Gene_locus Report for: canan-lipasA Candida antarctica (Trichosporon oryzae) (yeast) CalA Lipase AComment Ericsson, D.J., Kasrayan, A., Johansson, P., Bergfors, T., Sandstrom, A.G., Backvall, J.E., Mowbray, S.L. X-Ray Structure of Candida Antarctica Lipase A Shows a Novel Lid Structure and a Likely Mode of Interfacial Activation. Sold under the name lipase novozym 735 Novocor AD L CalA promiscuous activity 36/96 substrates Martinez-Martinez et al. 2018 Relationship
(Below N is a link to NCBI taxonomic web page and E link to ESTHER at designed phylum.) There are 2 a/b hydrolases in Candida antarctica view in a: Table
Canar_LipB : canar-LipB Candida antarctica (Trichosporon oryzae) (yeast) Lipase B CALB, CALipB ,Novozyme 435
Sequence
References 12 more
Alejaldre L, Lemay-St-Denis C, Pelletier JN, Quaglia D Ref: Biochemistry, :, 2022 : PubMed ESTHER: Alejaldre_2022_Biochemistry__ PubMedSearch: Alejaldre 2022 Biochemistry PubMedID: 36580299 Gene_locus related to this paper: canan-lipasA Abstract Engineering studies of Candida (Pseudozyma) antarctica lipase A (CalA) have demonstrated the potential of this enzyme in the selective hydrolysis of fatty acid esters of different chain lengths. CalA has been shown to bind substrates preferentially through an acyl-chain binding tunnel accessed via the hydrolytic active site; it has also been shown that selectivity for substrates of longer or shorter chain length can be tuned, for instance by modulating steric hindrance within the tunnel. Here we demonstrate that, whereas the tunnel region is certainly of paramount importance for substrate recognition, residues in distal regions of the enzyme can also modulate substrate selectivity. To this end, we investigate variants that carry one or more substitutions within the substrate tunnel as well as in distal regions. Combining experimental determination of the substrate selectivity using natural and synthetic substrates with computational characterization of protein dynamics and of tunnels, we deconvolute the effect of key substitutions and demonstrate that epistatic interactions contribute to procuring selectivity toward either long-chain or short/medium-chain fatty acid esters. We demonstrate that various mechanisms contribute to the diverse selectivity profiles, ranging from reshaping tunnel morphology and tunnel stabilization to obstructing the main substrate-binding tunnel, highlighting the dynamic nature of the substrate-binding region. This work provides important insights into the versatility of this robust lipase toward diverse applications. Title: Suppression of water as a nucleophile in Candida antarctica lipase B catalysis Larsen MW, Zielinska DF, Martinelle M, Hidalgo A, Jensen LJ, Bornscheuer UT, Hult K Ref: Chembiochem, 11:796, 2010 : PubMed ESTHER: Larsen_2010_Chembiochem_11_796 PubMedSearch: Larsen 2010 Chembiochem 11 796 PubMedID: 20235107 Gene_locus related to this paper: canan-lipasA Abstract A water tunnel in Candida antarctica lipase B that provides the active site with substrate water is hypothesized. A small, focused library created in order to prevent water from entering the active site through the tunnel was screened for increased transacylation over hydrolysis activity. A single mutant, S47L, in which the inner part of the tunnel was blocked, catalysed the transacylation of vinyl butyrate to 20 mM butanol 14 times faster than hydrolysis. The single mutant Q46A, which has a more open outer end of the tunnel, showed an increased hydrolysis rate and a decreased hydrolysis to transacylation ratio compared to the wild-type lipase. Mutants with a blocked tunnel could be very useful in applications in which hydrolysis is unwanted, such as the acylation of highly hydrophilic compounds in the presence of water. Title: X-ray structure of Candida antarctica lipase A shows a novel lid structure and a likely mode of interfacial activation Ericsson DJ, Kasrayan A, Johansson P, Bergfors T, Sandstrom AG, Backvall JE, Mowbray SL Ref: Journal of Molecular Biology, 376:109, 2008 : PubMed ESTHER: Ericsson_2008_J.Mol.Biol_376_109 PubMedSearch: Ericsson 2008 J.Mol.Biol 376 109 PubMedID: 18155238 Gene_locus related to this paper: canan-lipasA Inhibitor(s) related to this paper: Tetraethylene-glycol Abstract In nature, lipases (EC 3.1.1.3) catalyze the hydrolysis of triglycerides to form glycerol and fatty acids. Under the appropriate conditions, the reaction is reversible, and so biotechnological applications commonly make use of their capacity for esterification as well as for hydrolysis of a wide variety of compounds. In the present paper, we report the X-ray structure of lipase A from