Fusing catalase to an alkane-producing enzyme maintains enzymatic activity by converting the inhibitory byproduct H_2O_2 to the cosubstrate O2

摘要

Biologically produced alkanes represent potential renewable alternatives to petroleum-derived chemicals. A cyanobacterial pathway consisting of acyl-Acyl Carrier Protein reductase and an aldehydedeformylating oxygenase (ADO) converts acyl-Acyl Carrier Proteins into corresponding n-1 alkanes via aldehyde intermediates in an oxygen-dependent manner (Km for O_2, 84 ± 9 μM). In vitro, ADO turned over only three times, but addition of more ADO to exhausted assays resulted in additional product formation. While evaluating the peroxide shunt to drive ADO catalysis, we discovered that ADO is inhibited by hydrogen peroxide (H_2O_2) with an apparent Ki of 16 ± 6 μM and that H_2O_2 inhibition is of mixed-type with respect to O_2. Supplementing exhausted assays with catalase (CAT) restored ADO activity, demonstrating that inhibition was reversible and dependent on H_2O_2, which originated from poor coupling of reductant consumption with alkane formation. Kinetic analysis showed that long-chain (C14-C18) substrates follow Michaelis-Menten kinetics, whereas short and medium chains (C8-C12) exhibit substrate inhibition. A bifunctional protein comprising an N-terminal CAT coupled to a C-terminal ADO (CAT-ADO) prevents H_2O_2 inhibition by converting it to the cosubstrate O_2. Indeed, alkane production by the fusion protein is observed upon addition of H_2O_2 to an anaerobic reaction mix. In assays, CAT-ADO turns over 225 times versus three times for the native ADO, and its expression in Escherichia coli increases catalytic turnovers per active site by fivefold relative to the expression of native ADO. We propose the term "protection via inhibitor metabolism" for fusion proteins designed to metabolize inhibitors into noninhibitory compounds.