Substrate Report for: TrioctanoinGeneral
Target
References: Search PubMed for references concerning: Trioctanoin 6 more
Jadeja D, Dogra N, Arya S, Singh G, Kaur J Ref: Journal of Cellular Biochemistry, 117:390, 2016 : PubMed ESTHER: Jadeja_2016_J.Cell.Biochem_117_390 PubMedSearch: Jadeja 2016 J.Cell.Biochem 117 390 PubMedID: 26212120 Gene_locus related to this paper: myctu-Rv2970c Abstract LipN (Rv2970c) belongs to the Lip family of M. tuberculosis H37Rv and is homologous to the human Hormone Sensitive Lipase. The enzyme demonstrated preference for short carbon chain substrates with optimal activity at 45 degrees C/pH 8.0 and stability between pH 6.0-9.0. The specific activity of the enzyme was 217 U/mg protein with pNP-butyrate as substrate. It hydrolyzed tributyrin to di- and monobutyrin. The active-site residues of the enzyme were confirmed to be Ser216, Asp316, and His346. Tetrahydrolipstatin, RHC-80267 and N-bromosuccinimide inhibited LipN enzyme activity completely. Interestingly, Trp145, a non active-site residue, demonstrated functional role to retain enzyme activity. The enzyme was localized in cytosolic fraction of M. tuberculosis H37Rv. The enzyme was able to synthesize ester of butyric acid, methyl butyrate, in presence of methanol. LipN was able to hydrolyze 4-hydroxyphenylacetate to hydroquinone. The gene was not expressed in in-vitro growth conditions while the expression of rv2970c gene was observed post 6h of macrophage infection by M. tuberculosis H37Ra. Under individual in-vitro stress conditions, the gene was expressed during acidic stress condition only. These findings suggested that LipN is a cytosolic, acid inducible carboxylesterase with no positional specificity in demonstrating activity with short carbon chain substrates. It requires Trp145, a non active site residue, for it's enzyme activity. J. Cell. Biochem. 117: 390-401, 2016. (c) 2015 Wiley Periodicals, Inc. Title: Purification, characterization and kinetic properties of the rabbit gastric lipase Moreau H, Gargouri Y, Lecat D, Junien JL, Verger R Ref: Biochimica & Biophysica Acta, 960:286, 1988 : PubMed ESTHER: Moreau_1988_Biochim.Biophys.Acta_960_286 PubMedSearch: Moreau 1988 Biochim.Biophys.Acta 960 286 PubMedID: 3382677 Abstract Rabbit gastric lipase was purified from an acetonic powder of rabbit stomach fundus. 25 mg of pure rabbit gastric lipase (glycerol ester hydrolase, EC 3.1.1.3) was obtained from 30 rabbit stomachs after ammonium sulfate fractionation, Sephadex G-100 gel filtration and cation exchange (mono S column) using a fast protein liquid chromatography (FPLC) system. The pure enzyme obtained was resistant to acidic pH conditions, and had specific activities of 1200, 850 and 280 U/mg, using, respectively, short- (tributyroylglycerol (TC4)), medium- (trioctanoyl- to tridecanoylglycerol (TC8-TC10)) and long-chain (soybean oil) triacylglycerols. The amino-acid composition was determined, and the first 30 N-terminal amino-acid residues were sequenced. Interfacial denaturation and catalytic properties on triacylglycerol emulsions were studied. Rabbit gastric lipase turned out to be structurally and kinetically very similar to human gastric lipase. Title: Kinetic assay of human gastric lipase on short- and long-chain triacylglycerol emulsions Gargouri Y, Pieroni G, Riviere C, Sauniere JF, Lowe PA, Sarda L, Verger R Ref: Gastroenterology, 91:919, 1986 : PubMed ESTHER: Gargouri_1986_Gastroenterology_91_919 PubMedSearch: Gargouri 1986 Gastroenterology 91 919 PubMedID: 3743968 Abstract Under optimal conditions, assay for pure human gastric lipase was carried out with short- and long-chain triacylglycerol emulsions. Maximal specific activities of 1160 and 620 U/mg were obtained with tributyrin and soybean emulsion, respectively. We observed that with a tributyrin substrate, bovine serum albumin or bile salts must