scholarly journals Two Distinct Alcohol Dehydrogenases Participate in Butane Metabolism by Pseudomonas butanovora

2002 ◽  
Vol 184 (7) ◽  
pp. 1916-1924 ◽  
Author(s):  
Alisa S. Vangnai ◽  
Daniel J. Arp ◽  
Luis A. Sayavedra-Soto
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Alcohol Dehydrogenase ◽  
Primary Alcohol ◽  
Leader Sequence ◽  
Butane Oxidation ◽  
Oxidation Activity ◽  
Alcohol Dehydrogenases ◽  
Cell Extracts ◽  
Pseudomonas Butanovora

ABSTRACT The involvement of two primary alcohol dehydrogenases, BDH and BOH, in butane utilization in Pseudomonas butanovora (ATCC 43655) was demonstrated. The genes coding for BOH and BDH were isolated and characterized. The deduced amino acid sequence of BOH suggests a 67-kDa alcohol dehydrogenase containing pyrroloquinoline quinone (PQQ) as cofactor and in the periplasm (29-residue leader sequence). The deduced amino acid sequence of BDH is consistent with a 70.9-kDa, soluble, periplasmic (37-residue leader sequence) alcohol dehydrogenase containing PQQ and heme c as cofactors. BOH and BDH mRNAs were induced whenever the cell's 1-butanol oxidation activity was induced. When induced with butane, the gene for BOH was expressed earlier than the gene for BDH. Insertional disruption of bdh or boh affected adversely, but did not eliminate, butane utilization by P. butanovora. The P. butanovora mutant with both genes boh and bdh inactivated was unable to grow on butane or 1-butanol. These cells, when grown in citrate and incubated in butane, developed butane oxidation capability and accumulated 1-butanol. The enzyme activity of BOH was characterized in cell extracts of the P. butanovora strain with bdh disrupted. Unlike BDH, BOH oxidized 2-butanol. The results support the involvement of two distinct NAD+-independent, PQQ-containing alcohol dehydrogenases, BOH (a quinoprotein) and BDH (a quinohemoprotein), in the butane oxidation pathway of P. butanovora.

scholarly journals Catalytic and Molecular Properties of the Quinohemoprotein Tetrahydrofurfuryl Alcohol Dehydrogenase from Ralstonia eutropha Strain Bo

2001 ◽  
Vol 183 (6) ◽  
pp. 1954-1960 ◽  
Author(s):  
Grit Zarnt ◽  
Thomas Schräder ◽  
Jan R. Andreesen
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Alcohol Dehydrogenase ◽  
Tertiary Structure ◽  
Ralstonia Eutropha ◽  
Signal Sequence ◽  
Substrate Inhibition ◽  
Catalytic Efficiency ◽  
Gene Encoding

ABSTRACT The quinohemoprotein tetrahydrofurfuryl alcohol dehydrogenase (THFA-DH) from Ralstonia eutropha strain Bo was investigated for its catalytic properties. The apparentk cat/Km andK i values for several substrates were determined using ferricyanide as an artificial electron acceptor. The highest catalytic efficiency was obtained with n-pentanol exhibiting a k cat/Km value of 788 × 104 M−1 s−1. The enzyme showed substrate inhibition kinetics for most of the alcohols and aldehydes investigated. A stereoselective oxidation of chiral alcohols with a varying enantiomeric preference was observed. Initial rate studies using ethanol and acetaldehyde as substrates revealed that a ping-pong mechanism can be assumed for in vitro catalysis of THFA-DH. The gene encoding THFA-DH from R. eutropha strain Bo (tfaA) has been cloned and sequenced. The derived amino acid sequence showed an identity of up to 67% to the sequence of various quinoprotein and quinohemoprotein dehydrogenases. A comparison of the deduced sequence with the N-terminal amino acid sequence previously determined by Edman degradation analysis suggested the presence of a signal sequence of 27 residues. The primary structure of TfaA indicated that the protein has a tertiary structure quite similar to those of other quinoprotein dehydrogenases.

scholarly journals The complete amino acid sequence of three alcohol dehydrogenase alleloenzymes (AdhN-11, AdhS and AdhUF) from the fruitfly Drosophila melanogaster

1980 ◽  
Vol 187 (3) ◽  
pp. 875-883 ◽  
Author(s):  
D R Thatcher
Keyword(s):  
Drosophila Melanogaster ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Alcohol Dehydrogenase ◽  
Aspartic Acid ◽  
Structure Prediction ◽  
Secondary Structure Prediction ◽  
British Library ◽  
Horse Liver ◽  
And Staphylococcus Aureus

