Front Matter

4.7 Probing the role of structure-function relationships

in substrate selectivity

Identification of the relationship between primary amino acid sequence and enzyme

activity offers the opportunity to optimize enzyme performance through directed

mutagenesis. An understanding of the structural basis of substrate selectivity could

facilitate the production of a suite of customized lipases displaying desired substrate

specificities. Such enzymes could be useful as applied catalysts in applications such

as the harvesting of selected fatty acids from heterogeneous glycerides, or the trans-

fer of such fatty acids to acceptor molecules. In effect, mutagenesis represents an

alternative or adjunct to the screening of natural isolates for new enzyme activ-

ities. For these reasons, site-directed mutagenesis, coupled with computer-assisted

molecular modeling, was employed to explore structure-function relationships in the

Rhizopus-Rhizomucorfamily of enzymes, and to generate new, potentially useful,

lipases.

Much of this work was conducted using the prolipase gene, rather than the lipase

gene, because its reduced host toxicity, in conjunction with high activity, facilitated

direct screening of bacterial populations to identify potential mutants. The structural

model of the Rd enzyme was not available at the time this work was begun. However,

given the high degree of homology between the Rd and Rm lipases, the available

structure of the latter enzyme was used as a model. The Rd lipase structure was used

to guide these studies when it subsequently became available.

In both the Rd and Rm lipases, the substrate binding site is a shallow trough along

the surface of the enzyme, with the Ser-His-Asp catalytic triad at one end (Figure 4).

The side chains of the amino acids forming the walls of this trough are largely hy-

drophobic, and in some cases are highly conserved among the 1,3-specific lipases. It

was postulated that these amino acids were vital to the binding of the acyl chain of

fatty acid substrates, or the access of the fatty acids to the active site, and that muta-

tions at these sites could interfere with substrate binding and enzyme activity. Site-

directed mutagenesis was employed to make all possible amino acid substitutions at

individual locations along the substrate binding trough. The mutant DNAs were

introduced into bacteria and the substrate ranges of the resulting lipases were exam-

ined, with the goal of generating and recovering enzymes that possessed marked fatty

acid chain length specificity. Rhodamine screening media containing either triolein

(18 : 1 fatty acid glyceride), tricaprylin (8 : 0) or tributyrin (4 : 0) were employed to

identify changes in fatty acid selectivity. Within a factor of 2, the wild-type lipase

hydrolyzed long-, medium-, and short-chain fatty acid glyceride esters at the same

rates. Mutant lipases were isolated that exhibited between 2- and 12-fold enhance-

ments in their relative activities toward medium- or short-chain fatty acids (Klein et

al., 1997a). DNA sequence analysis was employed to identify the amino acid sub-

stitutions in these enzymes.

In these initial studies, single mutations were introduced into the lipase gene. The

recovery and characterization of modified enzymes allowed the identification of

amino acid positions involved in determining the fatty acid substrate specificity

of the enzyme. In subsequent work (Klein et al., 1997b), these single mutations

were combined to produce double-mutant lipases. In addition, molecular dynamics

80 4 Cloning, Mutagenesis, and Biochemical Properties