be added before the addition of the enzyme in order to prevent its irreversible interfacial denaturation. With long-chain triacylglycerols as substrate, a decrease with time in the rate of hydrolysis was associated with release of protonated long-chain fatty acids. The inhibitory effect of protonated fatty acids was also observed using tributyrin at pH 3.0. These observations support the conclusion that human gastric lipase shows no intrinsic specificity for short-chain triacylglycerols and that its apparent specificity is modulated by pH and presence of amphiphile in the incubation medium. Our conclusions support the view that, in the human, gastric lipolysis may play an important role in long-chain fat digestion. 6 less
Martinez-Martinez M, Coscolin C, Santiago G, Chow J, Stogios PJ, Bargiela R, Gertler C, Navarro-Fernandez J, Bollinger A and Ferrer M <32 more author(s)> Martinez-Martinez M, Coscolin C, Santiago G, Chow J, Stogios PJ, Bargiela R, Gertler C, Navarro-Fernandez J, Bollinger A, Thies S, Mendez-Garcia C, Popovic A, Brown G, Chernikova TN, Garcia-Moyano A, Bjergah GE, Perez-Garcia P, Hai T, Del Pozo MV, Stokke R, Steen IH, Cui H, Xu X, Nocek BP, Alcaide M, Distaso M, Mesa V, Pelaez AI, Sanchez J, Buchholz PCF, Pleiss J, Fernandez-Guerra A, Glockner FO, Golyshina OV, Yakimov MM, Savchenko A, Jaeger KE, Yakunin AF, Streit WR, Golyshin PN, Guallar V, Ferrer M (- 32) Ref: ACS Chemical Biology, 13:225, 2018 : PubMed ESTHER: Martinez-Martinez_2018_ACS.Chem.Biol_13_225 PubMedSearch: Martinez-Martinez 2018 ACS.Chem.Biol 13 225 PubMedID: 29182315 Gene_locus related to this paper: 9zzzz-a0a2k8jn75, 9zzzz-a0a2k8jt94, 9zzzz-a0a0g3fj44, 9zzzz-a0a0g3fh10, 9zzzz-a0a0g3fh03, 9bact-a0a1s5qkj8, 9zzzz-a0a0g3feh5, 9zzzz-a0a0g3fkz4, 9zzzz-a0a0g3fh07, 9zzzz-a0a0g3fh34, 9zzzz-a0a0g3fh31, 9bact-KY458167, alcbs-q0vqa3, 9bact-a0a1s5qki8, 9zzzz-a0a0g3feq8, 9zzzz-a0a0g3feh8, 9zzzz-a0a0g3fh19, 9bact-KY203037, 9bact-a0a1s5ql22, 9bact-a0a1s5qm34, 9bact-KY203034, 9bact-r9qzg0, 9bact-a0a1s5qly8, 9zzzz-a0a0g3fkz8, 9zzzz-a0a0g3feg9, 9zzzz-KY203033, 9zzzz-a0a0g3fes4, 9zzzz-a0a0g3fh42, 9bact-a0a1s5qlx2, 9zzzz-KY483651, 9bact-a0a1s5qmh4, 9zzzz-KY203032, 9zzzz-EH87, 9zzzz-a0a0g3fei1, 9zzzz-a0a0g3fet2, 9zzzz-KY483647, 9zzzz-EH82, 9zzzz-a0a0g3fe15, 9bact-KY203031, 9bact-t1w006, 9zzzz-a0a0g3fet6, 9bact-KY458164, geoth-g8myf3, 9bact-a0a1s5ql04, 9gamm-a0a1y0ihk7, 9bact-a0a1s5qly6, 9bact-a0a1s5qkg4, 9bact-a0a1s5qkm4, 9gamm-s5tv80, 9gamm-a0a0c4zhg2, 9zzzz-t1b379, 9gamm-KY483646, 9bact-KY458160, 9zzzz-a0a0g3fj57, 9gamm-s5t8349, 9arch-KY203036, 9bact-KY458168, 9zzzz-a0a0g3fes0, 9zzzz-t1be47, 9bact-KY458159, 9zzzz-a0a0g3fh39, 9bact-t1vzd5, 9prot-EH41, 9bact-Lip114, alcbs-q0vt77, 9bact-a0a1s5qke6, 9bact-a0a1s5qkf3, 9prot-SRP030024, 9gamm-s5t532, 9bact-a0a1s5qkl2, 9bact-a0a1s5qkk8, 9zzzz-KY203030, 9zzzz-t1d4I7, 9prot-KY019260, 9bact-a0a1s5qm38, 9arch-KY458161, 9prot-KY010302, 9zzzz-a0a0g3fl25, 9actn-KY010298, 9gamm-s5u059, 9bact-a0a1s5qmi7, 9bact-KY010297, 9bact-KY483642, 9bact-a0a1s5qkj1, 9bact-KY010299, 9bact-KY483648, alcbs-q0vtl7, 9bact-a0a1s5qf1, 9bact-a0a1s5qkg0, 9bact-a0a0h4tgu6, 9bact-MilE3, 9bact-LAE6, 9alte-MGS-MT1, 9bact-r9qzf7, 9gamm-k0c6t6, alcbs-q0vl36, alcbs-q0vlq1, alcbs-q0vq49, bacsu-pnbae, canar-LipB, canan-lipasA, geost-lipas, marav-a1u5n0, pseps-i7k8x5, staep-GEHD, symth-q67mr3, altma-s5cfn7, cycsp-k0c2b8, alcbs-q0vlk5, 9bact-k7qe48, 9bact-MGS-M1, 9bact-MGS-M2, 9bact-a0a0b5kns5, 9zzzz-a0a0g3fej4, 9zzzz-a0a0g3fj60, 9zzzz-a0a0g3fej0, 9zzzz-a0a0g3fj64, 9bact-a0a0b5kc16, 9zzzz-a0a0g3feg6, 9zzzz-a0a0g3feu6 Abstract Esterases receive special attention because their wide distribution in biological systems and environments and their importance for physiology and chemical synthesis. The prediction of esterases substrate promiscuity level from sequence data and the molecular reasons why certain such enzymes are more