Front Matter

chum candidumlipase II, was reversal of stereopreference observed when triolein

was used as substrate instead of trioctanoin. Instead, stereoselectivity depends

strongly on the quality of the interface, which was investigated by varying the sur-

face pressure in monomolecular films.

To identify further the selectivity-determining factors in substrate-lipase interac-

tion, syntheticsn-2 derivatives have been investigated, with the functional ester

group replaced by ether, benzylether, amide or phenyl residues (Paltauf and

Wagner, 1976; Stadler et al., 1995; Kovac et al., 1996). In both hydrolysis and es-

terification reactions, stereoselectivity depends strongly on the structure of these

substituents.

5.2 Structure information

Since 1990, when the first experimental lipase structures became available (Winkler

et al., 1990; Brady et al., 1990), the interaction of lipases with substrate-analogous

inhibitors was investigated to understand the structural basis of catalytic activity,

substrate specificity and stereoselectivity, and to identify crucial residues. Although

their sequences show no overall similarity, all microbial lipases are members of the

a/bhydrolase fold family (Brzozowski et al., 1991; Derewenda et al., 1992; Martinez

et al., 1992; Ollis et al., 1992; Grochulski et al., 1994; Uppenberg et al., 1995; Schrag

et al., 1997). Thea/bhydrolase fold consists of a central hydrophobic eight-stranded

b-sheet packed between two layers of amphiphilica-helices.a/bHydrolases have a

common catalytic mechanism of ester hydrolysis, which consists of five subsequent

steps (Cygler et al., 1994): after binding of the ester substrate, a first tetrahedral

intermediate is formed by nucleophilic attack of the catalytic serine, with the oxya-

nion stabilized by two or three hydrogen bonds, the so-called oxyanion hole. The

ester bond is cleaved and the alcohol moiety leaves the enzyme. In a last step,

the acyl enzyme is hydrolyzed. The nucleophilic attack by the catalytic serine is

mediated by the catalytic histidine and aspartic (or glutamic) acid. While the geo-

metry of the catalytic machinery is highly conserved, size and shape of the substrate

binding site vary considerably. In most lipases, a mobile element covers the catalytic

site in the inactive form of the lipase. This ’lid’ consists of one or two shorta-helices

linked to the body of the lipase by flexible structure elements. In the open, active

form of the lipase, the lid moves away and makes the binding site accessible to the

substrate.

At the time of this publication, structures of 11 microbial lipases have been pub-

lished in the Protein Data Bank PDB (Bernstein et al., 1977). They can be grouped in

five homologous families:

1. Candida antarcticalipase B (PDB entries 1TCA, 1TCB, 1TCC, 1LBS, 1LBT);

2. Pseudomonaslipases:P. cepacia(recently reclassified asBurkholderia cepacia)

(1LIP, 3LIP, 1OIL),P. glumae(1CVL, 1TAH);

3. Filamentous fungi lipases:Rhizomucor miehei(1TGL, 3TGL, 4TGL, 5TGL),

Rhizopus(1LGY, 1TIC),Humicola lanuginosa(1TIB),Penicillium camembertii

(1TIA)

86 5 Molecular Basis of Specificity and Stereoselectivity of Microbial Lipases