Rachel N. Austin

The genome sequence of Desulfatibacillum alkenivorans AK-01: a blueprint for anaerobic alkane oxidation

Desulfatibacillum alkenivorans AK-01 serves as a model organism for anaerobic alkane biodegradation because of its distinctive biochemistry and metabolic versatility. The D. alkenivorans genome provides a blueprint for understanding the genetic systems involved in alkane metabolism including substrate activation, CoA ligation, carbon-skeleton rearrangement and decarboxylation. Genomic analysis suggested a route to regenerate the fumarate needed for alkane activation via methylmalonyl-CoA and predicted the capability for syntrophic alkane metabolism, which was experimentally verified. Pathways involved in the oxidation of alkanes, alcohols, organic acids and n-saturated fatty acids coupled to sulfate reduction and the ability to grow chemolithoautotrophically were predicted. A complement of genes for motility and oxygen detoxification suggests that D. alkenivorans may be physiologically adapted to a wide range of environmental conditions. The D. alkenivorans genome serves as a platform for further study of anaerobic, hydrocarbon-oxidizing microorganisms and their roles in bioremediation, energy recovery and global carbon cycling.

Alkane-oxidizing metalloenzymes in the carbon cycle

This review examines the metalloenzymes that catalyze the oxidation of alkanes in the environment. The focus of the review is on what is known about the relative abundances of these metalloenzymes, their metal ion requirements, and their reaction mechanisms. The relative significance of these reactions in the global transformation of alkanes is discussed.

Parallel and Competitive Pathways for Substrate Desaturation, Hydroxylation, and Radical Rearrangement by the Non-heme Diiron Hydroxylase AlkB

A purified and highly active form of the non-heme diiron hydroxylase AlkB was investigated using the diagnostic probe substrate norcarane. The reaction afforded C2 (26%) and C3 (43%) hydroxylation and desaturation products (31%). Initial C-H cleavage at C2 led to 7% C2 hydroxylation and 19% 3-hydroxymethylcyclohexene, a rearrangement product characteristic of a radical rearrangement pathway. A deuterated substrate analogue, 3,3,4,4-norcarane-d(4), afforded drastically reduced amounts of C3 alcohol (8%) and desaturation products (5%), while the radical rearranged alcohol was now the major product (65%). This change in product ratios indicates a large kinetic hydrogen isotope effect of ∼20 for both the C-H hydroxylation at C3 and the desaturation pathway, with all of the desaturation originating via hydrogen abstraction at C3 and not C2. The data indicate that AlkB reacts with norcarane via initial C-H hydrogen abstraction from C2 or C3 and that the three pathways, C3 hydroxylation, C3 desaturation, and C2 hydroxylation/radical rearrangement, are parallel and competitive. Thus, the incipient radical at C3 either reacts with the iron-oxo center to form an alcohol or proceeds along the desaturation pathway via a second H-abstraction to afford both 2-norcarene and 3-norcarene. Subsequent reactions of these norcarenes lead to detectable amounts of hydroxylation products and toluene. By contrast, the 2-norcaranyl radical intermediate leads to C2 hydroxylation and the diagnostic radical rearrangement, but this radical apparently does not afford desaturation products. The results indicate that C-H hydroxylation and desaturation follow analogous stepwise reaction channels via carbon radicals that diverge at the product-forming step.

Cage Escape Competes with Geminate Recombination during Alkane Hydroxylation by the Diiron Oxygenase AlkB