promiscuous than others, remain to be elucidated. This limits the surveillance of the sequence space for esterases potentially leading to new versatile biocatalysts and new insights into their role in cellular function. Here we performed an extensive analysis of the substrate spectra of 145 phylogenetically and environmentally diverse microbial esterases, when tested with 96 diverse esters. We determined the primary factors shaping their substrate range by analyzing substrate range patterns in combination with structural analysis and protein-ligand simulations. We found a structural parameter that helps ranking (classifying) promiscuity level of esterases from sequence data at 94% accuracy. This parameter, the active site effective volume, exemplifies the topology of the catalytic environment by measuring the active site cavity volume corrected by the relative solvent accessible surface area (SASA) of the catalytic triad. Sequences encoding esterases with active site effective volumes (cavity volume/SASA) above a threshold show greater substrate spectra, which can be further extended in combination with phylogenetic data. This measure provides also a valuable tool for interrogating substrates capable of being converted. This measure, found to be transferred to phosphatases of the haloalkanoic acid dehalogenase superfamily and possibly other enzymatic systems, represents a powerful tool for low-cost bioprospecting for esterases with broad substrate ranges, in large scale sequence datasets. Title: Characterization of LipN (Rv2970c) of Mycobacterium Tuberculosis H37Rv and its Probable Role in Xenobiotic Degradation Jadeja D, Dogra N, Arya S, Singh G, Kaur J Ref: Journal of Cellular Biochemistry, 117:390, 2016 : PubMed ESTHER: Jadeja_2016_J.Cell.Biochem_117_390 PubMedSearch: Jadeja 2016 J.Cell.Biochem 117 390 PubMedID: 26212120 Gene_locus related to this paper: myctu-Rv2970c Abstract LipN (Rv2970c) belongs to the Lip family of M. tuberculosis H37Rv and is homologous to the human Hormone Sensitive Lipase. The enzyme demonstrated preference for short carbon chain substrates with optimal activity at 45 degrees C/pH 8.0 and stability between pH 6.0-9.0. The specific activity of the enzyme was 217 U/mg protein with pNP-butyrate as substrate. It hydrolyzed tributyrin to di- and monobutyrin. The active-site residues of the enzyme were confirmed to be Ser216, Asp316, and His346. Tetrahydrolipstatin, RHC-80267 and N-bromosuccinimide inhibited LipN enzyme activity completely. Interestingly, Trp145, a non active-site residue, demonstrated functional role to retain enzyme activity. The enzyme was localized in cytosolic fraction of M. tuberculosis H37Rv. The enzyme was able to synthesize ester of butyric acid, methyl butyrate, in presence of methanol. LipN was able to hydrolyze 4-hydroxyphenylacetate to hydroquinone. The gene was not expressed in in-vitro growth conditions while the expression of rv2970c gene was observed post 6h of macrophage infection by M. tuberculosis H37Ra. Under individual in-vitro stress conditions, the gene was expressed during acidic stress condition only. These findings suggested that LipN is a cytosolic, acid inducible carboxylesterase with no positional specificity in demonstrating activity with short carbon chain substrates. It requires Trp145, a non active site residue, for it's enzyme activity. J. Cell. Biochem. 