scholarly journals SSoNΔ andSsoNΔlong: two thermostable esterases from the same ORF in the archaeonSulfolobus solfataricus?

Archaea ◽  
2006 ◽  
Vol 2 (2) ◽  
pp. 109-115 ◽  
Author(s):  
Luigi Mandrich ◽  
Margherita Pezzullo ◽  
Mosè Rossi ◽  
Giuseppe Manco
Keyword(s):  
Sequence Similarity ◽  
Hormone Sensitive Lipase ◽  
Divergent Evolution ◽  
High Sequence Similarity ◽  
Ancestral Gene ◽  
Reading Frame ◽  
Family Analysis ◽  
N Terminus ◽  
Metabolic Conditions ◽  
Hormone Sensitive

Previously, we reported from theSulfolobus solfataricusopen reading frame (ORF) SSO2517 the cloning, overexpression and characterization of an esterase belonging to the hormone-sensitive lipase (HSL) family and apparently having a deletion at the N-terminus, which we namedSsoNΔ. Searching the recently reportedSulfolobus acidocaldariusgenome by sequence alignment, using SSO2517 as a query, allowed identity of a putative esterase (ORF SAC1105) sharing high sequence similarity (82%) with SSO2517. This esterase displays an N-terminus and total length similar to other known esterases of the HSL family. Analysis of the upstream DNA sequence of SS02517 revealed the possibility of expressing a longer version of the protein with an extended N-terminus; however, no clear translation signal consistent with a longer protein version was detected. This new version of SSO2517 was cloned, over-expressed, purified and characterized. The resulting protein, namedSsoNΔlong, was 15-fold more active with the substratep-nitrophenyl hexanoate thanSsoNΔ. Furthermore,SsoNΔlong andSsoNΔ displayed different substrate specificities for triacylglycerols. These results and the phylogenetic relationship betweenS. solfataricusandS. acidocaldariussuggest a common origin of SSO2517 and SAC1105 from an ancestral gene, followed by divergent evolution. Alternatively, a yet-to-be discovered mechanism of translation that directs the expression ofSsoNΔlong under specific metabolic conditions could be hypothesized.

scholarly journals Overexpression and properties of a new thermophilic and thermostable esterase from Bacillus acidocaldarius with sequence similarity to hormone-sensitive lipase subfamily

1998 ◽  
Vol 332 (1) ◽  
pp. 203-212 ◽  
Author(s):  
Giuseppe MANCO ◽  
Elena ADINOLFI ◽  
Francesca M. PISANI ◽  
Gianluca OTTOLINA ◽  
Giacomo CARREA ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Active Form ◽  
Enantiomeric Excess ◽  
Biochemical Properties ◽  
Catalytic Triad ◽  
Hormone Sensitive Lipase ◽  
High Sequence Identity ◽  
Reading Frame ◽  
Hormone Sensitive ◽  
Overexpression System

We previously purified a new esterase from the thermoacidophilic eubacterium Bacillus acidocaldarius whose N-terminal sequence corresponds to an open reading frame (ORF3) reported to show homology with the mammalian hormone-sensitive lipase (HSL)-like group of the esterase/lipase family. To compare the biochemical properties of this thermophilic enzyme with those of the homologous mesophilic and psychrophilic members of the HSL group, an overexpression system in Escherichia coli was established. The protein, expressed in soluble and active form at 10 mg/l E. coli culture, was purified to homogeneity and characterized biochemically. The enzyme, a 34 kDa monomeric protein, was demonstrated to be a B´-type carboxylesterase (EC 3.1.1.1) on the basis of substrate specificity and the action of inhibitors. Among the p-nitrophenyl (PNP) esters tested the best substrate was PNP-exanoate with Km and kcat values of 11±2 µM (mean±S.D., n = 3) and 6610±880 s-1 (mean±S.D., n = 3) respectively at 70 °C and pH 7.1. In spite of relatively high sequence identity with the mammalian HSLs, the psychrophilic MoraxellaTA144lipase 2 and the human liver arylacetamide deacetylase, no lipase or amidase activity was detected. A series of substrates were tested for enantioselectivity. Substantial enantioselectivity was observed only in the resolution of (±)-3-bromo-5-(hydroxymethyl)-Δ2-isoxazoline, where the (R)-product was obtained with an 84% enantiomeric excess at 36% conversion. The enzyme was also able to synthesize acetyl esters when tested in vinyl acetate and toluene. Inactivation by diethylpyrocarbonate, diethyl-p-nitrophenyl phosphate, di-isopropylphosphofluoridate (DFP) and physostigmine, as well as labelling with [3H]DFP, supported our previous suggestion of a catalytic triad made up of Ser-His-Asp. The activity–stability–temperature relationship is discussed in relation to those of the homologous members of the HSL group.

