Bacteria readily oxidize alkanes utilizing the metalloenzyme alkane hydroxylase (AlkB) and thus the actions of AlkB represent one of nature's best defenses against the environmental effects of oil spills. Mammalian fatty acids are also desaturated by integral membrane fatty acid desaturases that are structurally quite similar to AlkB. It is not understood why AlkB catalyzes the addition of an OH group to an alkane while desaturases convert single C-C bonds to double C=C bonds. AlkB and soluble methane monooxygenase (sMMO) both catalyze the transformation of inert C-H bonds using a diiron catalyst but AlkB has a diiron catalyst with primarily nitrogen atoms as ligands while sMMO has an oxygen-rich coordination site. The coordination environment is predicted to influence the electronic structure and hence reactivity but the electronic structure of AlkB has not been characterized. The long- term objective of this research effort is to understand how the structure of AlkB determines the chemistry it affects and to use this knowledge to deepen our understanding of how biology selectively activates molecular oxygen and catalyzes the oxidation of inert hydrocarbons. An associated objective is to understand how structurally very similar biological motifs select between catalyzing the hydroxylation of alkanes or their desaturation. The primary goal of the specific research proposed is obtain a well-diffracting crystal of AlkB that will enable us to determine the three-dimensional structure of this enzyme. Related goals are to spectroscopically characterize the ground state and reactive intermediates and to begin explore AlkBs and related metalloenzymes from additional microorganisms. These goals enable us to test the hypothesis that the structures of the active sites of all AlkBs and membrane-spanning desaturases are the same but that structural differences in the substrate binding pocket control substrate selectivity and catalyst reactivity. If successful, this work woul have an impact on bioinorganic chemistry and environmental chemistry. A three-dimensional structure of AlkB would help to answer many questions about the range of chemical motifs that biology can use to oxidize alkanes. It would also answer questions about some of the chemical processes utilized in the environmental response to oil spills.
This project focuses on understanding how the enzyme that catalyzes the first step in the transformation of oil to carbon dioxide works. It will also explore how this enzyme, which is structurally similar to enzymes important in human lipid metabolism, functions in comparison with that important class of enzymes as well.
|Austin, Rachel Narehood; Saito, Mak A (2014) Metals in marine biochemistry. Metallomics 6:1105-6|
|Austin, Rachel Narehood; Kenney, Grace E; Rosenzweig, Amy C (2014) Perspective: what is known, and not known, about the connections between alkane oxidation and metal uptake in alkanotrophs in the marine environment. Metallomics 6:1121-5|
|Naing, Swe-Htet; Parvez, Saba; Pender-Cudlip, Marilla et al. (2013) Substrate specificity and reaction mechanism of purified alkane hydroxylase from the hydrocarbonoclastic bacterium Alcanivorax borkumensis (AbAlkB). J Inorg Biochem 121:46-52|
|Austin, Rachel N; Luddy, Kate; Erickson, Karla et al. (2008) Cage escape competes with geminate recombination during alkane hydroxylation by the diiron oxygenase AlkB. Angew Chem Int Ed Engl 47:5232-4|
|Chakrabarty, Sarmistha; Austin, Rachel N; Deng, Dayi et al. (2007) Radical intermediates in monooxygenase reactions of rieske dioxygenases. J Am Chem Soc 129:3514-5|
|Rozhkova-Novosad, Elena A; Chae, Jong-Chan; Zylstra, Gerben J et al. (2007) Profiling mechanisms of alkane hydroxylase activity in vivo using the diagnostic substrate norcarane. Chem Biol 14:165-72|
|Austin, Rachel N; Deng, Dayi; Jiang, Yongying et al. (2006) The diagnostic substrate bicyclohexane reveals a radical mechanism for bacterial cytochrome P450 in whole cells. Angew Chem Int Ed Engl 45:8192-4|
|Bertrand, Erin; Sakai, Ryo; Rozhkova-Novosad, Elena et al. (2005) Reaction mechanisms of non-heme diiron hydroxylases characterized in whole cells. J Inorg Biochem 99:1998-2006|