Today I want to tell you about a useful enzyme that was characterized mostly by a group of Johann Heider from Marburg University with the support of my PhD student (former) Agnieszka Winiarska. The enzyme is a Zn-dependent alcohol dehydrogenase, namely benzyl alcohol dehydrogenase from A. aromaticum (our favorite microbe for digging up strange enzymes.) We published a paper on the enzyme in Applied Microbiology and Biotechnology.
The enzyme oxidizes primary alcohols to aldehydes (or reduces aldehydes to alcohol) which is nothing special on its own. What is interesting about this enzyme is the fact that it naturally exhibits affinity for both NAD+ and NADP+. What is even more interesting is that both cofactors work best at different pHs. The enzyme is more active with NADP+/NADPH at the acidic range compared to best working at neutral or slightly basic conditions with NAD+/NADH couple.
And here is where Alphafold2 and its database of ready structures come to be very handy - without too much effort we just docked NADP into the active site of the AF model and easily identified an unusual mutation not seen in other enzymes of this class. Close to the phosphate group of the cofactor, there is His217 - which may get positively charged at lower pH thus increasing the enzyme's affinity for phosphorylated cofactor. Thanks to the AF database, we were able to conduct this in 1 day. I think I would never stumble on that mutation (not so obvious from the seq alignment) as normally I would be too lazy to run homology modeling just to make a nice figure for the paper.
Due to its double affinity for cofactor, the enzyme was essential in the development of activity assay for AOR in reverse reaction of H2-dependent reduction of benzoic acid to benzaldehyde. With NADPH we were able to form a coupled assay enabling continuous detection of AOR activity with UV-vis. While when NAD+ was used we had a cascade reaction in which AOR was both reducing acid and making NADH, thus pushing reduction toward alcohol.
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