Front Matter

pocket at the bottom of the active site crevice, and the two dibutyl-carbamoyl chains

pointing towards the surface of the protein. It was concluded that the size of thesn-3

binding pocket would be responsible for the preference of cutinase for short-chain

fatty acids atsn-3 position (Mannesse et al., 1995). The chain length specificity of

cutinase for thesn-1 andsn-2 chain, however, could not be explained.

5.3 Modeling and engineering of fatty acid specificity

Using molecular modeling and subsequent verification by site-directed mutagenesis,

the relationships between fatty acid chain length specificity and shape or physico-

chemical properties of binding sites have been investigated. Interesting properties of

lipases are specificities toward unsaturated fatty acid chains and length of saturated

chains.

Geotrichum candidumlipase I (GCL I) has been shown to have a unique prefer-

ence for long-chaincis(D-9) unsaturated acyl chains. This preference is lacking in

the highly homologous lipase II of the same organism. By comparing sequence and

specificity of hybrids between these two lipases, amino acids were identified which

mediate recognition of unsaturated acyl chains (Holmquist et al., 1997). Crucial

residues are located at the entrance of the active site and at the bottom of the active

site cavity of GCL I. Replacing four residues of GCL I by the corresponding residues

from GCL II led to a specificity profile similar to that of GCL II. The reverse muta-

tions in GCL II only partially recoveredcis(D-9) specificity, however.

The family of filamentous fungi lipases has been most extensively investigated for

structure-function relationship of chain length specificity. Based on the X-ray struc-

ture of a complex ofRhizomucor mieheilipase withn-hexylphosphonate ethyl ester

(Brzozowski et al., 1991), binding of the scissile fatty acid of triacylglycerol sub-

strates have been modeled forRhizopus, Rhizomucor mieheiandHumicola lanugi-

nosalipase (Vasel et al., 1993; Lawson et al., 1994; Norin et al., 1994; Klein et al.,

1997; Pleiss et al., 1998). The binding pocket ofRhizomucor mieheilipase (RML) is

a shallow bowl with a long axis of 18 A ̊, and a width of 4.5 A ̊at its base, and 6 A ̊at the

protein surface (Figure 1). The scissile fatty acid up to C 8 binds to a cleft at the

bottom of the binding pocket. Its bottom is formed by side chains of residues

P177, H108 and the catalytic S144. It is lined by V205 and D91 on its left- and

right-hand side, respectively. From C 8 to C 10 , the fatty acid chain rises at the

wall of the binding pocket and enters a hydrophobic crevice up to C 18 (length

9.5 A ̊), which runs parallel to the surface of the protein. Its width of 5.5 A ̊ at the

bottom gives the fatty acid room for movement. Its bottom is formed by residues

P209 and P210, its left- and right-hand wall by L208 and F94/F213, respectively.

Location and properties of the scissile fatty acid binding site has been supported by

creating mutants of lipases fromRhizopusandHumicola lanuginosawith altered

fatty acid chain length profile (Joerger and Haas, 1994; Atomi et al., 1996; Marti-

nelle et al., 1996; Klein et al., 1997; see also Chapter 4). Experimental effects of

mutations in the scissile fatty acid binding site ofRhizopus delemarandRhizopus

oryzaelipases (Table 1) can be explained by the position of the residue and its inter-

action with the fatty acid chain (Figure 2). V205 lines the cleft at the bottom of the

88 5 Molecular Basis of Specificity and Stereoselectivity of Microbial Lipases