scholarly journals Structure and Sequence Conservation of hao Cluster Genes of Autotrophic Ammonia-Oxidizing Bacteria: Evidence for Their Evolutionary History

2005 ◽  
Vol 71 (9) ◽  
pp. 5371-5382 ◽  
Author(s):  
David J. Bergmann ◽  
Alan B. Hooper ◽  
Martin G. Klotz
Keyword(s):  
Gene Cluster ◽  
Sequence Similarity ◽  
Molecular Ecology ◽  
Gene Clusters ◽  
Nitrite Reduction ◽  
Divergent Evolution ◽  
Ammonia Oxidizing Bacteria ◽  
High Sequence Similarity ◽  
Hydroxylamine Oxidoreductase ◽  
Transfer Event

ABSTRACT Comparison of the organization and sequence of the hao (hydroxylamine oxidoreductase) gene clusters from the gammaproteobacterial autotrophic ammonia-oxidizing bacterium (aAOB) Nitrosococcus oceani and the betaproteobacterial aAOB Nitrosospira multiformis and Nitrosomonas europaea revealed a highly conserved gene cluster encoding the following proteins: hao, hydroxylamine oxidoreductase; orf2, a putative protein; cycA, cytochrome c 554; and cycB, cytochrome c m 552. The deduced protein sequences of HAO, c 554, and c m 552 were highly similar in all aAOB despite their differences in species evolution and codon usage. Phylogenetic inference revealed a broad family of multi-c-heme proteins, including HAO, the pentaheme nitrite reductase, and tetrathionate reductase. The c-hemes of this group also have a nearly identical geometry of heme orientation, which has remained conserved during divergent evolution of function. High sequence similarity is also seen within a protein family, including cytochromes c m 552, NrfH/B, and NapC/NirT. It is proposed that the hydroxylamine oxidation pathway evolved from a nitrite reduction pathway involved in anaerobic respiration (denitrification) during the radiation of the Proteobacteria. Conservation of the hydroxylamine oxidation module was maintained by functional pressure, and the module expanded into two separate narrow taxa after a lateral gene transfer event between gamma- and betaproteobacterial ancestors of extant aAOB. HAO-encoding genes were also found in six non-aAOB, either singly or tandemly arranged with an orf2 gene, whereas a c 554 gene was lacking. The conservation of the hao gene cluster in general and the uniqueness of the c 554 gene in particular make it a suitable target for the design of primers and probes useful for molecular ecology approaches to detect aAOB.

scholarly journals New Thermophilic and Thermostable Esterase with Sequence Similarity to the Hormone-Sensitive Lipase Family, Cloned from a Metagenomic Library

2005 ◽  
Vol 71 (2) ◽  
pp. 817-825 ◽  
Author(s):  
Jin-Kyu Rhee ◽  
Dae-Gyun Ahn ◽  
Yeon-Gu Kim ◽  
Jong-Won Oh
Keyword(s):  
Amino Acid ◽  
Lipolytic Activity ◽  
Sequence Similarity ◽  
Acyl Chain ◽  
Metagenomic Library ◽  
Environmental Dna ◽  
Hormone Sensitive Lipase ◽  
Functional Screening ◽  
E Coli ◽  
Hormone Sensitive

