Fusing catalase to an alkane-producing enzyme maintains enzymatic activity by converting the inhibitory byproduct H2O2 to the cosubstrate O2

Abstract

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 acyl-Acyl Carrier Proteins into corresponding n-1 alkanes via aldehyde intermediates in an oxygen-dependent manner (K(m) 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(2)O(2)) with an apparent K(i) of 16 ± 6 µM and that H(2)O(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(2)O(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(2)O(2) inhibition by converting it to the cosubstrate O(2). Indeed, alkane production by the fusion protein is observed upon addition of H(2)O(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.

Fig. 1.

ADO is inactivated after three catalytic turnovers by H₂O₂ during in vitro enzyme reactions. (A) ADO catalytic turnovers after adding more NADPH (+NADPH), +FNR, +Fd, the entire ETC (+ETC; NADPH, FNR, and Fd), or more ADO (+ADO) to the reaction after 15 min. (B) ADO catalytic turnovers in the absence or presence of catalase. (C) ADO catalytic turnovers after adding more ADO protein or catalase to the reaction after 15 min. (D) ADO catalytic turnovers in the presence of catalase or H₂O₂ with NADPH, FNR, and Fd or with PMS and NADH reducing systems. All reactions contained 200 µM 18 carbon aldehyde (18-ALD). All data are the mean ± SD (n = 3).

Fig. 2.

Mixed-type inhibition of ADO by H₂O₂ with respect to O₂. (A) ADO activity in the presence of various concentrations of H₂O₂ in 50, 250, and 1,250 µM O₂ atmospheres. (B) Double reciprocal plot of ADO activity versus oxygen concentration in the presence of various amounts of H₂O₂. (C) ADO activity under ambient atmosphere (control), in 100% argon (–O₂) after degassing, or in air after degassing (open). After 15 min under argon, air was reintroduced and activity was restored (–O₂, then open). (D) ADO activity in various concentrations of O₂. All reactions used NADPH, FNR, and Fd with 200 µM 18-ALD. All data are the mean ± SD (n = 3).

Fig. 3.

Improved ADO performance by physical tethering to catalase. (A) Schematic representation of the CAT–ADO fusion protein made by fusing E. coli (Ec) catalase and P. marinus (Pm) ADO with a 20 amino acid linker. (B) Catalase activity of ADO, a commercial catalase preparation (catalase), and the CAT–ADO fusion protein. (C) ADO catalytic turnovers of ADO (ADO) and CAT–ADO in the absence (CAT–ADO) or presence (CAT–ADO + 1 mM H₂O₂) of H₂O₂. (D) ADO catalytic turnovers in anaerobic reactions of ADO (ADO) and CAT–ADO in the presence or absence of water or H₂O₂. Assays for C and D used NADPH, FNR, and Fd with 200 µM 18-ALD. (E) In vivo alkane production per ADO active site of ADO and CAT–ADO when expressed in E. coli with an AAR. All data are the mean ± SD (n = 3). **P < 0.01 by t test.

Fig. 4.

ADO reaction kinetics with various aldehyde substrates. (A) ADO specific activity with octadecenal (18:1-ALD), octadecanal (18-ALD), hexadecanal (16-ALD), and tetradecanal (14-ALD) as substrates. (B) ADO specific activity with dodecanal (12-ALD), decanal (10-ALD), and octanal (8-ALD) as substrates. (C) Alkane products formed in substrate competition assays. Substrates are labeled as two numbers, x/y, where x is always 200 µM 18-ALD and y is the chain length of the other aldehyde substrate, also at 200 µM. The control reaction contains 400 µM 18-ALD. All reactions used NADPH, FNR, and Fd. All data are mean ± SD (n = 3).