino acid sequences of members of the mammalian 3β-hydroxysteroid dehydrogenase–plant dihydroflavonol 4-reductase superfamily but not to the amino acid sequences of members of the aldo-keto reductase superfamily or to the amino acid sequence of an aldehyde reductase previously isolated from the same organism (K. Kita, K. Matsuzaki, T. Hashimoto, H. Yanase, N. Kato, M. C.-M. Chung, M. Kataoka, and S. Shimizu, Appl. Environ. Microbiol. 62:2303–2310, 1996). The ARII protein was overproduced inEscherichia coli about 2,000-fold compared to the production in the original yeast cells. The enzyme expressed inE. coli was purified to homogeneity and had the same catalytic properties as ARII purified from S. salmonicolor. To examine the contribution of the dinucleotide-binding motif G19-X-X-G22-X-X-A25, which is located in the N-terminal region, during ARII catalysis, we replaced three amino acid residues in the motif and purified the resulting mutant enzymes. Substrate inhibition of the G19→A and G22→A mutant enzymes by 4-COBE did not occur. The A25→G mutant enzyme could reduce 4-COBE when NADPH was replaced by an equimolar concentration of NADH.

scholarly journals Sequence Analysis and Initial Characterization of Two Isozymes of Hydroxylaminobenzene Mutase from Pseudomonas pseudoalcaligenes JS45

2000 ◽  
Vol 66 (7) ◽  
pp. 2965-2971 ◽  
Author(s):  
John K. Davis ◽  
George C. Paoli ◽  
Zhongqi He ◽  
Lloyd J. Nadeau ◽  
Charles C. Somerville ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Genomic Library ◽  
Amino Acid Sequences ◽  
Open Reading Frames ◽  
Pcr Analysis ◽  
Pseudomonas Pseudoalcaligenes ◽  
E Coli ◽  
Specific Activities ◽  
Reading Frames

ABSTRACT Pseudomonas pseudoalcaligenes JS45 grows on nitrobenzene by a partially reductive pathway in which the intermediate hydroxylaminobenzene is enzymatically rearranged to 2-aminophenol by hydroxylaminobenzene mutase (HAB mutase). The properties of the enzyme, the reaction mechanism, and the evolutionary origin of the gene(s) encoding the enzyme are unknown. In this study, two open reading frames (habA and habB), each encoding an HAB mutase enzyme, were cloned from a P. pseudoalcaligenes JS45 genomic library and sequenced. The open reading frames encoding HabA and HabB are separated by 2.5 kb and are divergently transcribed. The deduced amino acid sequences of HabA and HabB are 44% identical. The HAB mutase specific activities in crude extracts of Escherichia coli clones synthesizing either HabA or HabB were similar to the specific activities of extracts of strain JS45 grown on nitrobenzene. HAB mutase activity in E. coli extracts containing HabB withstood heating at 85�C for 10 min, but extracts containing HabA were inactivated when they were heated at temperatures above 60�C. HAB mutase activity in extracts of P. pseudoalcaligenesJS45 grown on nitrobenzene exhibited intermediate temperature stability. Although both the habA gene and thehabB gene conferred HAB mutase activity when they were separately cloned and expressed in E. coli, reverse transcriptase PCR analysis indicated that only habA is transcribed in P. pseudoalcaligenes JS45. A mutant strain derived from strain JS45 in which the habA gene was disrupted was unable to grow on nitrobenzene, which provided physiological evidence that HabA is involved in the degradation of nitrobenzene. A strain in which habB was disrupted grew on nitrobenzene. Gene Rv3078 of Mycobacterium tuberculosisH37Rv encodes a protein whose deduced amino acid sequence is 52% identical to the HabB amino acid sequence. E. colicontaining M. tuberculosis gene Rv3078 cloned into pUC18 exhibited low levels of HAB mutase activity. Sequences that exhibit similarity to transposable element sequences are present between habA and habB, as well as downstream ofhabB, which suggests that horizontal gene transfer resulted in acquisition of one or both of the hab genes.