ABSTRACT A gene coding for a thermostable esterase was isolated by functional screening of Escherichia coli cells that had been transformed with fosmid environmental DNA libraries constructed with metagenomes from thermal environmental samples. The gene conferring esterase activity on E. coli grown on tributyrin agar was composed of 936 bp, corresponding to 311 amino acid residues with a molecular mass of 34 kDa. The enzyme showed significant amino acid similarity (64%) to the enzyme from a hyperthermophilic archaeon, Pyrobaculum calidifontis. An amino acid sequence comparison with other esterases and lipases revealed that the enzyme should be classified as a new member of the hormone-sensitive lipase family. The recombinant esterase that was overexpressed and purified from E. coli was active above 30°C up to 95°C and had a high thermal stability. It displayed a high degree of activity in a pH range of 5.5 to 7.5, with an optimal pH of approximately 6.0. The best substrate for the enzyme among the p-nitrophenyl esters (C4 to C16) examined was p-nitrophenyl caproate (C6), and no lipolytic activity was observed with esters containing an acyl chain length of longer than 10 carbon atoms, indicating that the enzyme is an esterase and not a lipase.

scholarly journals The Synechocystis sp. PCC 6803 open reading frame slr0201 that is homologous to sdhC from Archaea codes for a [2Fe-2S] protein

2021 ◽  
Author(s):  
Fusheng Xiong ◽  
Russell LoBrutto ◽  
Wim F. J. Vermaas
Keyword(s):  
Sequence Similarity ◽  
Immunoblot Analysis ◽  
Open Reading Frame ◽  
Heterodisulfide Reductase ◽  
Redox Titration ◽  
High Sequence Similarity ◽  
Reading Frame ◽  
Midpoint Potential ◽  
Synechocystis Sp ◽  
Pcc 6803

A hypothetical protein encoded by Synechocystis sp. PCC 6803 open reading frame slr0201 shows high sequence similarity to the C subunit of a group of unusual succinate dehydrogenases found in some archaeal species. Slr0201 was originally annotated as HdrB, the B subunit of heterodisulfide reductase, but appears to be SdhC instead. This protein was overexpressed in E. coli by cloning the PCR-derived slr0201 open reading frame into a pET16b-based expression vector. The overproduced Slr0201 accumulated predominantly in inclusion bodies with an apparent molecular mass of 33 kDa. The protein contained at least one [2Fe-2S] cluster based on UV-visible absorbance and CD spectra and EPR spectroscopy, in conjunction with stoichiometric analysis of protein-bound iron and sulfur content. Redox titration showed a midpoint potential (Em) of + 17 mV at pH 7.0, which is consistent with Slr0201 serving a role in transferring electrons between succinate and plastoquinone. Slr0201 was also overproduced in Synechocystis sp. PCC 6803 by introducing an additional, His-tagged slr0201 into the Synechocystis genome replacing psbA3, creating the slr0201+-His overexpression strain. Immunoblot analysis shows that Slr0201 is membrane-associated in the wild type. However, in the Slr0201+-His strain, immunoreaction occurred in both the membrane and soluble fractions, possibly as a consequence of processing near the N-terminus. The results obtained with Slr0201 are discussed in the light of one of the cyanobacterial SdhB subunits, which shares redox commonalities with archaeal SdhB.

scholarly journals Glutathione S-transferase class Kappa: characterization by the cloning of rat mitochondrial GST and identification of a human homologue

1996 ◽  
Vol 319 (3) ◽  
pp. 749-754 ◽  
Author(s):  
Sally E PEMBLE ◽  
Anthony F WARDLE ◽  
John B TAYLOR
Keyword(s):  
Dna Sequences ◽  
Protein Sequence ◽  
Sequence Similarity ◽  
Open Reading Frame ◽  
Glutathione S Transferase ◽  
Reading Frame ◽  
Sequence Identity ◽  
Methionine Residue ◽  
N Terminus ◽  
Related Proteins