Candida antarctica, solved by single isomorphous replacement with anomalous scattering, and refined to 2.2-A resolution. The structure is the first from a novel family of lipases. Contrary to previous predictions, the fold includes a well-defined lid as well as a classic alpha/beta hydrolase domain. The catalytic triad is identified as Ser184, Asp334 and His366, which follow the sequential order considered to be characteristic of lipases; the serine lies within a typical nucleophilic elbow. Computer docking studies, as well as comparisons to related structures, place the carboxylate group of a fatty acid product near the serine nucleophile, with the long lipid tail closely following the path through the lid that is marked by a fortuitously bound molecule of polyethylene glycol. For an ester substrate to bind in an equivalent fashion, loop movements near Phe431 will be required, suggesting the primary focus of the conformational changes required for interfacial activation. Such movements will provide virtually unlimited access to solvent for the alcohol moiety of an ester substrate. The structure thus provides a basis for understanding the enzyme's preference for acyl moieties with long, straight tails, and for its highly promiscuous acceptance of widely different alcohol and amine moieties. An unconventional oxyanion hole is observed in the present structure, although the situation may change during interfacial activation. 12 less
Alejaldre L, Lemay-St-Denis C, Pelletier JN, Quaglia D Ref: Biochemistry, :, 2022 : PubMed ESTHER: Alejaldre_2022_Biochemistry__ PubMedSearch: Alejaldre 2022 Biochemistry PubMedID: 36580299 Gene_locus related to this paper: canan-lipasA Abstract Engineering studies of Candida (Pseudozyma) antarctica lipase A (CalA) have demonstrated the potential of this enzyme in the selective hydrolysis of fatty acid esters of different chain lengths. CalA has been shown to bind substrates preferentially through an acyl-chain binding tunnel accessed via the hydrolytic active site; it has also been shown that selectivity for substrates of longer or shorter chain length can be tuned, for instance by modulating steric hindrance within the tunnel. Here we demonstrate that, whereas the tunnel region is certainly of paramount importance for substrate recognition, residues in distal regions of the enzyme can also modulate substrate selectivity. To this end, we investigate variants that carry one or more substitutions within the substrate tunnel as well as in distal regions. Combining experimental determination of the substrate selectivity using natural and synthetic substrates with computational characterization of protein dynamics and of tunnels, we deconvolute the effect of key substitutions and demonstrate that epistatic interactions contribute to procuring selectivity toward either long-chain or short/medium-chain fatty acid esters. We demonstrate that various mechanisms contribute to the diverse selectivity profiles, ranging from reshaping tunnel morphology and tunnel stabilization to obstructing the main substrate-binding tunnel, highlighting the dynamic nature of the substrate-binding region. This work provides important insights into the versatility of this robust lipase toward diverse applications. Title: Transesterification of a Tertiary Alcohol by Engineered Candida antarctica Lipase A Lofgren J, Gorbe T, Oschmann M, Svedendahl Humble M, Backvall JE Ref: Chembiochem, 20:1438, 2019 : PubMed ESTHER: Lofgren_2019_Chembiochem_20_1438 PubMedSearch: Lofgren 2019 Chembiochem 20 1438 PubMedID: 30676685 Gene_locus related to this paper: canan-lipasA Abstract Tertiary alcohols are known to be challenging substrates for applications in asymmetric synthesis due to their complexity and steric hinderance. The occurrence of tertiary alcohols and their esters in nature indicates the presence of natural biocatalytic synthetic routes for their preparation. Lipase A from Candida antarctica (CalA) is a hydrolase that has previously been shown to catalyze the transesterification of racemic 2-phenylbut-3-yn-2-ol at a low rate. In this work, the activity of that enzyme was improved by protein engineering through a semi-rational design strategy. An enzyme library was created and screened for transesterification activity towards racemic 2-phenylbut-3-yn-2-ol in an organic solvent. One successful enzyme variant (L367G) showed a tenfold increased reaction rate compared to the wild-type enzyme, while maintaining a high enantioselectivity. Title: Holistic engineering of Cal-A lipase chain-length selectivity identifies triglyceride binding hot-spot Quaglia D, Alejaldre L, Ouadhi