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Structure-function lipase relationships in low water media

Objetivo

- To understand the role of water in substrate binding, conformational changes and catalysis of lipases.
- To study the design of the active site of two lipolytic enzymes at different hydration levels.
- To produce lipase mutants altering electrostatic properties and active site environment.
- To correlate kinetic and structural changes with the theoretical data.
The lipase from Pseudomonas glumae (PGL) and the Fusarium solani pisi cutinase (FSC) were chosen as examples of lipolytic enzymes with and without a `flap', respectively, for studies on the role of water on the mechanism of reaction. The production of PGL has been achieved but the enzyme adhered to hydrophobic surfaces due to the presence of rhamnolipids and the studies with the purified enzyme have been limited. These rhamnolipids are produced by the Pseudomonas host in large quantities and co-elute with the enzyme in many chromatographic steps. While the samples of enzyme finally obtained are free from contaminating proteins, residual molecules of rhamnolipids present cannot be removed without loss of enzyme acitivity.
The production and purification of wild type and variants of cutinase has been performed.
It has been shown that electrostatic effects stemming from the charged residues (mainly located on the protein surface) highly contribute to protein function. The titration state of a given residue depends in a complex manner on the presence or absence of other charged residues in the environment of the residue in question. A TITRA program has been developed that allow for a detailed visualization of electrostatic potentials and computation of pK values for proteins.The sequences of 6 fungal cutinases have been compared with those of FSC revealing a strong sequence homology (40-50% identity). 3D models of these enzymes have been construct based on the homology with FSC and a likely enzymatic mechanism has been proposed for the pH activity profile of FSC explaining both the onset and disappearance of enzyme activity.
A generic novel SW-tool has been generated that allow the NMR user to link NMR assignment data with X-ray protein crystallographic data.
Electrostatic and hydrophobic mutations on the protein surface have been found to induce the formation of new crystal packings
The characterization of the cutinase oxyanion hole has been done allowing to identify the Ser42 Ogamma as the third component. The proper orientation of Ser42 Ogamma is maintained in the proper orientation by Asn 84 Ndelta2.
FSC dynamics have been investigated using different variants showing that surface loops are the major flexible protein regions, particularly those building up the active site groove, whereas the elements of the proteins scaffold were shown to be more rigid.
In order to obtain information on the relationship between structure and selectivity, the resolution of cutinase covalently inhibited by a four carbon phosphonate triglyceride analogue has been carried out at 2.3 Å. One of the important results from this work is the finding that the conformation of the covalent enzyme-triglyceride analog complex is very close to that of the free enzyme. This lack of major conformational changes in the enzyme helps us in our interpretation of the effects of substrate binding to the enzyme in terms of water distribution at the enzyme surface. Another result of this work is the detailed understanding of the acyl-chain binding mode into the active site binding cleft. The assymetric unit consists of two protein molecules arranged head to head, with their substrate binding sites facing each other. It is tempting to establish a correlation between the selectivity of lipases for triglycerides of certain length and the depth of their active site.
Atomic resolution (1.0 Å) of cutinase using synchrotron radiation sources allows to precisely identify hydrogen atoms in the electron density map providing the first direct evidence on the protonation state of the catalytic histidine. The analysis point out a need for revision of some well-established rules of protein chemistry. Very fast Laue data collection conditions on crystals of native and mutant cutinase have been set up using synchrotron sources.
Structural effects of two solvents (methanol and acetonitrile) on the 3-D structure of cutinase have been tested showing a rapid deactivation of the enzyme. Acetonitrile, which is unable to form hydrogen bond, gives an increase of Tyr residues (especially Tyr 119 and Tyr 191) exposed to the surface. Methanol, which is able to form hydrogen bonds gives very small changes in the exposed aromatic residues. Acetonitrile deactivation is totally reversible when it is irreversible with methanol.
The kinetics of the two enzymes (PGL and FSC) have been studied in different reaction systems under different hydration levels.