We have isolated a cDNA clone that encodes rat glutathione S-transferase (GST) subunit 13, a GST originally isolated from rat liver mitochondrial matrix by Harris, Meyer, Coles and Ketterer [(1991) Biochem. J. 278, 137–141]. The 896 bp cDNA contains an open reading frame of 678 bp encoding a deduced protein sequence of which the first 33 residues (excluding the initiation methionine residue) correspond to the N-terminal sequence reported by Harris et al. Hence like many other nuclear-encoded, mitochondrially located proteins, there is no cleavable mitochondrial presequence at the N-terminus. GST subunit 13 was originally placed into the Theta class of GSTs on the basis of sequence identity at the N-terminus; however, this is the only identity with the Theta class and in fact GST subunit 13 shows little sequence similarity to any of the known GST classes. Most importantly it lacks the SNAIL/TRAIL motif that has so far been a characteristic of soluble GSTs, although it does possess a second motif (FGXXXXVXXVDGXXXXXF) reported for GST-related proteins (Koonin, Mushegian, Tatusov, Altschul, Bryant, Bork and Valencia [(1994) Protein Sci. 3, 2045–2054]. Southern and Northern blot analyses of rat DNA and mRNA are consistent with GST subunit 13's being the product of a single hybridizing gene locus. Searches of EST databases identified numerous similar human DNA sequences and a single pig sequence. We have derived a human cDNA sequence from these EST sequences which shows a high nucleotide similarity (77%) to rat GST subunit 13. The largest open reading frame is identical in length with subunit 13 and yields a deduced protein sequence identity of 70%. Most unusually the 3´ non-coding nucleotide sequence identity is also 77%. We conclude that these cDNAs belong to a novel GST class hereby designated Kappa, with the rat GST subunit 13 gene designated rGSTK1 and the human gene being called hGSTK1.

Human mucin gene MUC5AC: organization of its 5′-region and central repetitive region

2001 ◽  
Vol 358 (3) ◽  
pp. 763-772 ◽  
Author(s):  
Fabienne ESCANDE ◽  
Jean-Pierre AUBERT ◽  
Nicole PORCHET ◽  
Marie-Pierre BUISINE
Keyword(s):  
Central Region ◽  
Tandem Repeats ◽  
Sequence Similarity ◽  
Repetitive Region ◽  
High Sequence Similarity ◽  
Ancestral Gene ◽  
Chromosome 11P15 ◽  
Repeat Array ◽  
Mucin Gene ◽  
Von Willebrand

Human mucin gene MUC5AC is clustered with MUC2, MUC5B and MUC6 on chromosome 11p15.5. We report here the full length cDNA sequence upstream of the repetitive region of human MUC5AC. We have also determined the sequence of its large central tandem repeat array. The 5′-region reveals high degree of sequence similarity with MUC2 and MUC5B and codes for 1336 amino acids organized into a signal peptide, four pro-von Willebrand factor-like D domains (D1, D2, D′ and D3) and a short domain which connects to the central repetitive region. In the central region, 17 major domains have been identified. Nine code for cysteine-rich domains (Cys-domains 1–9) and exhibit high sequence similarity to the cysteine-rich domains described in the central region of MUC2 and MUC5B. Cys-domains 1–5 are interspersed by domains enriched with serine, threonine, and proline residues. Cys-domains 1–9 are interspersed by four domains (TR1–TR4) composed of various numbers of MUC5AC-type repeats. Southern-blot analyses reveal allelic variations both in length and nucleotide sequence. The length polymorphism which is due to variable numbers of tandem repeats is located in TR1 and TR4, whereas a mutation polymorphism detected with TaqI is located in Cys-domain 6. In this study, the organization of MUC5AC has been entirely elucidated showing extensive similarity to the other chromosome 11p15 MUC genes, particularly MUC5B, and providing additional arguments for common evolution from a single ancestral gene.