S, Rousseau O, Pelletier JN Ref: PLoS ONE, 14:e0210100, 2019 : PubMed ESTHER: Quaglia_2019_PLoS.One_14_e0210100 PubMedSearch: Quaglia 2019 PLoS.One 14 e0210100 PubMedID: 30640952 Gene_locus related to this paper: canan-lipasA Abstract Through the application of a region-focused saturation mutagenesis and randomization approach, protein engineering of the Cal-A enzyme was undertaken with the goal of conferring new triglyceride selectivity. Little is known about the mode of triglyceride binding to Cal-A. Engineering Cal-A thus requires a systemic approach. Targeted and randomized Cal-A libraries were created, recombined using the Golden Gate approach and screened to detect variants able to discriminate between long-chain (olive oil) and short-chain (tributyrin) triglyceride substrates using a high-throughput in vivo method to visualize hydrolytic activity. Discriminative variants were analyzed using an in-house script to identify predominant substitutions. This approach allowed identification of variants that exhibit strong discrimination for the hydrolysis of short-chain triglycerides and others that discriminate towards hydrolysis of long-chain triglycerides. A clear pattern emerged from the discriminative variants, identifying the 217-245 helix-loop-helix motif as being a hot-spot for triglyceride recognition. This was the consequence of introducing the entire mutational load in selected regions, without putting a strain on distal parts of the protein. Our results improve our understanding of the Cal-A lipase mode of action and selectivity. This holistic perspective to protein engineering, where parts of the gene are individually mutated and the impact evaluated in the context of the whole protein, can be applied to any protein scaffold. Title: Determinants and prediction of esterase substrate promiscuity patterns 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: Rationale behind the near-ideal catalysis of Candida antarctica lipase A (CAL-A) for highly concentrating omega-3 polyunsaturated fatty acids into monoacylglycerols He Y, Li J, Kodali S, Chen B, Guo Z Ref: Food Chem, 219:230, 2017 : PubMed ESTHER: He_2017_Food.Chem_219_230 PubMedSearch: He 2017 Food.Chem 219 230 PubMedID: 27765222 Gene_locus related to this paper: canan-lipasA Abstract Dramatic decline in the quality and quantity of omega-3 PUFAs from marine resource demands new environmental-friendly technology to produce high quality omega-3 PUFAs concentrates in a better bioavailable form. Accordingly this work demonstrated an exceptionally highly efficient non-aqueous approach that non-regiospecific and non omega-3 PUFAs preferential Candida antarctica lipase A (CAL-A), functioning as a near-ideal biocatalyst, is capable to directly concentrate omega-3 PUFAs from 20% to 30% in oils to up to >90% in monoacylglycerols form through one step reaction. The rationale behind the experimental observation is justified and the catalytic property and specificity of an ideal enzyme tackling this task are defined. High selectivity and efficiency, excellent reusability of biocatalyst, general applicability for concentrating omega-3 PUFAs from both fish and microalgae oils, simple process for product recovery (e.g. by short path distillation), make this novel approach a highly industrially relevant and with potential application in food and drug industries. Title: Elucidation of a key position for acyltransfer activity in Candida parapsilosis lipase/acyltransferase (CpLIP2) and in Pseudozyma antarctica lipase A (CAL-A) by rational design Jan AH, Subileau M, Deyrieux C, Perrier V, Dubreucq E Ref: Biochimica & Biophysica Acta, 1864:187, 2016 : PubMed ESTHER: Jan_2016_Biochim.Biophys.Acta_1864_187 PubMedSearch: Jan 2016 Biochim.Biophys.Acta 1864 187 PubMedID: 26602447 Gene_locus related to this paper: canan-lipasA, candc-CduLAc, canpa-LIP2 Abstract Performing transesterifications in aqueous media is becoming a priority challenge in lipid biotechnology in order to develop more eco-friendly and efficient biocatalytic processes in systems containing both polar and apolar substrates. In this context, our group has explored for several years the high potential of the lipase/acyltransferase CpLIP2 from Candida parapsilosis and of several of its homologs, that catalyze efficiently acyltransfer reactions in lipid/water media with high water activity (aw>0.9). The discovery of a new member of this group, CduLAc from Candida dubliniensis, with a higher acyltransferase activity than CpLIP2, has provided a new insight on structure-function relationships in this group. Indeed, the comparison of sequences and 3D models, especially of CpLIP2 