Fusing catalase to an alkane-producing enzyme maintains enzymatic activity by converting the inhibitory byproduct H2O2 to the cosubstrate O2 Carl Andre1, Sung Won Kim, Xiao-Hong Yu, and John Shanklin2 Biosciences Department, Brookhaven National Laboratory, Upton, NY 11973 Edited by Rodney B. Croteau, Washington State University, Pullman, WA, and approved December 31, 2012 (received for review October 29, 2012) Biologically produced alkanes represent potential renewable alter- enzyme was determined as part of a structural genomics effort natives to petroleum-derived chemicals. A cyanobacterial pathway (Protein Data Bank 2OC5, Joint Center for Structural Genom- consisting of acyl–Acyl Carrier Protein reductase and an aldehyde- ics). ADO converts aldehydes to n-1 alkanes with the aldehyde C1 deformylating oxygenase (ADO) converts acyl–Acyl Carrier Pro- released as formic acid (8, 9). Electrons required for the reaction teins into corresponding n-1 alkanes via aldehyde intermediates can be provided by NADPH via ferredoxin-NADP reductase in an oxygen-dependent manner (Km for O2,84± 9 μM). In vitro, (FNR), and ferredoxin (Fd) (7), or via the chemical mediator ADO turned over only three times, but addition of more ADO to phenazine methosulfate (PMS) (10). A proposed reaction scheme exhausted assays resulted in additional product formation. While for ADO (adapted from ref. 9) is depicted below where R evaluating the peroxide shunt to drive ADO catalysis, we discov- represents Acyl: ered that ADO is inhibited by hydrogen peroxide (H2O2) with an apparent Ki of 16 ± 6 μM and that H2O2 inhibition is of mixed-type with respect to O2. Supplementing exhausted assays with catalase (CAT) restored ADO activity, demonstrating that inhibition was reversible and dependent on H2O2, which originated from poor coupling of reductant consumption with alkane formation. Kinetic – PLANT BIOLOGY analysis showed that long-chain (C14 C18) substrates follow ADO has been reported to catalyze mechanistically distinct – Michaelis Menten kinetics, whereas short and medium chains oxygen-dependent (8) and oxygen-independent (10) production – (C8 C12) exhibit substrate inhibition. A bifunctional protein com- of alkanes, although there is some uncertainty regarding the – prising an N-terminal CAT coupled to a C-terminal ADO (CAT ADO) anaerobic mechanism (11) and a recent publication has provided prevents H2O2 inhibition by converting it to the cosubstrate O2. evidence for only oxygen-dependent activity (12). In vitro, ADO Indeed, alkane production by the fusion protein is observed upon exhibits poor catalytic activity and so far has yielded only 3–5 – addition of H2O2 to an anaerobic reaction mix. In assays, CAT ADO catalytic turnovers per active site, regardless of the assay system turns over 225 times versus three times for the native ADO, and its (9, 10) posing a serious barrier to its further study. Escherichia coli expression in increases catalytic turnovers per ac- An in-depth biochemical characterization of ADO is crucial if fi tive site by vefold relative to the expression of native ADO. We we wish to understand and improve this enzyme, and we embarked “ ” propose the term protection via inhibitor metabolism for fusion on a study to optimize the in vitro ADO assay and determine its proteins designed to metabolize inhibitors into noninhibitory kinetic parameters. Unexpectedly we found that ADO is reversibly compounds. inhibited by hydrogen peroxide (H2O2). Catalase, when included in enzyme reactions, was able to either prevent or relieve H2O2 adlehyde decarbonylase | diiron enzyme | dinuclear iron | inhibition. A fusion protein encoding catalase and ADO domains enzyme regulation separated by a flexible linker increases enzyme catalytic turnover by approximately two orders of magnitude relative to ADO alone he production of renewable liquid biofuels is an industrial by converting its inhibitor (H2O2) into a cosubstrate (O2). Timperative, and many methods and fuel types are being ex- plored. Alkanes are major constituents of our current fossil-de- Results rived fuels and also appear in nature as products of biosynthesis. Previous reports have found that ADOs are capable of ∼3–5 Therefore, alkanes represent attractive targets for engineering catalytic turnovers (9, 10, 12). We observed a very similar result large-scale biofuel production. Early studies of alkane biosynthesis with Prochlorococcus marinus ADO, using Zea mays ferredoxin described the conversion of fatty aldehydes (ALDs) to alkanes by NADP(H) oxidoreductase (FNR), Anabaena sp. pyridinum chlor- integral membrane proteins (1–3), and the ECERIFERUM1 ochromate (PCC) 7120 vegetative Fd, and NADPH to supply (CER1) gene of Arabidopsis has been found to encode this ac- electrons (Fig. 1A), except that our reactions appeared to be tivity (4–6). Unfortunately, the details of alkane biosynthesis have exhausted by 15 min, whereas the previously reported incuba- remained unresolved due to the difficulties associated with bio- tions were for many hours (9, 10). However, supplementing an chemical analyses of membrane proteins. exhausted reaction with additional ADO caused more product The recent discovery (7) of a soluble aldehyde-deformylating oxygenase (ADO, formerly known as aldehyde decarbonylase) in cyanobacteria that converts free aldehydes to the corresponding Author contributions: C.A. and J.S. designed research; C.A. and X.