scholarly journals An Oligoribonuclease Gene inStreptomyces griseus

2000 ◽  
Vol 182 (16) ◽  
pp. 4647-4653 ◽  
Author(s):  
Yasuo Ohnishi ◽  
Yoko Nishiyama ◽  
Rie Sato ◽  
Shogo Kameyama ◽  
Sueharu Horinouchi
Keyword(s):  
Sequence Similarity ◽  
Streptomyces Griseus ◽  
Aerial Mycelium ◽  
Morphological Development ◽  
Developmentally Regulated ◽  
High Sequence Similarity ◽  
S1 Nuclease ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
Nuclease Mapping

ABSTRACT In Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) serves as a microbial hormone that switches on many genes required for streptomycin production and morphological development. An open reading frame (Orf1) showing high sequence similarity to oligoribonucleases of various origins is present just downstream of adpA, one of the A-factor-dependent genes. Orf1 was named OrnA (oligoribonuclease A) because it showed 3′-to-5′ exo-oligoribonuclease activity, releasing [32P]CMP from ApCpC[32P]pC used as a substrate. Reverse transcription-PCR and S1 nuclease mapping analyses revealed that ornA was transcribed from two promoters; one was a developmentally regulated, A-factor-dependent promoter in front of adpA, and the other was a constitutive promoter in front of the ornA coding sequence. Transcription ofornA was thus additively enhanced at the initiation stage for secondary metabolism and aerial mycelium formation.ornA-disrupted strains grew slowly and scarcely formed aerial mycelium. ornA homologues were distributed in a wide variety of Streptomyces species, including S. coelicolor A3(2), as determined by Southern hybridization analysis. Disruption of the ornA homologue in S. coelicolor A3(2) also caused phenotypes similar to those of theS. griseus ΔornA strains. The OrnA oligoribonucleases inStreptomyces species are therefore not essential but play an important role in vegetative growth and in the initiation of differentiation.

scholarly journals An esterase from Escherichia coli with a sequence similarity to hormone-sensitive lipase

1998 ◽  
Vol 332 (1) ◽  
pp. 75-80 ◽  
Author(s):  
Shigenori KANAYA ◽  
Tomoyoshi KOYANAGI ◽  
Eiko KANAYA
Keyword(s):  
Amino Acid ◽  
Chain Length ◽  
Sequence Similarity ◽  
Acyl Chain ◽  
Hydrolytic Activity ◽  
Catalytic Triad ◽  
Enzymic Activity ◽  
Hormone Sensitive Lipase ◽  
Acyl Chain Length ◽  
Hormone Sensitive

An esterase from Escherichia colithat is a member of the hormone-sensitive lipase (HSL) family was overproduced, purified and characterized. It is encoded by the ybaCgene and composed of 319 amino acid residues with an Mr of 36038. The enzymic activity was determined by using various p-nitrophenyl esters of fatty acids as a substrate at 25 °C and pH 7.1. The enzyme showed hydrolytic activity towards substrates with an acyl chain length of less than 8, whereas it showed little hydrolytic activity towards those with an acyl chain length of more than 10. In addition, it showed little hydrolytic activity towards trioleoylglycerol and cholesterol oleate. Determination of the kinetic parameters for the hydrolyses of the substrates from C2 to C8 indicates that C4 and C5 substrates are the most preferred. Close agreement between the Mr determined by SDS/PAGE (37000) and column chromatography (38000) suggests that the enzyme exists in a monomeric form. It is an acidic protein with a pI value of 4.1. The far-UV CD spectrum suggests that its helical content is 26.1%. Comparison of the amino acid sequence of this enzyme with those involved in the HSL family allows us to propose that Ser165, Asp262 and His292 constitute the catalytic triad of E. coliesterase.

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