The sequence of three alcohol dehydrogenase alleloenzymes from the fruitfly Drosophila melanogaster has been determined by the sequencing of peptides produced by trypsin, chymotrypsin, thermolysin, pepsin and Staphylococcus aureus-V8-proteinase digestion. The amino acid sequence shows no obvious homology with the published sequences of the horse liver and yeast enzymes, and secondary structure prediction suggests that the nucleotide-binding domain is located in the N-terminal half of the molecule. The amino acid substitutions between AdhN-11 (a point mutation of AdhF), AdhS and AdhUF alleloenzymes were identified. AdhN-11 alcohol dehydrogenase differed from the other two by a glycine-14-(AdhS and AdhUF)-to-aspartic acid substitution, the AdhS enzyme from AdhN-11 and AdhUF enzymes by a threonine-192-(AdhN-11 and AdhUF)-to-lysine (AdhS) substitution and the AdhUF enzyme was found to differ by an alanine-45-(AdhS and AdhN-11)-to-aspartic acid (AdhUF) charge substitution and a ‘silent’ asparagine-8-(AdhS and AdhN-11)-to-alanine (AdhUF) substitution. Detailed sequence evidence has been deposited as Supplementary Publication SUP 50107 (36 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies can be obtained on the terms indicated in Biochem. J. (1978) 169, 5.

scholarly journals A Mono-2-Ethylhexyl Phthalate Hydrolase from a Gordonia sp. That Is Able To Dissimilate Di-2-Ethylhexyl Phthalate

2006 ◽  
Vol 72 (4) ◽  
pp. 2394-2399 ◽  
Author(s):  
Tuguhiro Nishioka ◽  
Makoto Iwata ◽  
Takuya Imaoka ◽  
Maiko Mutoh ◽  
Yoshihiro Egashira ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Contaminated Soil ◽  
Primary Structure ◽  
Independent Group ◽  
Serine Hydrolases ◽  
Cell Extracts ◽  
Significant Homology ◽  
Serine Esterases ◽  
Ethylhexyl Phthalate

ABSTRACT Gordonia sp. strain P8219, a strain able to decompose di-2-ethylhexyl phthalate, was isolated from machine oil-contaminated soil. Mono-2-ethylhexyl phthalate hydrolase was purified from cell extracts of this strain. This enzyme was a 32,164-Da homodimeric protein, and it effectively hydrolyzed monophthalate esters, such as monoethyl, monobutyl, monohexyl, and mono-2-ethylhexyl phthalate. The Km and V max values for mono-2-ethylhexyl phthalate were 26.9 ± 4.3 μM and 18.1 ± 0.9 μmol/min · mg protein, respectively. The deduced amino acid sequence of the enzyme exhibited less than 30% homology with those of meta-cleavage hydrolases which are serine hydrolases but exhibited no significant homology with the sequences of serine esterases. The pentapeptide motif GXSXG, which is conserved in serine hydrolases, was present in the sequence. The enzymatic properties and features of the primary structure suggested that this enzyme is a novel enzyme belonging to an independent group of serine hydrolases.

scholarly journals Amino acid sequence homology in alcohol dehydrogenase

FEBS Letters ◽  
1973 ◽  
Vol 33 (1) ◽  
pp. 1-3 ◽  
Author(s):  
John Bridgen ◽  
Edith Kolb ◽  
J.Ieuan Harris
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Alcohol Dehydrogenase ◽  
Sequence Homology ◽  
Amino Acid Sequence Homology

scholarly journals Plasmid-Encoded asp Operon Confers a Proton Motive Metabolic Cycle Catalyzed by an Aspartate-Alanine Exchange Reaction

2002 ◽  
Vol 184 (11) ◽  
pp. 2906-2913 ◽  
Author(s):  
Keietsu Abe ◽  
Fumito Ohnishi ◽  
Kyoko Yagi ◽  
Tasuku Nakajima ◽  
Takeshi Higuchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Sequence Analysis ◽  
Amino Acid Sequence ◽  
Expression Vector ◽  
Alcaligenes Faecalis ◽  
E Coli ◽  
Cell Extracts ◽  
Membrane Spanning ◽  
Metabolic Cycle

