Page 400 - Cascade_Biocatalysis_Integrating_Stereoselective_and_Environmentally_Friendly_Reactions

376 17 Enzymatic Generation of Sialoconjugate Diversity

to the natural Neu5Ac substrate (1) and, correspondingly, an overall 2800-fold
reduced catalytic efficiency (as judged by k /K ) [33, 34].
cat M
17.2.2.3 Engineering of Promiscuous CSS Variants
In order to create CSS variants with improved efficiency for such sterically more
demanding N-acylated substrates, the Phe192/Phe193 pair appeared to be plausible
candidates for alteration. Mutant libraries constructed by simultaneous saturation
mutagenesis at the adjacent 192/193 positions with NDT degeneracy were screened
for improved catalytic efficiency against substrate analogs 14, 15,and 22 using the
pH-dependent high-throughput screening (HTS) assay protocol [33, 34]. Screening
compounds were chosen for their bio-orthogonal alkyne and azide functions that
post-synthetically can be conjugated and diversified by click chemistry at the
neosialooligosaccharide stage [50]. Sequencing of active hits identified the Phe192
site to have a profound influence for accommodating a bulky N-acyl moiety from the
substrate [34]. Particularly, the double mutant F192S/F193Y showed the best overall
improvement (of 30-fold and 60-fold for 15 or 22, respectively), closely followed
by the single exchange F192S variant (Figure 17.4). A detailed kinetic analysis
revealed that the improvement in catalytic efficiency with the bulky substrate
analogs is largely caused by the superior substrate affinity and significantly higher
conversion rates of the mutant enzymes when compared to the wild-type CSS
(Table 17.2) [34].
In contrast, two recently reported mutants of the N. meningitidis CSS, namely
(S81R) and CSS(Q163A), which were inspired by analogy to CSS sequence vari-
ations found in other bacteria and assumed to confer higher substrate flexibility,

Table 17.2 Apparent steady-state kinetic parameters for wild-type CSS and mutant variants
for Neu5Ac (1) and bulky N-acyl analogs (14, 15,and 22) [34].

Substrate Parameter wt F192S F192S/F193Y

1 K (mm) 0.08 0.78 1.98
M
−1
k cat (s ) 3.24 4.65 4.86
−1
k /K (s −1 mm ) 40.5 6.0 2.5
cat M
Improvement 1 0.15 0.06
14 K (mm) 2.82 0.44 0.80
M
−1
k cat (s ) 1.02 4.42 5.32
−1
k /K (s −1 mm ) 0.36 10.0 6.6
cat M
Improvement 1 27.8 18.3
15 K M (mm) 4.75 0.32 0.37
−1
k (s ) 2.56 4.42 6.03
cat
−1
k /K M (s −1 mm ) 0.54 13.9 16.5
cat
Improvement 1 25.7 30.6
22 K M (mm) 4.29 0.41 0.34
−1
k (s ) 0.85 4.73 4.10
cat
−1
k /K M (s −1 mm ) 0.2 11.4 12.1
cat
Improvement 1 57.0 60.5