Biochemical characterization of two recombinant ferredoxin reductases from Alcanivorax borkumensis SK2 Biotechnology and Applied Biochemistry Afsaneh Teimoori, 1,2 Shahin Ahmadian, 1 ∗ and Armin Madadkar-Sobhani 1,3 1 Department of Biochemistry, Institute of Biochemistry and Biophysics (IBB), University of Tehran, Tehran, Iran 2 Department of Industrial and Environmental Biotechnology, Division of Industrial Microbiology, National Institute for Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran 3 Department of Life Sciences, Barcelona Supercomputing Center, Barcelona, Spain Abstract. Alcanivorax borkumensis strain SK2 is a cosmopolitan oil-degrading oligotrophic marine γ -proteobacterium that exclusively uses petroleum hydrocarbons as sources of carbon and energy. Its ubiquity and unusual physiology suggest its global importance in the removal of hydrocarbons from polluted marine systems. The genome of A. borkumensis SK2 was recently sequenced. Two ferredoxin–nicotinamide adenine dinucleotide phosphate (NADPH) reductase genes (ABO_0145 and ABO_0203) have been annotated for this bacterium. In the present study, the expression, purification, and kinetic properties of these two genes were explored by constructing the prokaryotic expression vectors (pET21a) for the first time. Isopropyl β -d-thiogalactoside (0.5 mM) was used for induction of exponentially growing cells (30 ◦ C, overnight). Most of the proteins were expressed in inclusion body. Partial purification of recombinant enzymes was performed by ion-exchange chromatography on a DEAE-sepharose column using only one linear gradient of sodium chloride ranging between 0 and 500 mM. The recombinant enzymes displayed reductase activity, which was optimal at pH 6.0 and 45 ◦ C. Ferredoxin–NADPH reductases exhibited several outstanding properties that made them excellent model proteins to address broad biological questions. This study serves as the basis for further investigations of the biotechnological potential of these enzymes. c  2012 International Union of Biochemistry and Molecular Biology, Inc. Volume 59, Number 6, November/December 2012, Pages 457–464 • E-mail: ahmadian@ibb.ut.ac.ir. Keywords: Alcanivorax borkumensis, alkane hydroxylation, marine bacterium, NADPH, purification, kinetic assays 1. Introduction The oceans comprise the world’s largest natural habitat, as more than 70% of the earth’s surface is covered by water. Con- sidering the high biodiversity in ocean waters, marine microor- ganisms are important players in the global carbon cycle. In their natural habitat, free-living marine bacteria are facing a low-nutrient and high-salt environment. To meet these chal- lenges, marine microorganisms are forced to effectively adapt their physiology to the prevailing environmental situation. The Abbreviations: FNR, ferredoxin reductase; IPTG, isopropyl β-d-1-thiogalactopyranoside; MTT, 3-(4,5 dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide; NADH, nicotinamide adenine dinucleotide; NADPH, nicotinamide adenine dinucleotide phosphate; ABO_0145, ordered locus name of ferredoxin–NADPH reductase gene from Alcanivorax borkumensis SK2; LB, Luria Bertani; IB, inclusion body; FNR145, gene product of ABO_0145; His6-tagged FNRs, ferredoxin reductases containing 6 histidines at C terminal of the enzymes; GMO, genetically modified organism. ∗ Address for correspondence: Shahin Ahmadian, PhD, Professor, Institute of Biochemistry and Biophysics (IBB), University of Tehran, Enghelab Avenue, P.O. Box: 13145-1384, Tehran, Iran, Tel.: (+9821) 61113380; Fax: (+9821) 66404680; e-mail: ahmadian@ibb.ut.ac.ir. Received 19 May 2012; accepted 22 September 2012 DOI: 10.1002/bab.1047 Published online 18 December 2012 in Wiley Online Library (wileyonlinelibrary.com) ubiquity of Alcanivorax borkumensis reflects its highly devel- oped ability to adapt to the varying conditions that it faces in different unpolluted and polluted marine environments. In recent years, particular attention has been paid to A. borku- mensis SK2, which can assimilate alkanes but is unable to me- tabolize sugars, amino acids, and most other common carbon sources. It was originally isolated from the North Sea but gradu- ally from different geographical locations and different layers of oceanic surface waters [1–4]. The marine bacterium Alcanivorax sp., which belongs to the γ -subclass of proteobacteria, can uti- lize n-alkanes as sole carbon and energy sources, and becomes predominant in crude-oil-containing seawater when nitrogen and phosphorus nutrients are supplemented. This bacterium has two genes, alkB1 and alkB2, encoding nonheme alkane hydroxylase and mediating the oxidation of n-alkanes. It also has three cytochrome P450 that oxidize various hydrocarbons, including not only n-alkanes but also acycloalkane and aro- matic hydrocarbons. An alkane hydroxylase system consists of three components: a monooxygenase (AlkB, cytochrome P450), a small iron–sulfur protein (rubredoxin [AlkG, RubA], ferredoxin [Fd]), and a flavin reductase (rubredoxin reductase [AlkT, RubB], Fd reductase). In the first system (AlkB–rubredoxin–rubredoxin reductase), rubredoxin and rubredoxin reductase are soluble 457