Front Matter

4. Other yeast lipases:Candida rugosa(1CRL,1LPM, 1LPN, 1LPO, 1LPP, 1LPS,

1TRH),Geotrichum candidum(1THG), and the structurally similarCandida

cylindraceacholesterol esterase (1CLE)

5. Fusarium solanicutinase (1AGY, 1CEX,1CUS, 2CUT, 1OXM, 1XZK, 1XZL,

1XZM, and 31 entries on mutants).

For each family, at least one structure of a complex with a substrate-analogous phos-

phonate, phosphate or sulfonate inhibitor is available. Comparison of open and

closed structures revealed the conformational changes which occur upon binding

of a substrate. For lipases of families 2, 3, and 4, the most prominent conformational

change is the opening of the lid;Fusarium solanicutinase seems not to have a lid,

while forCandida antarcticalipase B a closed structure has not yet been crystallized,

and thus a lid could not yet be assigned. In addition, the conformation of the

oxyanion hole differs between closed and open form inPseudomonasandCandida

rugosalipases, while the oxyanion hole is pre-formed in filamentous fungi lipases.

Apart from these conformational changes upon activation of the lipase, binding of a

triacylglycerol analogous inhibitor induces only minor changes of side chain con-

formation, as concluded from open structures with and without inhibitor forF. solani

cutinase andP. cepacialipase. This is different from binding of the two enantiomers

of a chiral menthyl ester analog toC. rugosalipase: conformational changes induced

by binding have been attributed to be the molecular reason for the high stereoselec-

tivity toward secondary alcohols (Cygler et al., 1994).

In all lipases, the substrate binding site is located inside a deep, elliptical pocket on

top of the centralb-sheet. Shape of the binding sites and binding of the scissile fatty

acid differ among the lipases. They have been assigned to three classes (Pleiss et al.,

1998): (1) lipases with a hydrophobic, crevice-like binding site located near the

protein surface (lipases fromRhizomucorandRhizopus); (2) lipases with a fun-

nel-like binding site (lipases fromCandida antarctica,Pseudomonasand cuti-

nase); and (3) lipases with a tunnel-like binding site (lipases fromCandida rugosa

andGeotrichum candidum).

Only for two microbial lipases,Pseudomonas cepacialipase andFusarium solani

cutinase, has the binding of triacylglyerol analogous inhibitors yet been studied. For

Pseudomonaslipase, the structure has been solved of a complex with one enantiomer

of a chiral substrate analogous inhibitors, (RC,SP)-1,2-dioctylcarbamoylglycero-3-O-

p-nitrophenyl octylphosphonate, covalently bound to the catalytic serine (Lang et al.,

1998). The inhibitor adopted a bent tuning fork conformation. Four binding pockets

for the triacylglycerol analogue were detected: the oxyanion hole, two hydrophobic

pockets and one more hydrophilic pocket, which accommodate the three substituents

of the inhibitor. Interaction with substrate is dominated by Van der Waals interac-

tions; in addition, a hydrogen bond to the carbonyl oxygen of thesn-2 chain con-

tributes to fixation of the position of the inhibitor. Since theSCenantiomer was not

experimentally resolved, the interaction between this less preferred enantiomer and

the lipase was modeled. Clashes between a carbonyl oxygen with the C-terminal

neighbor L287 of the catalytic histidine and I290 were identified as determinants

of stereoselectivity. In a complex ofFusarium solanicutinase with (R)-1,2-dibu-

tyl-carbamoylglycero-3-O-p-nitrophenylbutyl-phosphonate (Longhi et al., 1997),

the inhibitor also bound in a fork-like shape, with thesn-3 chain binding to a small

5.2 Structure information 87