Fusing catalase to an alkane-producing enzyme maintains enzymatic activity by converting the inhibitory byproduct H 2 O 2 to the cosubstrate O 2

• DOI: 10.1073/pnas.1218769110
• Publication Date: 2013-02-08T08:09:43Z
• Authors: Carl Andre; Sung Won Kim; Xiao-Hong Yu; John Shanklin
• Keywords: Alkane; Turnover number

Biologically produced alkanes represent potential renewable alternatives to petroleum-derived chemicals. A cyanobacterial pathway consisting of acyl–Acyl Carrier Protein reductase and an aldehyde-deformylating oxygenase (ADO) converts Proteins into corresponding n-1 via aldehyde intermediates in oxygen-dependent manner (K m for O 2 , 84 ± 9 µM). In vitro, ADO turned over only three times, but addition more exhausted assays resulted additional product formation. While evaluating the peroxide shunt drive catalysis, we discovered that is inhibited by hydrogen (H ) with apparent K i 16 6 µM H inhibition mixed-type respect . Supplementing catalase (CAT) restored activity, demonstrating was reversible dependent on which originated from poor coupling reductant consumption alkane Kinetic analysis showed long-chain (C14–C18) substrates follow Michaelis–Menten kinetics, whereas short medium chains (C8–C12) exhibit substrate inhibition. bifunctional protein comprising N-terminal CAT coupled a C-terminal (CAT–ADO) prevents converting it cosubstrate Indeed, production fusion observed upon anaerobic reaction mix. assays, CAT–ADO turns 225 times versus native ADO, its expression Escherichia coli increases catalytic turnovers per active site fivefold relative ADO. We propose term “protection inhibitor metabolism” proteins designed metabolize inhibitors noninhibitory compounds.