Page 353 - Cascade_Biocatalysis_Integrating_Stereoselective_and_Environmentally_Friendly_Reactions

15.3 Cascade Reactions for Assaying Transketolase Activity by In Vivo Selection 329

The relative specific activity data was converted to gray scale value. The specific
activity of TK toward 50 mM glycolaldehyde was used as 100% reference.
eco
a, 50 mM methanal; b, 50 mM glycolaldehyde; c, 50 mM d-glyceraldehyde; d,
50 mM d,l-glyceraldehyde; e, 50 mM d-erythrose; f, 50 mM l-threose;g,50 mM
d,l-2,4-dihydroxybutanal; h, 200 mM d-ribose; i, 200 mM ethanal; j, 200 mM 3-
hydroxypropanal; k, 200 mM propanal; l, 200 mM (R)-3,4-dihydroxybutanal; and m,
200 mM 4-hydroxybutanal.
To conclude, this pH-based assay holds promise as a very useful tool for the
screening of novel TK enzymes for their substrate tolerance and modification by
directed evolution that offers simplicity, speed, high sensitivity, and low cost.

15.3
Cascade Reactions for Assaying Transketolase Activity by In Vivo Selection

A current challenge is to modify the properties ofTKtoextend its synthetic
potential. The construction of large libraries of mutant TK variants would expand
the possibilities of modifying the substrate specificity of the enzyme. In vitro assays
are unsuitable for a primary screening of huge libraries of mutant enzymes because
each enzyme variant has first to be produced (and possibly even purified) separately
to determine its functional expression and catalytic properties. Various strategies
have been proposed for in vivo selection of enzymes, but none of them concern TK
as the target. Most of these assays aim at linking the catalytic activity of the enzyme
under study to the release of a survival factor that lends a growth advantage to the
host microorganism [36–39].
The principle of an in vivo selection test may enable the direct detection of
foreign TK activity such as that from yeast on nonnatural substrates in E. coli
host cells. This type of metabolic assay was developed for using specially designed
synthetic compounds (probes 16a and 16b) that combine a ketose moiety, potentially
recognized for selective cleavage by TK, and the side chain of a proteinogenic amino
acid [40, 41].
The cleavage of the C –C bond in probes 16a and 16b by TK releases an
3
2
α-hydroxyaldehyde, which can be converted by cellular metabolism in vivo into
an l-amino acid, which is required for growth by auxotrophic E. coli host cells
(Scheme 15.16). This principle has been validated for leucine or methionine E.
coli auxotrophs overproducing wild-type TK [40]. The specially designed probes,

O OH OH NH 2
TK
HO O HO
R R R
OH O
α(S)-Hydroxy
16a: R = CH CH(CH )
2 3 2 aldehyde L-Leu
16b: R = (CH ) SCH
2 2 3 L-Met
Scheme 15.16 Principle of ‘‘in vivo metabolism’’ detection of TK activity in E. coli aux-
otrophs from 16a,b.