-H.Y. performed re- n-1 alkane has reinvigorated the study of alkane biosynthesis. The search; C.A. and S.W.K. contributed new reagents/analytic tools; C.A. and X.-H.Y. ana- soluble ADO is homologous to a number of oxygen-dependent lyzed data; and C.A. and J.S. wrote the paper. soluble diiron enzymes, including methane monooxygenase (MMO) The authors declare no conflict of interest. and acyl–ACP desaturases, whereas the membrane-bound alde- This article is a PNAS Direct Submission. hyde decarbonylase CER1 is homologous to the oxygen-dependent 1Present address: BASF Plant Science, Research Triangle Park, NC 27709. membrane class of diiron desaturase enzymes (7). Cyanobacte- 2To whom correspondence may be addressed. E-mail: [email protected]. fi rial ADOs are readily puri ed from heterologous expression This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. systems, are active in vitro, and a crystal structure of the 1073/pnas.1218769110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1218769110 PNAS Early Edition | 1of6 Downloaded by guest on September 25, 2021 Table 1. Reversible Inhibition of ADO by H2O2 AB5 no addition 14 + NADPH ADO 12 Relative activity* after H2O2 4 +FNR ADO + catalase † +Fd incubation + ETC 10 3 + ADO 8 H2O2 (mM) Catalase Immediate 10 min 6 2 0No100± 5.1 83 ± 1.9 4 ADO turnovers ADO turnovers ± ± 1 1No191.9 5.8 0.3 2 10 No 4.7 ± 1.3 6.8 ± 1.3 0 0 0 Yes 100 ± 7.7 103 ± 4.9 0 10 20 30 01020301 Yes 96 ± 3.2 105 ± 13 reaction time (min) reaction time (min) 10 Yes 89 ± 4.1 74 ± 9.5 CD12 25 *Activity relative to 0 mM H2O2 controls. † control Time of H O incubation after which buffer or catalase was added. Reac- 10 2 2 + ADO 20 tions were initiated with substrate 1 min after the addition of catalase and 8 + catalase proceeded for 15 min. Values are the mean ± SD, n = 3. 15 6 10 4 mediated reactions (Fig. 1D). During in vitro reactions, H2O2 is ADO turnovers ADO turnovers 5 2 produced by reduction of O2 from uncoupled NADPH con- 0 0 sumption by the electron transport components (principally Fd) 0102030catalase --+--+ contained in the ADO reaction (Table 2). We note that the use H O 2 2 -+--+- reaction time (min) of PMS resulted in a similar accumulation of H2O2 and in- PMS + NADH hibition of ADO to that observed for FNR, Fd, and NADPH. Inhibition of ADO by H2O2 was investigated in more detail, and found to be dose dependent and saturable (Fig. 2A). A Fig. 1. ADO is inactivated after three catalytic turnovers by H2O2 during in vitro enzyme reactions. (A) ADO catalytic turnovers after adding more proposed mechanism for ADO under aerobic conditions includes + + + + NADPH ( NADPH), FNR, Fd, the entire ETC ( ETC; NADPH, FNR, and Fd), the formation of a peroxo intermediate after O2 binding to the + or more ADO ( ADO) to the reaction after 15 min. (B) ADO catalytic turn- diiron active site (8). We therefore hypothesized that H2O2 should overs in the absence or presence of catalase. (C) ADO catalytic turnovers compete with oxygen for binding to the diiron site. To test this, after adding more ADO protein or catalase to the reaction after 15 min. (D) H2O2 inhibition was measured under various O2 concentrations. ADO catalytic turnovers in the presence of catalase or H2O2 with NADPH, FNR, A and Fd or with PMS and NADH reducing systems. All reactions contained Fig. 2 shows that the degree of inhibition is inversely proportional 200 μM 18 carbon aldehyde (18-ALD). All data are the mean ± SD (n = 3). to the O2 concentration. Further analysis of these data by double reciprocal plot indicates mixed-type inhibition in which H2O2 binding can occur before or after O2 (Fig. 2B). The Ki for H2O2 A to accumulate (Fig. 1 ). In contrast, adding extra NADPH, Fd, canbecalculatedusingthedatapresentedinFig.2A if the Km for or FNR, or a combination of the three, did not result in addi- O2 is known. The Km for oxygen has not been reported, and there tional product accumulation (Fig. 1A). From this we conclude is a lack of clarity in the literature as to whether ADO requires that ADO becomes inactivated upon incubation with the other molecular oxygen for catalysis (10–12). We therefore first per- assay components. formed a rigorous experiment to test whether molecular oxygen is In an attempt to simplify the assay system, we tried to replace required for catalysis (Fig. 2C). A Schlenk line and BASF O2- the electron transport components NADPH, Fd, and FNR with scrubbing catalyst was used to exchange O2-free argon for air by hydrogen peroxide (H2O2).