117: 390-401, 2016. (c) 2015 Wiley Periodicals, Inc. Title: The beta5-Loop and Lid Domain Contribute to the Substrate Specificity of Pancreatic Lipase-related Protein 2 (PNLIPRP2) Xiao X, Lowe ME Ref: Journal of Biological Chemistry, 290:28847, 2015 : PubMed ESTHER: Xiao_2015_J.Biol.Chem_290_28847 PubMedSearch: Xiao 2015 J.Biol.Chem 290 28847 PubMedID: 26494624 Gene_locus related to this paper: human-PNLIP, human-PNLIPRP2 Abstract Pancreatic triglyceride lipase (PNLIP) is essential for dietary fat digestion in children and adults, whereas a homolog, pancreatic lipase-related protein 2 (PNLIPRP2), is critical in newborns. The two lipases are structurally similar, yet they have different substrate specificities. PNLIP only cleaves neutral fats. PNLIPRP2 cleaves neutral and polar fats. To test the hypothesis that the differences in activity between PNLIP and PNLIPRP2 are governed by surface loops around the active site, we created multiple chimeras of both lipases by exchanging the surface loops singly or in combination. The chimeras were expressed, purified, and tested for activity against various substrates. The structural determinants of PNLIPRP2 galactolipase activity were contained in the N-terminal domain. Of the surface loops tested, the lid domain and the beta5-loop influenced activity against triglycerides and galactolipids. Any chimera on PNLIP with the PNLIPRP2 lid domain or beta5-loop had decreased triglyceride lipase activity similar to that of PNLIPRP2. The corresponding chimeras of PNLIPRP2 did not increase activity against neutral lipids. Galactolipase activity was abolished by the PNLIP beta5-loop and decreased by the PNLIP lid domain. The source of the beta9-loop had minimal effect on activity. We conclude that the lid domain and beta5-loop contribute to substrate specificity but do not completely account for the differing activities of PNLIP and PNLIPRP2. Other regions in the N-terminal domain must contribute to the galactolipase activity of PNLIPRP2 through direct interactions with the substrate or by altering the conformation of the residues surrounding the hydrophilic cavity in PNLIPRP2. Title: Structure of human pancreatic lipase-related protein 2 with the lid in an open conformation Eydoux C, Spinelli S, Davis TL, Walker JR, Seitova A, Dhe-Paganon S, de Caro A, Cambillau C, Carriere F Ref: Biochemistry, 47:9553, 2008 : PubMed ESTHER: Eydoux_2008_Biochemistry_47_9553 PubMedSearch: Eydoux 2008 Biochemistry 47 9553 PubMedID: 18702514 Gene_locus related to this paper: human-PNLIPRP2 Abstract Access to the active site of pancreatic lipase (PL) is controlled by a surface loop, the lid, which normally undergoes conformational changes only upon addition of lipids or amphiphiles. Structures of PL with their lids in the open and functional conformation have required cocrystallization with amphiphiles. Here we report two crystal structures of wild-type and unglycosylated human pancreatic lipase-related protein 2 (HPLRP2) with the lid in an open conformation in the absence of amphiphiles. These structures solved independently are strikingly similar, with some residues of the lid being poorly defined in the electron-density map. The open conformation of the lid is however different from that previously observed in classical liganded PL, suggesting different kinetic properties for HPLRP2. Here we show that the HPLRP2 is directly inhibited by E600, does not present interfacial activation, and acts preferentially on substrates forming monomers or small aggregates (micelles) dispersed in solution like monoglycerides, phospholipids and galactolipids, whereas classical PL displays reverse properties and a high specificity for unsoluble substrates like triglycerides and diglycerides forming oil-in-water interfaces. These biochemical properties imply that the lid of HPLRP2 is likely to spontaneously adopt in solution the open conformation observed in the crystal structure. This open conformation generates a large cavity capable of accommodating the digalactose polar head of galactolipids, similar to that previously observed in the active site of the guinea pig PLRP2, but absent from the classical PL. Most of the structural and kinetic properties of HPLRP2 were found to be different from those of rat PLRP2, the structure of which was previously obtained with the lid in a