and CduLAc, with those of the phylogenetically related lipase A from Pseudozyma antarctica (CAL-A), allowed elucidating a key structural determinant of the acyltransferase activity: serine S369 in CpLIP2 and its equivalents E370 in CAL-A and A366 in CduLAc. Mutants obtained by rational design at this key position showed significant changes in acyltransfer activity. Whereas mutation S369E resulted in an increase in the hydrolytic activity of CpLIP2, S369A increased alcoholysis. More strikingly, the single E370A mutation in CAL-A drastically increased the acyltransferase activity of this enzyme, giving it the character of a lipase/acyltransferase. Indeed, this single mutation lowered the methanol concentration for which the initial rates of alcoholysis and hydrolysis are equal from 2M in CAL-A down to 0.3M in its mutant, while the exceptional stability of the parental enzyme toward alcohol and temperature was conserved. Title: Peculiar features of four enzymes of the CaLA superfamily in aqueous media: Differences in substrate specificities and abilities to catalyze alcoholysis Neang PM, Subileau M, Perrier V, Dubreucq E Ref: J Mol Catal B Enzym, 94:36, 2013 : PubMed ESTHER: Neang_2013_J.Mol.Catal.B.Enzym_94_36 PubMedSearch: Neang 2013 J.Mol.Catal.B.Enzym 94 36 Gene_locus related to this paper: aspor-q2uix9, canan-lipasA, canpa-LIP2, cantt-c5mag0 Abstract With 31% of identity with the Lipase A of Candida antarctica (CaLA), the promising lipase/acyltransferase from Candida parapsilosis (CpLIP2) can be classified in the original CaLA-like superfamily. Contrary to CaLA, CpLIP2 has the exceptional property to catalyze acyltransfer reactions preferentially to hydrolysis even in aqueous media with high thermodynamic activity of water (aw > 0.9). Two new enzymes, CtroL4 from Candida tropicalis and AflaL0 from Aspergillus flavus, homologous to the CaLA-like superfamily proteins were obtained by heterologous production and used for comparative functional characterization in aqueous media. The ability of the lipases to catalyze acyltransfer reaction in water was correlated with their degree of homology with CpLIP2, indicating a global sequence/function relationship that could be very useful for the selection of new biocatalysts of high industrial interest. The four enzymes exhibited different substrate specificity profiles, considering the length and the carbon chain unsaturation degree of the acyl group in the donor ester, and the class and the position of the hydroxyl group of the acyl accepting alcohol. Within the lipases sequences, peculiar variability in the (putative) substrate binding site was observed and will be further investigated for the elucidation of structure/function relationships. Title: Creation of a lipase highly selective for trans fatty acids by protein engineering Brundiek HB, Evitt AS, Kourist R, Bornscheuer UT Ref: Angew Chem Int Ed Engl, 51:412, 2012 : PubMed ESTHER: Brundiek_2012_Angew.Chem.Int.Ed.Engl_51_412 PubMedSearch: Brundiek 2012 Angew.Chem.Int.Ed.Engl 51 412 PubMedID: 22113941 Gene_locus related to this paper: canan-lipasA Abstract Sorting out: Protein engineering of lipase CAL-A led to the discovery of mutants with excellent chemoselectivity for the removal of trans and saturated fatty acids from partially hydrogenated vegetable oil. These fatty acids, identified as a major risk factor for human health, can now be removed by enzyme catalysis. Title: Protein engineering of alpha/beta-hydrolase fold enzymes Jochens H, Hesseler M, Stiba K, Padhi SK, Kazlauskas RJ, Bornscheuer UT Ref: Chembiochem, 12:1508, 2011 : PubMed ESTHER: Jochens_2011_Chembiochem_12_1508 PubMedSearch: Jochens 2011 Chembiochem 12 1508 PubMedID: 21506229 Gene_locus related to this paper: canan-lipasA Abstract The superfamily of alpha/beta-hydrolase fold enzymes is one of the largest known protein families, including a broad range of synthetically useful enzymes such as lipases, esterases, amidases, hydroxynitrile lyases, epoxide hydrolases and dehalogenases. This minireview covers methods developed for efficient protein engineering of these enzymes. Special emphasis is placed on the alteration of enzyme properties such as substrate range, thermostability and enantioselectivity for their application in biocatalysis. In addition, concepts for the investigation of the evolutionary relationship between the different members of this protein superfamily are covered, together with successful examples. Title: Directed evolution of an enantioselective lipase with broad substrate scope for hydrolysis of alpha-substituted esters Engstrom K, Nyhlen J, Sandstrom AG, Backvall JE Ref: Journal of