| |
| author |
Abigail Goldman
| | title |
Comparing the Kinetic Behavior of Aldo-Keto Reductases Isolated from Saccharomyces cerevisiae
| | abstract |
Aldo-keto reductases (AKRs) are a superfamily of enzymes that reduce carbonyls to alcohols in
a stereospecific manner, making them useful for biocatalysis. Previous studies of the α-amide
ketoreductase variant AKR 163 from the Saccharomyces cerevisiae strain SC108 have found that
it exhibits substrate inhibition. In particular, AKR 163 is only inhibited by substrates with
electron-withdrawing groups, such as ethyl-4-chloroacetoacetate (E4ClAA), ethyl-2-
fluoroacetoacetate (E2FAA), and ethyl pyruvate (EP). To test the generality of substrate
inhibition in AKRs, AKR 308, a variant of D-arabinose dehydrogenase (Ara1), was
overexpressed in E. coli and purified via glutathione affinity chromatography. In contrast to AKR
163, AKR 308 exhibited Michaelis-Menten kinetics with E4ClAA, E2FAA, and 2,3-
pentanedione (2,3-PD). AKR 308 displayed both substrate inhibition and cooperativity with EP,
with a Hill coefficient of 3.9. Overall, AKR 308 exhibited higher turnover numbers and greater
KM values than AKR 163 with these substrates, with kcat values between 1.5 and 7 s-1 and
KM values between 6.7 and 80 mM. AKR 308 also displayed distinct kinetic behavior from AKR
163 with ethyl acetoacetate (EAA) analogs containing alkyl substituents. Whereas increased
steric bulk at the C2 position decreased AKR 163's kcat, AKR 308-catalyzed ethyl-2-
ethylacetoacetate reduction displayed a higher kcat of 4.5 s-1 compared to 0.8 s-1 for EAA.
Substantial AKR 308 activity was not observed with 2,4-pentanedione or 1,1,1-trifluoro-2,4-
pentanedione, suggesting that β-ketone carbonyls are not effective AKR 308 substrates, even if
they have electron-withdrawing substituents. Fluorescence titrations yielded a KD value of 1.2 ±
0.2 μM for NADP+ binding to AKR 308, compared to a KD value of 0.5 ± 0.1 μM for NADP+
binding to AKR 163. This indicates that AKR 308 has slightly lower affinity for NADP+
compared to AKR 163. Substrate docking with Cresset Flare revealed that EP adopts
nonproductive poses in Ara1's active site that are more energetically favorable than the catalytic
pose, whereas E4ClAA and 2,3-PD do not. Allosteric pockets were generated using PASSer to
investigate potential mechanisms of EP's cooperativity, and a pocket that favors binding of EP
over E4ClAA and 2,3-PD was identified. However, this pocket has a low calculated probability
of being an allosteric site. These findings indicate that AKR 163's mechanism of substrate
inhibition is not general across yeast AKRs, as AKR 308's substrate inhibition appears to be
mediated by nonproductive pose formation rather than binding of substrate to the enzyme-
NADP+ complex. Furthermore, this study identifies conditions under which AKR 163 and AKR
308 would be most optimal for biocatalysis, demonstrating the importance of enzyme screening
studies.
| | school |
The College of Liberal Arts, Drew University
| | degree |
B.S. (2026)
|
| advisor |
Adam Cassano
|
| full text | AGoldman.pdf |
| |
