title: Substrate specificity and reaction mechanism of purified alkane hydroxylase from the hydrocarbonoclastic bacterium Alcanivorax borkumensis (AbAlkB).
abstract: An alkane hydroxylase from the marine organism Alcanivorax borkumensis (AbAlkB) was purified. The purified protein retained high activity in an assay with purified rubredoxin (AlkG), purified maize ferredoxin reductase, NADPH, and selected substrates. The reaction mechanism of the purified protein was probed using the radical clock substrates bicyclo[4.1.0]heptane (norcarane), bicyclo[3.1.0]hexane (bicyclohexane), methylphenylcyclopropane and deuterated and non-deuterated cyclohexane. The distribution of products from the radical clock substrates supports the hypothesis that purified AbAlkB hydroxylates substrates by forming a substrate radical. Experiments with deuterated cyclohexane indicate that the rate-determining step has a significant CH bond breaking character. The products formed from a number of differently shaped and sized substrates were characterized to determine the active site constraints of this AlkB. AbAlkB can catalyze the hydroxylation of a large number of aromatic compounds and linear and cyclic alkanes. It does not catalyze the hydroxylation of alkanes with a chain length longer than 15 carbons, nor does it hydroxylate sterically hindered C-H bonds.
authors: Department of Chemistry, Bates College, 5 Andrews Rd. Lewiston, ME 04240, USA.
authors: Swe-Htet
authors: SH
authors: Naing
authors: Saba
authors: S
authors: Parvez
authors: Marilla
authors: M
authors: Pender-Cudlip
authors: John T
authors: JT
authors: Groves
authors: Rachel N
authors: RN
authors: Austin
mesh_terms: D053521:Alcanivoraceae / Q000737:chemistry / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry* / Q000302:isolation & purification; D001673:Biodegradation, Environmental; D001643:Bridged Bicyclo Compounds / Q000737:chemistry; D003510:Cyclohexanes / Q000737:chemistry; D003521:Cyclopropanes / Q000737:chemistry; D042926:Cytochrome P-450 CYP4A / Q000737:chemistry* / Q000302:isolation & purification; D005609:Free Radicals / Q000737:chemistry; D006900:Hydroxylation; D008958:Models, Molecular; D009249:NADP / Q000737:chemistry; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000737:chemistry / Q000302:isolation & purification; D012416:Rubredoxins / Q000737:chemistry* / Q000302:isolation & purification; D013379:Substrate Specificity; D013729:Terpenes / Q000737:chemistry; D003313:Zea mays / Q000737:chemistry / Q000201:enzymology
chemical_list: D001426:Bacterial Proteins; D001643:Bridged Bicyclo Compounds; D003510:Cyclohexanes; D003521:Cyclopropanes; D005609:Free Radicals; D010940:Plant Proteins; D012416:Rubredoxins; D013729:Terpenes; C046059:bicyclo(3.1.0)hexane; C506365:Cyclohexane; D009249:NADP; D042926:Cytochrome P-450 CYP4A; C460459:norcarane