closed conformation. Our findings illustrate the essential role of the lid in determining the substrate specificity and the mechanism of action of lipases. Title: Optimization of a thermostable lipase from Bacillus stearothermophilus P1: overexpression, purification, and characterization Sinchaikul S, Sookkheo B, Phutrakul S, Pan FM, Chen ST Ref: Protein Expr Purif, 22:388, 2001 : PubMed ESTHER: Sinchaikul_2001_Protein.Expr.Purif_22_388 PubMedSearch: Sinchaikul 2001 Protein.Expr.Purif 22 388 PubMedID: 11483000 Gene_locus related to this paper: geost-lipas Inhibitor(s) related to this paper: 1-hexadecanesulfonyl-chloride Abstract An expression library was generated from a partial NcoI and HindIII digest of genomic DNA from the thermophilic bacterium, Bacillus stearothermophilus P1. The DNA fragments were cloned into the expression vector pQE-60 and transformed into Escherichia coli M15[EP4]. Sequence analysis of a lipase gene showed an open reading frame of 1254 nucleotides coding a 29-amino-acid signal sequence and a mature sequence of 388 amino acids. The expressed lipase was isolated and purified to homogeneity in a single chromatographic step. The molecular mass of the lipase was determined to be approximately 43 kDa by SDS-PAGE and mass spectrometry. The purified lipase had an optimum pH of 8.5 and showed maximal activity at 55 degrees C. It was highly stable in the temperature range of 30-65 degrees C. The highest activity was found with p-nitrophenyl ester-caprate as the synthetic substrate and tricaprylin as the triacylglycerol. Its activity was strongly inhibited by 10 mM phenylmethanesulfonyl fluoride and 1-hexadecanesulfonyl chloride, indicating that it contains a serine residue which plays a key role in the catalytic mechanism. In addition, it was stable for 1 h at 37 degrees C in 0.1% Chaps and Triton X-100. Title: A novel extracellular esterase from Bacillus subtilis and its conversion to a monoacylglycerol hydrolase Eggert T, Pencreac'h G, Douchet I, Verger R, Jaeger KE Ref: European Journal of Biochemistry, 267:6459, 2000 : PubMed ESTHER: Eggert_2000_Eur.J.Biochem_267_6459 PubMedSearch: Eggert 2000 Eur.J.Biochem 267 6459 PubMedID: 11029590 Gene_locus related to this paper: bacsu-LIPB Abstract A novel gene lipB, which encodes an extracellular lipolytic enzyme, was identified in the Bacillus subtilis genomic DNA sequence. We have cloned and overexpressed lipB in B. subtilis and Escherichia coli and have also purified the enzyme from a B. subtilis culture supernatant to electrophoretic homogeneity. Four different lipase assays were used to determine its catalytic activity: pH-stat, spectrophotometry, fluorimetry and the monomolecular film technique. LipB preferentially hydrolysed triacylglycerol-esters and p-nitrophenyl-esters of fatty acids with short chain lengths of <= 10 carbon atoms. Triolein, which is a typical substrate for true lipases, was not hydrolysed at all. These results led us to classify LipB as an esterase rather than a lipase. The catalytic triad of LipB consists of residues Ser78, Asp134, and His157 as demonstrated by amino-acid sequence alignments and site-directed mutagenesis. The nucleophile Ser78 is located in a lipase-specific consensus sequence, which is Ala-X-Ser-X-Gly for most Bacillus lipases. All other bacterial lipases contain a glycine residue instead of the alanine at position-2 with respect to the catalytic serine. We have investigated the role of this alanine residue by constructing LipB variant A76G, thereby restoring the lipase-specific consensus motif. When compared with LipB this variant showed a markedly reduced thermostability but an increased stability at pH 5-7. Determination of the specific activities of wild-type LipB and variant A76G using a monomolecular film of the substrate monoolein revealed an interesting result: the A76G substitution had