the American Chemical Society, 132:7038, 2010 : PubMed ESTHER: Engstrom_2010_J.Am.Chem.Soc_132_7038 PubMedSearch: Engstrom 2010 J.Am.Chem.Soc 132 7038 PubMedID: 20450151 Gene_locus related to this paper: canan-lipasA Abstract A variant of Candida antarctica lipase A (CalA) was developed for the hydrolysis of alpha-substituted p-nitrophenyl esters by directed evolution. The E values of this variant for 7 different esters was 45-276, which is a large improvement compared to 2-20 for the wild type. The broad substrate scope of this enzyme variant is of synthetic use, and hydrolysis of the tested substrates proceeded with an enantiomeric excess between 95-99%. A 30-fold increase in activity was also observed for most substrates. The developed enzyme variant shows (R)-selectivity, which is reversed compared to the wild type that is (S)-selective for most substrates. Title: Suppression of water as a nucleophile in Candida antarctica lipase B catalysis Larsen MW, Zielinska DF, Martinelle M, Hidalgo A, Jensen LJ, Bornscheuer UT, Hult K Ref: Chembiochem, 11:796, 2010 : PubMed ESTHER: Larsen_2010_Chembiochem_11_796 PubMedSearch: Larsen 2010 Chembiochem 11 796 PubMedID: 20235107 Gene_locus related to this paper: canan-lipasA Abstract A water tunnel in Candida antarctica lipase B that provides the active site with substrate water is hypothesized. A small, focused library created in order to prevent water from entering the active site through the tunnel was screened for increased transacylation over hydrolysis activity. A single mutant, S47L, in which the inner part of the tunnel was blocked, catalysed the transacylation of vinyl butyrate to 20 mM butanol 14 times faster than hydrolysis. The single mutant Q46A, which has a more open outer end of the tunnel, showed an increased hydrolysis rate and a decreased hydrolysis to transacylation ratio compared to the wild-type lipase. Mutants with a blocked tunnel could be very useful in applications in which hydrolysis is unwanted, such as the acylation of highly hydrophilic compounds in the presence of water. Title: Formation and hydrolysis of amide bonds by lipase A from Candida antarctica; exceptional features Liljeblad A, Kallio P, Vainio M, Niemi J, Kanerva LT Ref: Org Biomol Chem, 8:886, 2010 : PubMed ESTHER: Liljeblad_2010_Org.Biomol.Chem_8_886 PubMedSearch: Liljeblad 2010 Org.Biomol.Chem 8 886 PubMedID: 20135048 Gene_locus related to this paper: canan-lipasA Abstract Various commercial lyophilized and immobilized preparations of lipase A from Candida antarctica (CAL-A) were studied for their ability to catalyze the hydrolysis of amide bonds in N-acylated alpha-amino acids, 3-butanamidobutanoic acid (beta-amino acid) and its ethyl ester. The activity toward amide bonds is highly untypical of lipases, despite the close mechanistic analogy to amidases which normally catalyze the corresponding reactions. Most CAL-A preparations cleaved amide bonds of various substrates with high enantioselectivity, although high variations in substrate selectivity and catalytic rates were detected. The possible role of contaminant protein species on the hydrolytic activity toward these bonds was studied by fractionation and analysis of the commercial lyophilized preparation of CAL-A (Cat#ICR-112, Codexis). In addition to minor impurities, two equally abundant proteins were detected, migrating on SDS-PAGE a few kDa apart around the calculated size of CAL-A. Based on peptide fragment analysis and sequence comparison both bands shared substantial sequence coverage with CAL-A. However, peptides at the C-terminal end constituting a motile domain described as an active-site flap were not identified in the smaller fragment. Separated gel filtration fractions of the two forms of CAL-A both catalyzed the amide bond hydrolysis of ethyl 3-butanamidobutanoate as well as the N-acylation of methyl pipecolinate. Hydrolytic activity towards N-acetylmethionine was, however, solely confined to the fractions containing the truncated form of CAL-A. These fractions were also found to contain a trace enzyme impurity identified in sequence analysis as a serine carboxypeptidase. The possible role of catalytic impurities versus the function of CAL-A in amide bond hydrolysis is further discussed in the paper. Title: The crystal structure of lipase a from Candida Antarctica Brandt AM, Nymalm-Rejstrom Y, Airenne T, Salminen T Ref: Acta Crystallogr A Found Crystallogr, A64:C245, 2008 : PubMed ESTHER: Brandt_2008_Acta.Crystallogr.A.Found.Crystallogr_64_C245 PubMedSearch: Brandt 2008 Acta.Crystallogr.A.Found.Crystallogr 64 C245 Gene_locus related to this paper: canan-lipasA Inhibitor(s) related