The alkane hydroxylase AlkB ofPseudomonas putidaGPo1 is typical of a large class of membrane-spanning diiron oxygenases that catalyze hydroxylation, epoxidation, and desaturation reactions. These enzymes are of considerable interest due to their impact on global hydrocarbon metabolism, their potential for practical biocatalytic application, and the resulting inspiration for the design of synthetic biomimetic catalysts. Although the three-dimensional structures of AlkB or any closely related proteins are unknown, topology modeling has predicted a structure comprised of six membrane-spanning helices with the catalytic iron diad appended to the cytoplasmic termini of the helix bundle. M(ssbauer data and alanine scanning have suggested that the diiron binding site is histidine-rich, as found in hemerythrin, and in contrast to the predominantly carboxylate binding motifs found in the diiron hydroxylases sMMO and T4MOh. Through protein side-chain mutations, a long, hydrophobic substrate-binding channel within the bundle has been identified that is tuned to accept medium-length alkanes. AlkB was the first alkane hydroxylase shown to generate a longlived substrate carbon radical during catalysis, as revealed by diagnostic skeletal rearrangements of the hydrocarbon probe norcarane. Herein we report results for the AlkB hydroxylation reaction using a panel of radical-clock substrates that display intrinsic rearrangement rates spanning five orders of magnitude, from a moderately slow 2.8 0 10 s 1 for bicyclo[3.1.0]hexane to an ultrafast 10 s 1 for trans-1-methyl-2phenylcyclopropane. Significantly, the ratios of rearranged and unrearranged products (R/U) found for the three mostslowly rearranging substrates were all in the range of unity even though their rearrangement rates differed widely. To account for these unusual results we propose a new diffusional model of AlkB hydroxylation that involves radical cagelike active-site dynamics of the type observed for hemeand cobalamin-containing metalloproteins. AlkB from P. putida GPo1 was expressed in P. putida GPo12 in the manner we have previously described. GPo12 is a receptacle clone that has been stripped of its innate hydroxylases and dehydrogenases. This approach has the advantages of producing unambiguous protein expression and high activity for only the inserted hydroxylase gene, while showing otherwise negligible background oxidation. Substrates were oxidized in resting whole cells and in cell-free extracts because all attempts to isolate and purify AlkB to date have led to loss of activity. Results for the AlkBmediated oxygenation of the three alkane substrates, bicyclo[4.1.0]heptane (norcarane, 1), bicyclo[3.1.0]hexane (2), and bicyclo[2.1.0]pentane (3), are presented in Table 1. These simple alkanes were chosen because of their similar size, nearly spherical shape, highly analogous structures, and similar rearrangement chemistry (Scheme 1). The data for all three substrates showed large amounts of rearrangement products (> 50%) consistent with the involvement of discreet radical intermediates during the hydroxylation process. Further, the ratios of primary to secondary alcohols formed from norcarane and bicyclohexane are similar to the partition ratios observed for bona fide radical reactions for these substrates (ca. 2 and 10%, respectively). It is striking, however, that the ratios of rearranged products to unrearranged products (R/U) for these three substrates do not correlate with the 100-fold change in the radical rearrangement rate constants for bicyclo[2.1.0]pent-2yl (kr = 20 10 9 s ), 2-norcaranyl (kr = 2 0 10 8 s ), and bicyclo[3.1.0]hex-2-yl (kr = 2.8 0 10 7 s ). We found the average R/U values for the three substrates remarkably constant (1.6, 1.6, and 4.7), corresponding to apparent radical lifetimes of 0.78, 7.8, and 170 ns, respectively. Indeed, bicyclohexane, with the slowest rearrangement rate, displayed the most rearranged product and by far the longest radical lifetime. The same effect was observed when norcarane and bicyclohexane were oxidized as a mixture. By contrast, the ultrafast rearranging probe trans-1-methyl-2-phenylcyclopropane (kr = 10 11 s ) was confirmed to afford only rearranged products. Clearly, there is a discrepancy here between the observed results for the more-slowly rearranging substrates and expectations based on Arrhenius-type kinetic behavior [*] D. Deng, R. S. Buzdygon, Prof. J. T. Groves Department of Chemistry, Princeton University Princeton NJ 08544 (USA) Fax: (+1)609-258-0348 E-mail: raustin@bates.edu

Role of O-acetyltransferase in activation of oxidised metabolites of the genotoxic environmental pollutant 1-nitropyrene

