Reports: ND448781-ND4: Mechanism of Aldehyde Decarbonylase, a Novel Enzyme Involved in Hydrocarbon Biosynthesis

E. Neil G. Marsh, M.A., Sc.D. , University of Michigan


This award has provided valuable seed funding to initiate a new project aimed at the discovery and characterization of novel enzymes that may be useful in the area of biofuels biosynthesis.  The award funded a post-doctoral scientist, Dr. Bekir Eser to work full time on the project.  Dr. Eser worked very closely with a graduate student, Mr. Debasis Das, on the project whom he co-mentored with the P.I.  This resulted in a very productive research project that has led to the discovery of a new type of enzymatic reaction for generating alkanes, described below.  The project has generated several publications, presentations at scientific meetings and provided training and professional development for the graduate student and post-doctoral scientist.  It has led to a multinational collaborative project with scientists from 7 academic institutions in Europe, funded by the European Union and provided preliminary results to support further proposals to the National Science Foundation.


Biosynthetic pathways that generate hydrocarbons have become a subject of intense interest recently as their potential to generate new biofuels has been recognized.  Although hydrocarbons are simple molecules, the biosynthesis of molecules that lack any chemical functional groups is surprisingly challenging because biochemical reactions that remove functionality, such as decarboxylations, dehydrations and reduction of double bonds, invariably rely on the presence of adjacent functional groups to stabilize unfavorable transition states.  Therefore, in addition to their applications in biofuels production, enzymes involved in hydrocarbon biosynthesis are interesting because of the unusual and chemically difficult reactions they catalyze. 

We have focused on determining the mechanism of a a newly-discovered hydrocarbon-forming enzyme from cyanobacteria, aldehyde decarbonylase (cAD).  We have shown that it catalyzes a most unusual reaction:  the formal hydrolysis of long-chain fatty aldehydes to the corresponding alkanes and formate.  Most interestingly, although this enzyme is, structurally, a member of the family of non-heme di-iron oxygenases, the reaction occurs by a novel oxygen-independent mechanism that most likely involves free radicals. 


A crystal structure for cAD from Prochlorococcus marinus MIT9313 had previously been solved as part of a structural proteomics project, although no function had been assigned.  The structure revealed that cAD is member of the non-heme dinuclear iron oxygenase family of enzymes exemplified by methane monoxygenase, type I ribonucleotide reductase, and ferritin.  We first over-expressed cAD from Prochlorococcus. marinus MIT9313 in E. coli using a synthetic gene that allowing us to obtain ~ 100 mg quantities of enzyme.

Previously studied plant and algal ADs produce CO as the bi-product of alkane formation.  However all attempts to detect CO formation by cAD were negative.  We determined that formate was produced stoichiometrically as the co-product by derivatizing the products of reaction with 2-nitrophenyl hydrazine and subsequent analysis by reverse phase HPLC and mass spectrometry. When the reaction was performed in D2O, the product alkane contained deuterium.  Whereas when the reaction was performed with deuterated octadecanal the aldehyde proton was retained in formate.  Thus cAD produces alkanes by a very unusual and unprecedented reaction involving hydrolysis of aldehydes.

The reactions catalyzed by this structural class of di-iron enzymes invariably involve molecular oxygen and require an external reducing system in the form of ferredoxin, ferredoxin oxidoreductase and NADPH, to reduce the ‘resting’ di-ferric form of the enzyme to the active diferrous form at the start of each turn-over.  However the reaction catalyzed by cAD is redox neutral and we established that cAD does not require oxygen for activity.  Indeed the enzyme exhibited ~ 40 % higher activity when assayed under anaerobic conditions, possibly due to adventitious oxidation of the di-iron cluster to the inactive ferric form when the assays were performed aerobically.  Although oxygen is not required, the enzyme still exhibits an absolute requirement for a reducing system – a unique feature of this reaction and one which is not fully understood.  Furthermore, we showed that the reducing system, PMS/NADH appears to work better than the ferredoxin system, is catalytic in the reaction – multiple turn-overs were obtained with no NADH consumption.

By analogy with the reaction of di-iron enzymes with O2, we hypothesized that the first step in the cAD reaction may involve electron transfer from Fe(II) to the carbonyl group of the aldehyde.  To test this, we investigated the interaction of the substrate with the di-iron centre by EPR spectroscopy.  Experiments were conducted in the absence of the reducing system so that turn-over could not occur.  Addition of heptanal to cAD resulted in the appearance of a signal at g = 4.3 characteristic of a high-spin ferric ion.  This observation is consistent with Fe(II) transferring an electron to the substrate to generate Fe(III) and, presumably, a ketyl radical that is too unstable to observe directly.  Further support for the generation of an organic radical came from experiments in which the spin-trapping agent N-tert-Butyl-α-phenylnitrone (PBN) was included in the reaction.  The ferric signal is now accompanied by a characteristic signal for the PBN nitroxide radical-adduct at g = 2. 

We have also investigated the substrate range of cAD, which is of particular interest for potential use in biofuels applications.  Our experiments demonstrate that the enzyme is will convert aldehydes as short as heptanal to the corresponding alkanes.  However, kcat is extremely slow ~ 7 hour-1 (!); this is an important finding, with implications for biofuels applications.  We are currently working on ways to increase enzyme activity.


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