A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex

Brzozowski, A.M.; Derewenda, Urszula; Derewenda, Zygmunt S.; Dodson, Guy; Lawson, David M.; Turkenburg, J.P.; Björkling, Fredrik; Huge-Jensen, Birgitte; Patkar, Shamkant; Thim, Lars

doi:10.1038/351491a0

Cited by 1,108 publications

(668 citation statements)

References 23 publications

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“…The largest C a deviations from the native structure are provided by Ala 185 and 186, followed by Val 184 ( The absence of large conformational changes in the substrate binding region upon binding to a non-hydrolysable analogue, represents to date a feature unique to cutinase (Martinez et al, 1994; this study), contrary to the large movements that have been reported for covalently inhibited complexes of other lipases (Brzozowski et al, 1991;Derewenda et al, 1992;Cygler et al, 1994;Grochulski et al, 1994b;Egloff et al, 1995;Hermoso et al, 1996).…”

Section: Ca Atoms Of Leu 81 and Val 184 Is 1 A Greater In Both Molecmentioning

confidence: 63%

Crystal structure of cutinase covalently inhibited by a triglyceride analogue

et al. 1997

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Cutinase from Fusarium solani is a lipolytic enzyme that hydrolyses triglycerides efficiently. All the inhibited forms of lipolytic enzymes described so far are based on the use of small organophosphate and organophosphonate inhibitors, which bear little resemblance to a natural triglyceride substrate. In this article we describe. the crystal structure of cutinase covalently inhibited by (R)-1,2-dibutyl-carbamoylglycero-3-O-p-nitrophenyIbutyI-phosphonate, a triglyceride analogue mimicking the first tetrahedral intermediate along the reaction pathway. The structure, which has been solved at 2.3 A, reveals that in both the protein molecules of the asymmetric unit the inhibitor is almost completely embedded in the active site crevice. The overall shape of the inhibitor is that of a fork: the two dibutyl-carbamoyl chains point towards the surface of the protein, whereas the butyl chain bound to the phosphorous atom is roughly perpendicular to the sn-1 and sn-2 chains. The sn-3 chain is accommodated in a rather small pocket at the bottom of the active site crevice, thus providing a structural explanation for the preference of cutinase for short acyl chain substrates.

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Section: Ca Atoms Of Leu 81 and Val 184 Is 1 A Greater In Both Molecmentioning

confidence: 63%

Crystal structure of cutinase covalently inhibited by a triglyceride analogue

et al. 1997

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“…However, our knowledge of the molecular details of its substrate binding is poor. At the time of this writing, only 1 detailed structure of a lipase-inhibitor complex (Brzozowski et al, 1991; U. ) is currently available (Kinemage 1).…”

mentioning

confidence: 99%

“…miehei lipase is not clear (Derewenda & Sharp, 1993). Lipases are activated at hydrophobic surfaces (Sarda & Desnuelle, 1958) through movements of 1 or a few surface loops that bury the active site in the inactive conformation (Winkler et al, 1990;Brzozowski et al, 1991; U. Derewenda et al, , 1994van Tilbeurgh et al, 1992van Tilbeurgh et al, , 1993Grochulski et al, 1993).…”

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confidence: 99%

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Computer modeling of substrate binding to lipases from Rhizomucor miehei, Humicola lanuginosa, and Candida rugosa

et al. 1994

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The substrate-binding sites of the triacyl glyceride lipases from Rhizomucor miehei, Humicola Ianuginosa, and Candida rugosa were studied by means of computer modeling methods. The space around the active site was mapped by different probes. These calculations suggested 2 separate regions within the binding site. One region showed high affinity for aliphatic groups, whereas the other region was hydrophilic. The aliphatic site should be a binding cavity for fatty acid chains. Water molecules are required for the hydrolysis of the acyl enzyme, but are probably not readily accessible in the hydrophobic interface, in which lipases are acting. Therefore, the hydrophilic site should be important for the hydrolytic activity of the enzyme.Lipases from R. miehei and H. lanuginosa are excellent catalysts for enantioselective resolutions of many secondary alcohols. We used molecular mechanics and dynamics calculations of enzyme-substrate transition-state complexes, which provided information about molecular interactions important for the enantioselectivities of these reactions.

