Chemistry Reference
In-Depth Information
form a lipid aldehyde, which in turn reacts with 4-hydrazino-7-nitrobenzofurazan (NBD-
hydrazine) to form a strong fluorescent NBD-hydrazone. By this method, high-throughput
screening was employed and approximately 10 000 mutants were screened. Among them, 25
positives were detected to be able to synthesize phosphatidylinositol and its various isoforms.
This example has demonstrated not only the importance of correct prediction of amino acid
residues as the target for mutagenesis but also the importance of a proper detection system
for selecting or screening the desired mutants. Both are crucial for efficient modification
of an enzyme. To do this, an optimal combination of knowledge of physical, structural and
enzyme chemistry is necessary.
2.3.4.4
Examples for changing selectivity
Some enzymes recognize and act on multiple substrates while others carry out side reactions.
Protein engineering can also be applied to change the selectivity of enzymes. Porcine pan-
creatic PLA
2
has been modified for better selectivity towards negatively charged substrates
by site-directed mutagenesis.
32
PLA
2
, as described in Section 2.2.4.2, is a useful enzyme not
only in the food industry but also in the production of phospholipids of fine chemical grade
for drug delivery systems. The enzyme recognizes and hydrolyzes multiple phospholipids
with the head group being zwitterionic or negatively charged. The crystal structure of the
enzyme was determined and amino acid residues in the region for binding with the head
group of phospholipids were analyzed. In this region, two positively charged amino acid
groups, Arg53 and Arg43, are presumed responsible for binding favourably to negatively
charged phospholipids. Adjacent to this region was a negatively charged Glu46 that was fur-
ther identified as a residue that might unfavourably influence the binding with the negatively
charged head group of phospholipids. A mutant for exchanging the Glu to Lys (E46L) was
introduced by site-directed mutagenesis. This mutant was found indeed to be able to bind
more efficiently to substrates with the negatively charged head group.
Another example for changing the selectivity of enzymes by protein engineering is a
reduction of the hydrolyzing activity of a malto-oligotrehalose synthase over its synthesiz-
ing activity in order to increase the yield of trehalose production. Malto-oligosyltrehalose
synthase (EC 5.4.99.15, MTSase) catalyzes an intramolecular transglycosylation reaction to
produce a non-reducing malto-oligosyltrehalose by converting the
α
-1,4-glucosidic linkage
at the reducing end of malto-oligosaccharide to an
-1,1-glucosidic linkage. The malto-
oligosyltrehalose can then be hydrolyzed by malto-oligosyltrehalose trehalohydrolase (EC
3.2.1.141, MTHase) to produce trehalose, a widely used sugar as food preservative, stabilizers
and cosmetic ingredients. Besides the synthesizing reaction, MTSase also carries out the hy-
drolysis reaction of starch molecules to produce glucose instead of malto-oligosyltrehalose.
The ratio of hydrolysis over transglycosylation influences the yield of trehalose production.
One attempt to modify the enzyme in order to reduce the selectivity for hydrolysis was
reported by Fang
et al.
33-35
The enzyme was selected from
Sulfolobus solfataricus
. Enzyme
kinetic studies revealed possible amino acid residues (Asp228, Glu255 and Asp443) in the
active site, which are crucial for the catalytic reactions. Amino acid residues close to this site
(
α
,
α
1) were suggested as being responsible for selecting catalytic reactions towards
transglycosylation or towards hydrolysis. Therefore, site-directed mutagenesis at these re-
gions was chosen to try to enhance the selectivity towards transglycosylation. A mutant at
the
+
1 and
−
1 region, F405Y, was found to decrease hydrolysis. The exchange from Phe to Tyr
decreases the hydrophobic interactions between the enzyme molecule and the substrate.
+