PGL displays interfacial activation and for hydrolysis in water-acetonitrile medium the catalytic activity increases with the water concentration until a water content of 15-20% and then decreases suggesting that the medium is then less favourable for the enzyme to expose its catalytic site. The hydrophobicity of the medium is now sufficiently low for the 'lid' covering the active site to remain closed.
PGL and FSC have been immobilized on Accurel EP100 retaining approximately 20% of their original activities. Using immobilized PGL the synthesis of hexyl butyrate is high at very low water activity but as the water activity increases the activity decreases. Using FSC, studies have been done on the effect of solvents (water miscible or not) and water activity on catalytic activity. The catalytic activity decreases with increasing solvent polarity. From the obtained results it appeared that the solvating effect of solvents had to be accounted for. With FSC, depending on the initial water activity of the enzyme and the reaction medium, an autocatalytic effect can be obtained. The role of water on cutinase behaviour has also been evaluated in supercritical carbon dioxide. The Accurel EP 100 immobilized enzyme is able to catalyse hexanol-hexanoic acid synthesis under these conditions (45(C and 130 bar) and the reaction strongly depends on the water content of the system and is maximum at aw=0.76. The Accurel supported FSC has been tested in a continuous way in the solid gas system showing an important irreversible deactivation phenomenon when the water activity was increased upon 0.6. Kinetic studies have been compared for Accurel EP100 and Chromosorb P supported FSC. Accurel supported enzyme is strongly inhibited by high n-propanol thermodynamic activity under low hydration conditions when cutinase supported onto Chromosorb P is activated under the same conditions. In solid-gas system, the effect of water on the catalysis and the stability of the cutinase supported (on Chromosorb P) or not, have been studied and optimized. A non common catalytic behaviour has been obtained and an explanation based on the evolution of the polarity of the active site is proposed. A new solid-gas set-up able to operate under reduced pressure has been built in order to improve the effect of the activity of the different compounds and to increase the potential applications of lipolytic enzymes.
Structure-function relationships of FSC have been studied in reverse micelles. The stability of the enzyme was greater than previously reported in the presence of hexanol. In the complete absence of hexanol, for pentyl laurate synthesis for example, the enzyme exhibites a rapid deactivation, depending on the wo (and so to the size of the micelle water pool: smaller or larger than the cutinase) probably caused by the contact with the hydrophobic moiety of the reverse micelles. The renaturation of the enzyme is obtained after recovering in a new aqueous phase. The transesterification between esters of acetic acid and alcohols of different chain lengths has been studied with the cutinase microencapsulated in AOT reverse micelles. Using butyl acetate the highest specific activity is achieved with pentanol and the stabilization effect reported for hexanol is also obtained with pentanol and heptanol. The activity is very dependent of the acyl donor acid chain and rapidly decreases when the acyl carbon chain length increases.The EPR spectroscopy studies of active site labeled-cutinase in AOT microemulsions with varying wo values show that with all microemulsions except for very low water content, the mobility of the label is higher than in the aqueous solution with no dramatic changes on the active site conformation. The maximum of the enzyme activity does not correspond to a reduced active site mobility. As the water content of the system become higher, the mobility of the bound spin label further increases whereas the enzymatic activity droppes.
The cutinase has been immobilized on zeolites and used for triglyceride hydrolysis and butyl acetate-hexanol transesterification. The physico-chemical parameters and the kinetic parameters of the reaction have been optimized and the selectivity and stability evaluated. Cutinase immobilized on the NaY zeolite presents an increase on the turnover number and a decrease on the KM values with the increase of the solvent polarity used.
Transesterification between ethyl butyrate and butanol, pentanol and hexanol have been performed under microwave radiations giving a two to three fold enhancement or reduction of catalytic activity which is dependent on the hydration conditions of the enzyme.

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Régimen de financiación

CSC - Cost-sharing contracts

Coordinador

Université de la Rochelle
Aportación de la UE
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Dirección
23,Avenue Marillac
17042 La Rochelle
Francia

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Última actualización: 31 Diciembre 1997

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Permalink: https://cordis.europa.eu/project/id/BIO2943016/es

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