Oxidoreductase enzymes catalyze a wide range of valuable enantioselective oxidation and reduction reactions. However their cofactor dependence complicates their industrial use as the cofactors are valuable and they must be regenerated and recycled to support the biocatalytic cycle. There has been interest in the bioconjugation of cofactors with polymers in order to facilitate their recycling and recovery by taking advantage of the increased molecular weights. We have recently explored the impact of the PEGylation of NAD(H) on the steady state kinetics of a formate dehydrogenase and a lactate dehydrogenase (1). We found that PEGylation can affect the binding of the cofactor or the stabilization of the cofactor/substrate transition state, depending on the geometry of the enzymatic active site.<br/><br/>In order to eliminate the need to add, recycle, and separate cofactors from enzymatic reactions, we have fused oxidoreductase enzymes together using the spycatcher/spytag technology and then decorating the enzyme fusions with bioconjugated NAD(H) attached with PEG polymer chains. Thus the cofactors are available to both enzymes for recycling between the active sites and soluble cofactors are no longer mixed with the reactants and products. To demonstrate this approach, we have fused a formate dehydrogenase with a malate dehydrogenase and decorated it with PEGylated NAD(H) (2). This resulted in the creation of a novel formate:malate oxidoreductase. The new enzyme did not require the further addition of NAD(H) and it exhibited bi bi ping pong steady state kinetic behavior. The Km values of the fusion enzyme for reactants were the same as the parent oxidoreductases indicating that the kinetic behavior of the fusion proteins would be predictable.<br/><br/>We have extended this approach as we are increasing the extent of bioconjugation of the PEGylated NAD(H) to the fusion enzymes and expanding the oxidoreductases in the fusions to demonstrate this approach can be used to create additional new oxidoreductases with predictable, cofactorless bi bi ping pong kinetic behavior.

Conjugation of cofactors using polymeric swing arms enables creation of cofactorless ping pong enzymes with predictable kinetics

2019 Spring National Meeting

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