ABSTRACT Tetragenococcus halophila D10 catalyzes the decarboxylation of l-aspartate with nearly stoichiometric release of l-alanine and CO2. This trait is encoded on a 25-kb plasmid, pD1. We found in this plasmid a putative asp operon consisting of two genes, which we designated aspD and aspT, encoding an l-aspartate-β-decarboxylase (AspD) and an aspartate-alanine antiporter (AspT), respectively, and determined the nucleotide sequences. The sequence analysis revealed that the genes of the asp operon in pD1 were in the following order: promoter → aspD → aspT. The deduced amino acid sequence of AspD showed similarity to the sequences of two known l-aspartate-β-decarboxylases from Pseudomonas dacunhae and Alcaligenes faecalis. Hydropathy analyses suggested that the aspT gene product encodes a hydrophobic protein with multiple membrane-spanning regions. The operon was subcloned into the Escherichia coli expression vector pTrc99A, and the two genes were cotranscribed in the resulting plasmid, pTrcAsp. Expression of the asp operon in E. coli coincided with appearance of the capacity to catalyze the decarboxylation of aspartate to alanine. Histidine-tagged AspD (AspDHis) was also expressed in E. coli and purified from cell extracts. The purified AspDHis clearly exhibited activity of l-aspartate-β-decarboxylase. Recombinant AspT was solubilized from E. coli membranes and reconstituted in proteoliposomes. The reconstituted AspT catalyzed self-exchange of aspartate and electrogenic heterologous exchange of aspartate with alanine. Thus, the asp operon confers a proton motive metabolic cycle consisting of the electrogenic aspartate-alanine antiporter and the aspartate decarboxylase, which keeps intracellular levels of alanine, the countersubstrate for aspartate, high.

scholarly journals Diversity in Butane Monooxygenases among Butane-Grown Bacteria

1999 ◽  
Vol 65 (10) ◽  
pp. 4586-4593 ◽  
Author(s):  
Natsuko Hamamura ◽  
Ryan T. Storfa ◽  
Lewis Semprini ◽  
Daniel J. Arp
Keyword(s):  
Ethylene Oxide ◽  
Methane Oxidation ◽  
Environmental Isolate ◽  
Butane Oxidation ◽  
Butanol Production ◽  
Oxidation Activity ◽  
Physiological Level ◽  
Mycobacterium Vaccae ◽  
Pseudomonas Butanovora ◽  
Allyl Thiourea

ABSTRACT Butane monooxygenases of butane-grown Pseudomonas butanovora, Mycobacterium vaccae JOB5, and an environmental isolate, CF8, were compared at the physiological level. The presence of butane monooxygenases in these bacteria was indicated by the following results. (i) O2 was required for butane degradation. (ii) 1-Butanol was produced during butane degradation. (iii) Acetylene inhibited both butane oxidation and 1-butanol production. The responses to the known monooxygenase inactivator, ethylene, and inhibitor, allyl thiourea (ATU), discriminated butane degradation among the three bacteria. Ethylene irreversibly inactivated butane oxidation by P. butanovora but not by M. vaccae or CF8. In contrast, butane oxidation by only CF8 was strongly inhibited by ATU. In all three strains of butane-grown bacteria, specific polypeptides were labeled in the presence of [14C]acetylene. The [14C]acetylene labeling patterns were different among the three bacteria. Exposure of lactate-grown CF8 and P. butanovora and glucose-grownM. vaccae to butane induced butane oxidation activity as well as the specific acetylene-binding polypeptides. Ammonia was oxidized by all three bacteria. P. butanovora oxidized ammonia to hydroxylamine, while CF8 and M. vaccae produced nitrite. All three bacteria oxidized ethylene to ethylene oxide. Methane oxidation was not detected by any of the bacteria. The results indicate the presence of three distinct butane monooxygenases in butane-grown P. butanovora, M. vaccae, and CF8.

scholarly journals Molecular characterization and tissue distribution of ZO-2, a tight junction protein homologous to ZO-1 and the Drosophila discs-large tumor suppressor protein

1994 ◽  
Vol 124 (6) ◽  
pp. 949-961 ◽  
Author(s):  
LA Jesaitis ◽  
DA Goodenough
Keyword(s):  
Amino Acid ◽  
Tumor Suppressor ◽  
Amino Acid Sequence ◽  
Tight Junction ◽  
Mdck Cell ◽  
Sequence Information ◽  
Unique Region ◽  
Cell Extracts ◽  
Junction Protein ◽  
Discs Large

