Reaction mechanisms of non-heme diiron hydroxylases characterized in whole cells.
Whole cells expressing the non-heme diiron hydroxylases AlkB and toluene 4-monooxygenase (T4MO) were used to probe enzyme reaction mechanisms. AlkB catalyzes the hydroxylation of the radical clock substrates bicyclo[4.1.0]heptane (norcarane), spirooctane and 1,1-diethylcyclopropane, and does not catalyze the hydroxylation of the radical clocks 1,1-dimethylcyclopropane or 1,1,2,2-tetramethylcyclopropane. The hydroxylation of norcarane yields a distribution of products consistent with an "oxygen-rebound" mechanism for the enzyme in both the wild type Pseudomonas putida GPo1 and AlkB from P. putida GPo1 expressed in Escherichia coli. Evidence for the presence of a substrate-based radical during the reaction mechanism is clear. With norcarane, the lifetime of that radical varies with experimental conditions. Experiments with higher substrate concentrations yield a shorter radical lifetime (approximately 1 ns), while experiments with lower substrate concentrations yield a longer radical lifetime (approximately 19 ns). Consistent results were obtained using either wild type or AlkB-equipped host organisms using either "resting cell" or "growing cell" approaches. T4MO expressed in E. coli also catalyzes the hydroxylation of norcarane with a radical lifetime of approximately 0.07 ns. No radical lifetime dependence on substrate concentration was seen. Results from experiments with diethylcyclopropane, spirooctane, dimethylcyclopropane, and diethylcyclopropane are consistent with a restricted active site for AlkB.
Bertrand
Erin
E
Department of Chemistry, Bates College, 5 Andrews Rd. Lewiston, ME 04240, United States.
Sakai
Ryo
R
Rozhkova-Novosad
Elena
E
Moe
Luke
L
Fox
Brian G
BG
Groves
John T
JT
Austin
Rachel N
RN
D042926:Cytochrome P-450 CYP4A / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology; D006900:Hydroxylation; D019159:Nonheme Iron Proteins; D010105:Oxygenases / Q000378:metabolism*; D016958:Pseudomonas putida / Q000201:enzymology; D013379:Substrate Specificity; D013729:Terpenes / Q000378:metabolism
D019159:Nonheme Iron Proteins; D013729:Terpenes; D010105:Oxygenases; C068610:toluene-4-monooxygenase; D042926:Cytochrome P-450 CYP4A; C460459:norcarane