converted the esterase LipB into a monoacylglycerol hydrolase. Title: Purification and biochemical characterization of dog gastric lipase Carriere F, Moreau H, Raphel V, Laugier R, Benicourt C, Junien JL, Verger R Ref: European Journal of Biochemistry, 202:75, 1991 : PubMed ESTHER: Carriere_1991_Eur.J.Biochem_202_75 PubMedSearch: Carriere 1991 Eur.J.Biochem 202 75 PubMedID: 1935982 Gene_locus related to this paper: canfa-1lipg Abstract A lipase was found to be present in dog stomach which appeared to be more abundant in the fundic than in the pyloric mucosa. Dog gastric lipase was extracted by soaking the gastric tissue and further purified after cation exchange, anion exchange and gel-filtration using fast protein liquid chromatography. The amino-acid composition, N-terminal amino-acid sequence, substrate specificity, interfacial and kinetic behavior and inactivation by sulfhydryl reagents were determined and compared with those of human and rabbit gastric lipases. We report for the first time that a gastric lipase is 13 times more active on long-chain than on short-chain triacylglycerols at pH 4.0, reaching a maximal specific activity of 950 U/mg on Intralipide emulsion. Title: Purification, characterization and kinetic properties of the rabbit gastric lipase Moreau H, Gargouri Y, Lecat D, Junien JL, Verger R Ref: Biochimica & Biophysica Acta, 960:286, 1988 : PubMed ESTHER: Moreau_1988_Biochim.Biophys.Acta_960_286 PubMedSearch: Moreau 1988 Biochim.Biophys.Acta 960 286 PubMedID: 3382677 Abstract Rabbit gastric lipase was purified from an acetonic powder of rabbit stomach fundus. 25 mg of pure rabbit gastric lipase (glycerol ester hydrolase, EC 3.1.1.3) was obtained from 30 rabbit stomachs after ammonium sulfate fractionation, Sephadex G-100 gel filtration and cation exchange (mono S column) using a fast protein liquid chromatography (FPLC) system. The pure enzyme obtained was resistant to acidic pH conditions, and had specific activities of 1200, 850 and 280 U/mg, using, respectively, short- (tributyroylglycerol (TC4)), medium- (trioctanoyl- to tridecanoylglycerol (TC8-TC10)) and long-chain (soybean oil) triacylglycerols. The amino-acid composition was determined, and the first 30 N-terminal amino-acid residues were sequenced. Interfacial denaturation and catalytic properties on triacylglycerol emulsions were studied. Rabbit gastric lipase turned out to be structurally and kinetically very similar to human gastric lipase. Title: Kinetic assay of human gastric lipase on short- and long-chain triacylglycerol emulsions Gargouri Y, Pieroni G, Riviere C, Sauniere JF, Lowe PA, Sarda L, Verger R Ref: Gastroenterology, 91:919, 1986 : PubMed ESTHER: Gargouri_1986_Gastroenterology_91_919 PubMedSearch: Gargouri 1986 Gastroenterology 91 919 PubMedID: 3743968 Abstract Under optimal conditions, assay for pure human gastric lipase was carried out with short- and long-chain triacylglycerol emulsions. Maximal specific activities of 1160 and 620 U/mg were obtained with tributyrin and soybean emulsion, respectively. We observed that with a tributyrin substrate, bovine serum albumin or bile salts must be added before the addition of the enzyme in order to prevent its irreversible interfacial denaturation. With long-chain triacylglycerols as substrate, a decrease with time in the rate of hydrolysis was associated with release of protonated long-chain fatty acids. The inhibitory effect of protonated fatty acids was also observed using tributyrin at pH 3.0. These observations support the conclusion that human gastric lipase shows no intrinsic specificity for short-chain triacylglycerols and that its apparent specificity is modulated by pH and presence of amphiphile in the incubation medium. Our conclusions support the view that, in the human, gastric lipolysis may play an important role in long-chain fat digestion. |
