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 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 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.

HACRE1, a recently inserted copia-like retrotransposon of sunflower (Helianthus annuus L.)

Genome ◽  
2009 ◽  
Vol 52 (11) ◽  
pp. 904-911 ◽  
Author(s):  
M. Buti ◽  
T. Giordani ◽  
M. Vukich ◽  
L. Gentzbittel ◽  
L. Pistelli ◽  
...  
Keyword(s):  
Helianthus Annuus ◽  
Sequence Similarity ◽  
Protein Domain ◽  
Long Terminal Repeats ◽  
High Sequence Identity ◽  
Nonsense Mutations ◽  
Reading Frame ◽  
Sequence Identity ◽  
Isolation And Characterization ◽  
Copia Retrotransposon

In this paper we report on the isolation and characterization, for the first time, of a complete 6511 bp retrotransposon of sunflower. Considering its protein domain order and sequence similarity to other copia elements of dicotyledons, this retrotransposon was assigned to the copia retrotransposon superfamily and named HACRE1 ( Helianthus annuus copia-like retroelement 1). HACRE1 carries 5′ and 3′ long terminal repeats (LTRs) flanking an internal region of 4661 bp. The LTRs are identical in their sequence except for two deletions of 7 and 5 nucleotides in the 5′ LTR. Based on the sequence identity of the LTRs, HACRE1 was estimated to have inserted within the last ∼84 000 years. The isolated sequence contains a complete open reading frame with only one complete reading frame. The absence of nonsense mutations agrees with the very high sequence identity between LTRs, confirming that HACRE1 insertion is recent. The haploid genome of sunflower (inbred line HCM) contains about 160 copies of HACRE1. This retrotransposon is expressed in leaflets from 7-day-old plantlets under different light conditions, probably in relation to the occurrence of many putative light-related regulatory cis-elements in the LTRs. However, sequenced cDNAs show less variability than HACRE1 genomic sequences, indicating that only a subset of this family is expressed under these conditions.

scholarly journals Domain-structure analysis of recombinant rat hormone-sensitive lipase

1996 ◽  
Vol 319 (2) ◽  
pp. 411-420 ◽  
Author(s):  
Torben ØSTERLUND ◽  
Birgitta DANIELSSON ◽  
Eva DEGERMAN ◽  
Juan Antonio CONTRERAS ◽  
Gudrun EDGREN ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Domain Structure ◽  
Expression System ◽  
Guanidine Hydrochloride ◽  
Catalytic Triad ◽  
Water Soluble ◽  
Hormone Sensitive Lipase ◽  
Structural Domain ◽  
Hydrolase Fold ◽  
Hormone Sensitive

Hormone-sensitive lipase (HSL) plays a key role in lipid metabolism and overall energy homoeostasis, by controlling the release of fatty acids from stored triglycerides in adipose tissue. Lipases and esterases form a protein superfamily with a common structural fold, called the α/β-hydrolase fold, and a catalytic triad of serine, aspartic or glutamic acid and histidine. Previous alignments between HSL and lipase 2 of Moraxella TA144 have been extended to cover a much larger part of the HSL sequence. From these extended alignments, possible sites for the catalytic triad and α/β-hydrolase fold are suggested. Furthermore, it is proposed that HSL contains a structural domain with catalytic capacity and a regulatory module attached, as well as a structural N-terminal domain unique to this enzyme. In order to test the proposed domain structure, rat HSL was overexpressed and purified to homogeneity using a baculovirus/insect-cell expression system. The purification, resulting in > 99% purity, involved detergent solubilization followed by anion-exchange chromatography and hydrophobic-interaction chromatography. The purified recombinant enzyme was identical to rat adipose-tissue HSL with regard to specific activity, substrate specificity and ability to serve as a substrate for cAMP-dependent protein kinase. The recombinant HSL was subjected to denaturation by guanidine hydrochloride and limited proteolysis. These treatments resulted in more extensive loss of activity against phospholipid-stabilized lipid substrates than against water-soluble substrates, suggesting that the hydrolytic activity can be separated from recognition of lipid substrates. These data support the concept that HSL has at least two major domains.

scholarly journals Growth Hormone and Dexamethasone Stimulate Lipolysis and Activate Adenylyl Cyclase in Rat Adipocytes by Selectively Shifting Giα2 to Lower Density Membrane Fractions*

Endocrinology ◽  
1999 ◽  
Vol 140 (3) ◽  
pp. 1219-1227 ◽  
Author(s):  
Rupert Guk-Chor Yip ◽  
H. Maurice Goodman
Keyword(s):  
Adenylyl Cyclase ◽  
Active Form ◽  
Competitive Inhibitor ◽  
Hormone Sensitive Lipase ◽  
Western Analysis ◽  
Tissue Extracts ◽  
Dense Membrane ◽  
Hormone Sensitive ◽  
A Chain ◽  
Rat Adipose Tissue