to this paper: Pentaethylene-glycol Abstract Lipases are widely used as catalysts in industrial applications due to their thermostability and enantioselectivity. Candida antarctica lipase A (CAL-A) is highly thermostable in organic solvents and has therefore become a frequently used catalyst in chemical and pharmaceutical industry. CAL-A shows some unusual properties, which makes it a highly attractive enzyme. CAL-A is the only known lipase to have Sn2-preference towards triglycerides. It is able to hydrolyze sterically hindered alcohols, both secondary and tertiary alcohols. In addition, it shows a high chemoselectivity for the N-acylation of beta-amio esters, which makes CAL-A an important catalyst in the production of enantiopure amino acids. We have determined the crystal structure of CAL-A at 2.1 A resolution. CAL-A exhibits a typical alpha/beta hydrolase fold, consisting of a central beta-sheet and surrounding alpha-helices. The active site pocket is formed like a deep L-shaped tunnel covered by a lid that regulates the interfacial activation. Residues Ser184, Asp334 and His366 form the catalytic triad at the bottom of the pocket. Title: X-ray structure of Candida antarctica lipase A shows a novel lid structure and a likely mode of interfacial activation Ericsson DJ, Kasrayan A, Johansson P, Bergfors T, Sandstrom AG, Backvall JE, Mowbray SL Ref: Journal of Molecular Biology, 376:109, 2008 : PubMed ESTHER: Ericsson_2008_J.Mol.Biol_376_109 PubMedSearch: Ericsson 2008 J.Mol.Biol 376 109 PubMedID: 18155238 Gene_locus related to this paper: canan-lipasA Inhibitor(s) related to this paper: Tetraethylene-glycol Abstract In nature, lipases (EC 3.1.1.3) catalyze the hydrolysis of triglycerides to form glycerol and fatty acids. Under the appropriate conditions, the reaction is reversible, and so biotechnological applications commonly make use of their capacity for esterification as well as for hydrolysis of a wide variety of compounds. In the present paper, we report the X-ray structure of lipase A from Candida antarctica, solved by single isomorphous replacement with anomalous scattering, and refined to 2.2-A resolution. The structure is the first from a novel family of lipases. Contrary to previous predictions, the fold includes a well-defined lid as well as a classic alpha/beta hydrolase domain. The catalytic triad is identified as Ser184, Asp334 and His366, which follow the sequential order considered to be characteristic of lipases; the serine lies within a typical nucleophilic elbow. Computer docking studies, as well as comparisons to related structures, place the carboxylate group of a fatty acid product near the serine nucleophile, with the long lipid tail closely following the path through the lid that is marked by a fortuitously bound molecule of polyethylene glycol. For an ester substrate to bind in an equivalent fashion, loop movements near Phe431 will be required, suggesting the primary focus of the conformational changes required for interfacial activation. Such movements will provide virtually unlimited access to solvent for the alcohol moiety of an ester substrate. The structure thus provides a basis for understanding the enzyme's preference for acyl moieties with long, straight tails, and for its highly promiscuous acceptance of widely different alcohol and amine moieties. An unconventional oxyanion hole is observed in the present structure, although the situation may change during interfacial activation. Title: Enzymatic removal of carboxyl protecting groups. 1. Cleavage of the tert-butyl moiety Schmidt M, Barbayianni E, Fotakopoulou I, Hohne M, Constantinou-Kokotou V, Bornscheuer UT, Kokotos G Ref: J Org Chem, 70:3737, 2005 : PubMed ESTHER: Schmidt_2005_J.Org.Chem_70_3737 PubMedSearch: Schmidt 2005 J.Org.Chem 70 3737 PubMedID: 15845019 Gene_locus related to this paper: canan-lipasA Abstract [reaction: see text] A recent discovery that a certain amino acid motif (GGG(A)X-motif) in lipases and esterases determines their activity toward tertiary alcohols prompted us to investigate the use of these biocatalysts in the mild and selective removal of tert-butyl protecting groups in amino acid derivatives and related compounds. An esterase from Bacillus subtilis (BsubpNBE) and lipase A from Candida antarctica (CAL-A) were identified as the most active enzymes, which hydrolyzed a range of tert-butyl esters of protected amino acids (e.g., Boc-Tyr-O(t)Bu, Z-GABA-O(t)Bu, Fmoc-GABA-O(t)Bu) in good to high yields and left Boc, Z, and Fmoc-protecting groups intact. Other Papers | ||||||||||||||||||||||||||||||||
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