J. Mol. Biol. (1990) 215, 215-216

Crystallization and Preliminary X-ray Study of a Recombinant Cutinase from Fusarium solani pisi Chantal Abergel, Chrislaine Martinezt, Juan Fontecilla-Camps Christian Cambillaut Laboratoire de Cristallogr.aphie et Cristallisationldes Macromoldcules Biologiques URA-1296 CNRS, Facultd de Mddecine Nord Bd. P. Dramard, 13326 Marseille Cedex 15, France

Pieter de Geus and Marc Lauwereys Plant Genetic Systems N.V., J. Plateaustraat, 22, Gent, B-9000, Belgium (Received 23 April 1990; accepted 6 June 1990) Recombinant cutinase from Fusarium solani pisi is expressed and excreted with very high yields in Escherichia coli cultures. Cutinase was crystallized at 20°C using the vapour diffusion technique, with polyethylene glycol 6000 as precipitant. Best crystals were obtained at pH 7"0 with polyethylene glycol at 15 to 20%. They are monoclinic, with space group P21 and cell dimensions a = 35-1 A, b = 67-4 A, c = 37"05 A and fl = 94"0°; they diffract beyond 1-5 A resolution. The asymmetric unit contains one molecule of 22,000 Da ( V m = 1"98 A3/Da; 38 % water).

modification (Kolattukudy, 1984). The results of these studies suggest that cutinase belongs to the class of serine esterases, containing the classic catalytic triad (Ser, His and Asp). The stretch Gly-Tyr-Ser-Gln-Gly, containing the active-site serine, matches the consensus sequence Gly-X-Ser-X-Gly found in all serine esterase structures known to date (Blow, 1990). There is even stronger homology with the consensus sequence Gly-(Tyr or His)-Ser-X-Gly commonly present in lipases (Blow, 1990; Antonian, 1988). In addition, the fungal cutinases hydrolyse fatty acid esters and emulsified trioleyl glycerol as efficiently as porcine pancreatic lipase, although with a distinct kinetic behaviour. Furthermore, classical lipolytic enzymes have a low but measurable hydrolytic activity on cutin. The question then arises as to whether all these similarities between lipases and fungal cutinase, both with respect to catalytic and mechanism of action, point perhaps to another example of convergence of three-dimensional structures, very like the case of trypsin and subtilisin. In order to establish the relationship between lipases and cutinases more clearly, and to gain insight in the mechanism that enables these enzymes to function at the lipid/water interface, we started the structure determination of the recombinant fungal cutinase. This should eventually allow

Cutinases are a group of hydrolytic enzymes first identified in the extracellular culture fluids of fungal plant pathogens capable of degrading the insoluble lipid polyester matrix, i.e. cutin, which covers the surface parts of plants (Kolattukudy, 1985). They play a role in facilitating the attack of the pathogen on the plant and as such contribute to the pathogen's virulence. Recent experiments with transformed fungi have confirmed this view in a very convincing way (Dickman et al., 1989). Cutinases from several phytopathogenic fungi have been extensively characterized (Kolattukudy, 1984), and the primary sequence of three of them has been deduced from cloned eDNA (Ettinger et al., 1987). They constitute a group of homologous enzymes with a molecular weight of around 22,000, with highly conserved stretches. These include four invariant cysteine residues, which form two disulphide bridges. As no meaningful homology with other known protein structures has been found, this seems to imply a distinct tertiary fold, common for the fungal cutinases. Several active-site residues of Fusarium solani f. sp pisi cutinase have been identified by chemical

t Authors to whom all correspondence should be addressed. 0022-2836]90] 180215-02 $03.00/0

215

© 1990 Academic Press Limited

C. Abergel et al.

216

Table 1 Data collection statistics Resolution (A) ~ - 1.52 oo-2.76 2"76-2" 19 2-19- l "92 1-92-1"74 1-74-1.62 1-62-1-52

Independent reflections 21,942 4439 4334 4095 3824 3402 1848

U n i q u e d a t a (%)

Redundancy

R(%)t

83 99'5 98"2 92"4 87 77'5 48

2'3 2"7 2-5 2"3 2"15 2 1.6

4-41 3-42 4-81 6-21 7-89 9-97 12-93

l/al 35 77 44 30 18 12 7"7

M Nh

M Nh

"~R = Z Z II,. - (1).l/Z Z

Crystallization and preliminary X-ray study of a recombinant cutinase from Fusarium solani pisi.

Recombinant cutinase from Fusarium solani pisi is expressed and excreted with very high yields in Escherichia coli cultures. Cutinase was crystallized...
171KB Sizes 0 Downloads 0 Views