scholarly journals Identification and Characterization of thefis Operon in Enteric Bacteria

1998 ◽  
Vol 180 (22) ◽  
pp. 5932-5946 ◽  
Author(s):  
Michael B. Beach ◽  
Robert Osuna
Keyword(s):  
Amino Acid ◽  
Genomic Sequence ◽  
Erwinia Carotovora ◽  
Enteric Bacteria ◽  
Integration Host Factor ◽  
Amino Acid Sequences ◽  
Reading Frame ◽  
E Coli

ABSTRACT The small DNA binding protein Fis is involved in several different biological processes in Escherichia coli. It has been shown to stimulate DNA inversion reactions mediated by the Hin family of recombinases, stimulate integration and excision of phage λ genome, regulate the transcription of several different genes including those of stable RNA operons, and regulate the initiation of DNA replication at oriC. fis has also been isolated from Salmonella typhimurium, and the genomic sequence of Haemophilus influenzae reveals its presence in this bacteria. This work extends the characterization of fis to other organisms. Very similar fis operon structures were identified in the enteric bacteria Klebsiella pneumoniae, Serratia marcescens, Erwinia carotovora, andProteus vulgaris but not in several nonenteric bacteria. We found that the deduced amino acid sequences for Fis are 100% identical in K. pneumoniae, S. marcescens,E. coli, and S. typhimurium and 96 to 98% identical when E. carotovora and P. vulgaris Fis are considered. The deduced amino acid sequence forH. influenzae Fis is about 80% identical and 90% similar to Fis in enteric bacteria. However, in spite of these similarities, the E. carotovora, P. vulgaris, and H. influenzae Fis proteins are not functionally identical. An open reading frame (ORF1) precedingfis in E. coli is also found in all these bacteria, and their deduced amino acid sequences are also very similar. The sequence preceding ORF1 in the enteric bacteria showed a very strong similarity to the E. coli fis P region from −53 to +27 and the region around −116 containing an ihfbinding site. Both β-galactosidase assays and primer extension assays showed that these regions function as promoters in vivo and are subject to growth phase-dependent regulation. However, their promoter strengths vary, as do their responses to Fis autoregulation and integration host factor stimulation.

scholarly journals Roles of the C-Terminal Amino Acids of Non-Hexameric Helicases: Insights from Escherichia coli UvrD

2021 ◽  
Vol 22 (3) ◽  
pp. 1018
Author(s):  
Hiroaki Yokota
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Single Molecule ◽  
Underlying Mechanism ◽  
Amino Acid Sequences ◽  
E Coli ◽  
X Ray Crystallography ◽  
Terminal Amino

Helicases are nucleic acid-unwinding enzymes that are involved in the maintenance of genome integrity. Several parts of the amino acid sequences of helicases are very similar, and these quite well-conserved amino acid sequences are termed “helicase motifs”. Previous studies by X-ray crystallography and single-molecule measurements have suggested a common underlying mechanism for their function. These studies indicate the role of the helicase motifs in unwinding nucleic acids. In contrast, the sequence and length of the C-terminal amino acids of helicases are highly variable. In this paper, I review past and recent studies that proposed helicase mechanisms and studies that investigated the roles of the C-terminal amino acids on helicase and dimerization activities, primarily on the non-hexermeric Escherichia coli (E. coli) UvrD helicase. Then, I center on my recent study of single-molecule direct visualization of a UvrD mutant lacking the C-terminal 40 amino acids (UvrDΔ40C) used in studies proposing the monomer helicase model. The study demonstrated that multiple UvrDΔ40C molecules jointly participated in DNA unwinding, presumably by forming an oligomer. Thus, the single-molecule observation addressed how the C-terminal amino acids affect the number of helicases bound to DNA, oligomerization, and unwinding activity, which can be applied to other helicases.

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.

Sign in / Sign up

Export Citation Format

Share Document