Mechanistic studies on non-heme iron monooxygenase catalysis: epoxidation, aldehyde formation and demethylation by the .omega.-hydroxylation system of Pseudomonas oleovorans

Abstract

In previous work we have established that the "w-hydroxylation" system of P. oleouorans readily converts terminal olefins to the corresponding 1,2-oxides and does so stereoselectively. We also demonstrated loss of olefin configuration during enzymatic epoxidation, a result inconsistent with a concerted epoxidation mechanism ( J . Am. Chem. SOC. 1977,99, 2017-2024. Since loss of olefin configuration is unprecedented for monooxygenase-catalyzed epoxidations, these studies have been confirmed with isolated enzymes and further extended in order to probe the mechanism of non-heme iron monooxygenase catalysis. Enzymatic epoxidation of both cis-and trans-1-deuterio-1-octene proceeds with about 70% inversion of the olefinic configuration, with corresponding results being obtained for the two olefins. As we reported in a preliminary communication (Bio/ Technology 1983, I , 677-686), the w-hydroxylation system also produces aldehydes from olefins. Aldehyde formation exhibits the reaction characteristics expected for the usual oxygenase pathway. Deuterium migration from C-1 to C-2 occurs in formation of aldehyde from olefin, although loss of deuterium also occurs. The w-hydroxylation system was found to efficiently catalyze 0-demethylation of heptyl methyl ether, the first demonstration of such activity for a non-heme iron monooxygenase of this type. Taken together, the results provide support for a two-step mechanism involving enzyme-generated species with cationic and/or radical character, which accounts for the stereoselectivity, configurational loss, substrate specificity, formation of aldehydes with deuterium migration, and demethylation activity exhibited by this enzyme system.The mechanism by which oxygenases catalyze the insertion of molecular oxygen into organic molecules has been the subject of intense scrutiny in recent years, due to the importance of these enzymes in detoxification, oncogenesis, biosynthesis, and metabolism in general. However, while the P-450 and the flavin-containing monooxygenases have been intensively studied at the molecular level, the state of our understanding of the molecular basis of non-heme iron monooxygenase catalysis is, by comparison, poor indeed. Non-heme iron monooxygenases comprise a large class of enzymes which, in addition to the P. oleouorans system discussed here, includes the phenylalanine, tyrosine, and tryptophan monoxygenases as well as squalene epoxidase.1,2The non-heme iron monooxygenase system from P. oleouorans which catalyzes terminal methyl group hydroxylation of alkanes and fatty acids was first shown by Coon and co-workers to consist of three protein components: rubredoxin, a flavoprotein reductase, and non-heme iron m o n~x y g e n a s e .~-~~ In previous work from (1) Comprehensive reviews on oxygenases can be found in: (a) "The Enzymes", 3rd ed.; Boyer, P. D., Ed.; Academic Press: New York, 1975; vol. 12. (b) 'Molecular Mechanisms of Oxygen Activation"; Hayaishi, O., Ed.; Academic Press: New York, 1974.

Identification of an Amino Acid Position That Determines the Substrate Range of Integral Membrane Alkane Hydroxylases

“…However, recombinants containing pCom8-GPo1-alkB-W55S or pCom8-GPo1-alkB-W55C did not grow on C 14 or C 16 , even after induction with dicyclopropylketone and in the presence of 0.01% rhamnolipids or Triton X-100. In combination with the observation that we were not able to obtain substrate range mu- tants with GPo1, this result indicates that GPo1 and derived recombinants lack an uptake system for alkanes longer than C 13 . Mutagenesis of the M. tuberculosis H37Rv AH.…”

“…Growth experiments using baffled Erlenmeyer flasks showed that P. putida GPo1 is able to grow well on alkanes ranging from C 6 to C 13 , with growth rates ranging from 0.49 h Ϫ1 (doubling time, 1.7 h) for C 6 to 0.018 h Ϫ1 (doubling time, 40 h) for C 13 (Table 2). Selection experiments to obtain mutants of P. putida GPo1 able to grow on alkanes longer than C 13 failed also in the presence of a gratuitous inducer of the alk genes, dicyclopropylketone (8), and/or biosurfactants to facilitate alkane uptake by the strain, such as rhamnolipids (0.01%) or Triton X-100 (0.1%), failed to facilitate alkane uptake by this strain.…”

“…The P. putida GPo1 AH catalyzes the hydroxylation of linear and branched aliphatic, alicyclic, and alkylaromatic compounds (7,20,31); oxidation of terminal alcohols to the corresponding aldehydes; demethylation of branched methyl ethers; sulfoxidation of thioethers; and epoxidation of terminal olefins (12,13,18,19) and allyl alcohol derivatives (6). One of the substrate range studies was used to estimate the approximate dimensions of the substrate-binding site (31).…”

Monooxygenases involved in the n-alkanes metabolism by Rhodococcus sp. BCP1: molecular characterization and expression of alkB gene

“…1.11 P. putida GPo1 is able to oxidize linear alkanes ranging from n-pentane to n-dodecane by virtue of the AlkB hydroxylase system [170] and can oxidize also butane and propane, even though the degradation rate is much slower [165]. In addition to the hydroxylation of aliphatic and alicyclic, the AlkB system has been shown to catalyze: oxidation of terminal alcohols to the corresponding aldehydes; demethylation of branched methyl ethers; sulfoxidation of thioethers, and epoxidation of terminal olefins [171][172][173]. Methane, ethane, or alkanes longer than C 13 are not oxidized.…”

An alternative and convenient synthesis of oct-7-enal, a naturally-occurring aldehyde isolated from the Japanese thistle Cirsium dipsacolepis

“…The unsaturated aldehyde has been synthesized in widely different scales and diverse purposes, by dihydroxylation of one of the double bonds of 1,8-nonadiene, followed by oxidative fission of the resulting diol, 7,8 as well as by partial hydroboration-oxidation and further oxidation of 1,7-octadiene 9 and biochemical oxidation of this diene with Pseudomonas oleovorans monooxygenase. 10 Also, copper (I) and lead (IV)-catalyzed oxidative ring opening of cyclooctanol 11 were employed for its synthesis, as well as copper (I)-assisted conjugate addition of 4-pentenylmagnesium bromide to acrolein diethyl acetal, followed by acid hydrolysis of the resulting enol ether, 12 isomerization of cyclooctene oxide employing solid acids and bases, 13 and isomerization of 2,7-octadien-1-ol on copper, chromium and zinc composite catalysts at 180-250 ºC 14 or on a copper catalyst. 15 However, these approaches are not exempt from serious drawbacks, such as low yields, 7 use of harsh conditions, 13,14 requirement of expensive starting materials, 7,10,12 use of special or not readily available catalysts or co-factors, 10 inconvenient separation conditions, 14 such as preparative HPLC, 7 and the concomitant production of unwanted byproducts, 13 sometimes the aldehyde 8 being only a minor product.…”