Abstract GH, in the presence of glucocorticoid, produces a delayed increase in lipolysis in rat adipose tissue, but the biochemical mechanisms that account for this action have not been established. Other lipolytic agents rapidly activate adenylyl cyclase (AC) and the resulting production of cAMP initiates a chain of reactions that culminates in the activation of hormone-sensitive lipase. We compared responses of segments of rat epididymal fat or isolated adipocytes to 30 ng/ml GH and 0.1 μg/ml dexamethasone (Dex) with 0.1 ng/ml isoproterenol (ISO), which evoked a similar increase in lipolysis. All measurements were made during the fourth hour after the addition of GH+Dex or immediately after the addition of ISO to cells or tissues that had been preincubated for 3 h without hormone. Although no significant increases in cAMP were discernible in homogenates of GH+Dex-treated tissues, RP-cAMPS (RP-adenosine 3′5′-phosphothioate), a competitive inhibitor of cAMP, was equally effective in decreasing lipolysis induced by GH+Dex or ISO. The proportion of PKA that was present in the active form was determined by measuring the incorporation of 32P from[γ -32P]ATP into kemptide in the absence and presence of saturating amounts of cAMP. GH+Dex and ISO produced similar increases in protein kinase A activity in tissue extracts. Treatment with GH+Dex did not change the total forskolin-stimulated AC present in either a crude membrane pellet sedimented at 16K × g or a less dense membrane pellet sedimented at 100K × g, but doubled the AC activity in the 16K pellet when assayed in the absence of forskolin. To evaluate possible effects on G proteins, pellets obtained from centrifugation of adipocyte homogenates at 16K × g and 100K × g were solubilized and subjected to PAGE and Western analysis. GH+Dex decreased Giα2 by 44% (P < 0.02) in the 16K pellets and increased it by 52% (P < 0.01) in the 100K pellets. Gsα in the 16K pellet was unaffected by GH+Dex and was decreased (P < 0.05) in the 100K pellet. Sucrose density fractionation of the 16K pellets revealed a similar GH+Dex-dependent shift of Giα2 to less dense fractions as determined by both Western analysis and[ 32P]NAD ribosylation catalyzed by pertussis toxin. No such changes were seen in the distribution of Gsα or 5′-nucleotidase. Colchicine (100 μm) blocked the GH+Dex-dependent shift of Giα2 from the 16K to the 100K pellet and blocked the lipolytic effects of GH+Dex, but not those of ISO. We conclude that by modifying the relationship between AC and Giα2, GH+Dex relieves some inhibition of cAMP production and consequently increases lipolysis.

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 Species-specific alternative splicing generates a catalytically inactive form of human hormone-sensitive lipase

1997 ◽  
Vol 328 (1) ◽  
pp. 137-143 ◽  
Author(s):  
Henrik LAURELL ◽  
Jacques GROBER ◽  
Cécile VINDIS ◽  
Thierry LACOMBE ◽  
Michèle DAUZATS ◽  
...  
Keyword(s):  
Adipose Tissue ◽  
Alternative Splicing ◽  
Cell Lines ◽  
Catalytic Triad ◽  
Hormone Sensitive Lipase ◽  
Human Adipocytes ◽  
Brown Adipose ◽  
Specific Alternative ◽  
Hormone Sensitive ◽  
Species Specific

Hormone-sensitive lipase (HSL) catalyses the rate-limiting step of adipose tissue lipolysis. The enzyme is also expressed in steroidogenic tissues, mammary gland, muscle tissues and macrophages. A novel HSL mRNA termed hHSL-S, 228 bp shorter than the full-length HSL mRNA, was detected in human adipocytes. hHSL-S mRNA results from the in-frame skipping of exon 6, which encodes the serine residue of the catalytic triad. The corresponding 80 kDa protein was identified in human adipocytes after immunoprecipitation. The truncated protein expressed in COS cells showed neither lipase nor esterase activity but was phosphorylated by cAMP-dependent protein kinase. hHSL-S mRNA was found in all human tissues expressing HSL, except brown adipose tissue from newborns. It represented approx. 20% of total HSL transcripts in human subcutaneous adipocytes. No alternative splicing was detected in other mammals. Human and mouse three-exon HSL minigenes transfected into primate and rodent cell lines reproduced the splicing pattern of the endogenous HSL genes. Analysis of hybrid human/mouse minigenes transfected into human cell lines showed that cis-acting elements responsible for the skipping of human exon 6 were restricted to a 247 bp region including exon 6 and the first 19 nt of intron 6. Moreover, divergence in exonic splicing elements between mouse and human was shown to be critical for the species-specific alternative splicing.

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