ZO-1 is a 210-225-kD peripheral membrane protein associated with cytoplasmic surfaces of the zonula occludens or tight junction. A 160-kD polypeptide, designated ZO-2, was found to coimmunoprecipitate with ZO-1 from MDCK cell extracts prepared under conditions which preserve protein associations (Gumbiner, B., T. Lowenkopf, and D. Apatira. 1991. Proc. Natl. Acad. Sci. USA. 88: 3460-3464). We have isolated ZO-2 from MDCK cell monolayers by bulk coimmunoprecipitation with ZO-1 followed by electroelution from preparative SDS-PAGE gel slices. Amino acid sequence information obtained from a ZO-2 tryptic fragment was used to isolate a partial cDNA clone from an MDCK library. The deduced amino acid sequence revealed that canine ZO-2 contains a region that is very similar to sequences in human and mouse ZO-1. This region includes both a 90-amino acid repeat domain of unknown function and guanylate kinase-like domains which are shared among members of the family of proteins that includes ZO-1, erythrocyte p55, the product of the lethal(1)discs-large-1 (dlg) gene of Drosophila, and a synapse-associated protein from rat brain, PSD-95/SAP90. The dlg gene product has been shown to act as a tumor suppressor in the imaginal disc of the Drosophila larva, although the functions of other family members have not yet been defined. A polyclonal antiserum was raised against a unique region of ZO-2 and found to exclusively label the cytoplasmic surfaces of tight junctions in MDCK plasma membrane preparations, indicating that ZO-2 is a tight junction-associated protein. Immunohistochemical staining of frozen sections of whole tissue demonstrated that ZO-2 localized to the region of the tight junction in a number of epithelia, including liver, intestine, kidney, testis, and arterial endothelium, suggesting that this protein is a ubiquitous component of the tight junction. Double-label immunofluorescence microscopy performed on cryosections of heart, a nonepithelial tissue, revealed the presence of ZO-1 but no ZO-2 staining at the fascia adherens, a specialized junction of cardiac myocytes which has previously been shown to contain ZO-1 (Itoh, M., S. Yonemura, A. Nagafuchi, S. Tsukita, and Sh. Tsukita. 1991. J. Cell Biol. 115:1449-1462). Thus it appears that ZO-2 is not a component of the fascia adherens, and that unlike ZO-1, this protein is restricted to the epithelial tight junction.

scholarly journals Purification, Characterization, Gene Cloning, and Expression of a Novel Alcohol Dehydrogenase with Anti-Prelog Stereospecificity from Candida parapsilosis

2007 ◽  
Vol 73 (11) ◽  
pp. 3759-3764 ◽  
Author(s):  
Yao Nie ◽  
Yan Xu ◽  
Xiao Qing Mu ◽  
Hai Yan Wang ◽  
Ming Yang ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Alcohol Dehydrogenase ◽  
Gene Cloning ◽  
Structural Gene ◽  
Candida Parapsilosis ◽  
Cloning And Expression ◽  
Gene Cloning And Expression ◽  
Biochemical Activity ◽  
Short Chain Dehydrogenase

ABSTRACT An alcohol dehydrogenase from Candida parapsilosis CCTCC M203011 was characterized along with its biochemical activity and structural gene. The amino acid sequence shows similarity to those of the short-chain dehydrogenase/reductases but no overall identity to known proteins. This enzyme with unusual stereospecificity catalyzes an anti-Prelog reduction of 2-hydroxyacetophenone to (S)-1-phenyl-1,2-ethanediol.

scholarly journals Cloning and expression of the gene for periplasmic poly(vinyl alcohol) dehydrogenase from Sphingomonas sp. strain 113P3, a novel-type quinohaemoprotein alcohol dehydrogenase

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 1941-1949 ◽  
Author(s):  
Rie Hirota-Mamoto ◽  
Ryoko Nagai ◽  
Shinjiro Tachibana ◽  
Masaaki Yasuda ◽  
Akio Tani ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Alcohol Dehydrogenase ◽  
Vinyl Alcohol ◽  
Ralstonia Eutropha ◽  
Blot Hybridization ◽  
Amino Acid Sequences ◽  
Cloning And Expression ◽  
Poly Vinyl Alcohol ◽  
Dot Blot

A gene for periplasmic poly(vinyl alcohol) (PVA) dehydrogenase (PVADH) was cloned, based on the N-terminal amino acid sequence of the purified PVADH from Sphingomonas sp. 113P3 and the sequence of the gene for PVADH (pvaA, GenBank accession no. AB190288). The recombinant PVADH tagged with hexahistidine was expressed in Escherichia coli and purified to homogeneity. The recombinant enzyme had the same characteristics as the purified enzyme from Sphingomonas sp. strain 113P. In addition to PVA, the recombinant PVADH could oxidize glycols such as polypropylene glycols and 1,3-butane/cyclohexanediol and 2,4-pentanediol, but neither primary nor secondary alcohols. The amino acid sequence of the recombinant PVADH showed similarity with those of PVADH from Pseudomonas sp. strain VM15C, putative PVADHs from Azoarcus sp. EbN1, and Xanthomonas species (54–25 % identity), and the quinohaemoprotein alcohol dehydrogenases (QH-ADHs) from Comamonas testosteroni, Ralstonia eutropha and Pseudomonas putida (25–29 % identity). PVADHs from strains 113P3 and VM15C have a conserved superbarrel domain (SD), probable PQQ-binding amino acids in the SD and a haem-binding domain (HBD) (they should be designated QH-PVADHs), but the positions of the amino acid sequences for the HBD and SD are the reverse of those of QH-ADHs. A protein structure of QH-PVADHs is proposed. Results of dot-blot hybridization and RT-PCR indicated that the three genes encoding oxidized PVA hydrolase, PVADH and cytochrome c are expressed constitutively and form an operon.

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