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“…This triad is covered by a surface loop or 'lid' [14,23], which has been suggested to move during interfacial activation [14]. This assumption has recently been directly supported by the crystal structure determination of a complex of a triacyl glycerol lipase from the fungus, Rlti~onttrcor midtei, with the enzyme inhibitor rr-hexylphosphonate ethyl ester, where the active site is exposed by the movement of the helical lid [24]. Other amino acids involved in pig lipase activity are present in rat pancreatic lipases such as one of the histidines 75 or 1% (positions 77 and 158 in the rat sequence), the ethoxyformylation of which results in the loss of activity toward triacylglycerol hydrolysis [26].…”

Section: Anzino Acid Sryirertcementioning

confidence: 99%

Primary structure of rat pancreatic lipase mRNA

Wicker‐Planquart

Puigserver

1992

FEBS Letters

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The sequence of a rat pancreatic lipasc mRNA was dctrrmincd. The data have been assigned the followingaccession number, X61925, in the EMBIdata library. The total length of the mcsscngcr is 1531 nuclcotidcs. plus a poly(A) stretch of about 60 nucleotidcs. A 72-nuclcotida 5'-noncoding region is followed by a I4 19-n ucleotidcs open reading frame which encodes a protein of473 amino acids, including the I7 amino acid signal pcptidc. The mature enzyme (456 rcsiducs) has 6 additional C-terminal amino acids, as compared with the amino acid scqucncc of pig (direct amino acid sequence). dog. man snd rat isocnzymc rrom Cicnbank. M583G9 (all deduced from the nuclcotidc scqucncc). A higher degree of homology cxisls bctwccn the amino ucid scqucncc of rat mature cnzymc with those of dog (88%), pig (75%) and man (75%) than with that of rat isolipasc (74%).Rat pancreas lipasr; Nuclcotidc sequencePancreatic lipase (triacylglycerol acyl hydrolase, EC 3.1. I .3) the physiological function of which is to hydrolyze dietary triacylglycerols in the duodenum, plays an important role in fat metabolism. The enzyme prcferentially splits the esters of long-chain fatty acids at positions 1 and 3, producing mainly 2-monoacylglycerol and free fatty acids. and shows considerably higher activity against insoluble emulsified substrates than against soluble ones. The first step of the catalysis is an adsorption of the enzyme to the water-lipid interface [I]. The presence of various amphiphiles such as bile salts, by accumulating at the interface of emulsified particles, hinders lipase adsorption and completely abolishes lipase activity [2,3]. To overcome such an inhibition, pancreatic lipase must bind another protein, colipase, which, by adsorbing to the amphiphile covered interfaces, allows lipasc to gain access to the substrate [4].We have recently reported the nucleotide sequence of rat pancreatic colipase mRNA [S]. In this paper, we determined the nucleotide sequence of a rat pancreatic lipase cDNA and its deduced amino acid sequence (accession number in the EMBL Data Library, X61925). The comparison of the polypeptide chain sequence identity with another from rat (Genbank M58369), obtained in Dr. Mark E. Lowe's Laboratory (Washington University School of Medicine, St. Louis, USA), as well as with those from other species, will help, in conjunction with site-specific mutagenesis, in defining residues essential for interaction with colipase and lipids. We also report the partial nucleotide sequence of another cDNA clone. which is half-length and presents unambiguously some minor changes, This may reflect the cloning of 2 closely related, perhaps allelic, pancreatic lipase mRNAs. The construction of a rat pancreatic cDNA library in pUC9 has been reported [6]. Rat &t I I library was from Clontcch (Calif'ornia, USA). cDNA inserts of interest from the phage library were subcloncd into dephosphorylatcd and EcoRl-digested pUCI8 plasmids Tar prcparutive growth. Rat pancreatic libraries were screened with a canine pancrtxtic lipasc cDNA [7], radiolab...

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A model for interfacial activation in lipases from the structure of a fungal lipase-inhibitor complex

Cited by 1,108 publications

References 23 publications

Crystal structure of cutinase covalently inhibited by a triglyceride analogue

Crystal structure of cutinase covalently inhibited by a triglyceride analogue

Computer modeling of substrate binding to lipases from Rhizomucor miehei, Humicola lanuginosa, and Candida rugosa

Primary structure of rat pancreatic lipase mRNA

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