The genotoxic environmental contaminant 1-nitropyrene is metabolised in mammalian systems by pathways more complex than the straightforward nitroreduction which accounts for most of its biological activity in bacteria. In order to evaluate the role of O-acetyltransferase (OAT) activity in generation of genotoxic intermediates from 1-nitropyrene, the mutagenicity of the major primary oxidised metabolites of 1-nitropyrene was characterised in the Ames Salmonella typhimurium plate incorporation assay with strain TA98, and with variants of TA98 deficient (TA98/1,8-DNP6) or enhanced (YG1024) in O-acetyltransferase. 1-Nitropyren-3-ol was more mutagenic in the absence than in the presence of S9, while 1-nitropyren-4-ol, 1-nitropyren-6-ol and 1-nitropyren-8-ol required S9 for maximum expression of mutagenicity. 1-Nitropyren-4-ol (176 rev/nmol without S9, 467 rev/nmol with S9 in TA98) and 1-nitropyren-6-ol (13 rev/nmol without S9, 266 rev/nmol with S9 in TA98) were overall the most potent nitropyrenol isomers assayed. 1-Acetamidopyren-8-ol and 1-acetamidopyrene 4,5-quinone were only minimally active. 1-Acetamidopyren-3-ol exhibited direct-acting mutagenicity. 1-Acetamidopyren-6-ol, previously shown to be a major contributor to mutagenicity in the urines of rats dosed with 1-nitropyrene (Ball et al., 1984b), was confirmed as a potent (359 rev/nmol) S9-dependent mutagen. Both the direct-acting and the S9-dependent mutagenicity of all the compounds studied was enhanced in the OAT-overproducing strain and much diminished (though not always entirely lost) in the OAT-deficient strain, showing that OAT amplifies expression of the genotoxicity of these compounds. 1-Acetamidopyren-6-ol required both S9 and OAT activity in order to exhibit any mutagenicity; this finding strongly implicates N-hydroxylation followed by O-esterification, as opposed to further S9-catalyzed ring oxidation, as a major route of activation for urinary metabolites of 1-nitropyrene.

The Enigmatic P450 Decarboxylase OleT Is Capable of, but Evolved To Frustrate, Oxygen Rebound Chemistry

OleT is a cytochrome P450 enzyme that catalyzes the removal of carbon dioxide from variable chain length fatty acids to form 1-alkenes. In this work, we examine the binding and metabolic profile of OleT with shorter chain length (n ≤ 12) fatty acids that can form liquid transportation fuels. Transient kinetics and product analyses confirm that OleT capably activates hydrogen peroxide with shorter substrates to form the high-valent intermediate Compound I and largely performs C-C bond scission. However, the enzyme also produces fatty alcohol side products using the high-valent iron oxo chemistry commonly associated with insertion of oxygen into hydrocarbons. When presented with a short chain fatty acid that can initiate the formation of Compound I, OleT oxidizes the diagnostic probe molecules norcarane and methylcyclopropane in a manner that is reminiscent of reactions of many CYP hydroxylases with radical clock substrates. These data are consistent with a decarboxylation mechanism in which Compound I abstracts a substrate hydrogen atom in the initial step. Positioning of the incipient substrate radical is a crucial element in controlling the efficiency of activated OH rebound.

Synthesis and properties of novel substituted 4,5,6,7-tetrahydroindenes and selected metal complexes

The synthesis of a complete series of substituted 4,5,6,7-tetrahydroindenes (4,5,6,7-tetrahydroindene, 1; 1-methyl-, 1,3-dimethyl- and 1,2,3-trimethyl-4,5,6,7-tetrahydroindene, 2-4) is reported. The synthesis of ferrocenes (8-11) from these substituted cyclopentadienes is described. The electrochemistry of these ferrocenes indicates that these complexes are more readily oxidized than ferrocene and that the effect of methyl or alkyl substituents on the ease of oxidation is additive. A single crystal X-ray structure of two of the ferrocenes, bis(1,3-dimethyl-4,5,6,7-tetrahydroindenyl)iron(II), 10, and bis(1,2,3-trimethyl-4,5,6,7-tetrahy-droindenyl)iron(II), 11, indicates that steric hindrance causes the alkyl substituents to be bent away from the plane of the cyclopentadienyl ring. However, the structures differ in that the cyclopentadienyl rings in 10 are staggered wheras those in 11 are eclipsed. The synthesis of the cyclopentadienyltricarbonylmethyl compounds of molybdenum and tungsten from 3 and 4 is also described.

Substrate specificity and reaction mechanism of purified alkane hydroxylase from the hydrocarbonoclastic bacterium Alcanivorax borkumensis (AbAlkB).

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.

Spin coupling in distorted high-valent Fe(IV)-porphyrin radical complexes

In order to study structural influences on the interaction of Fe(IV) (S=1) and porphyrin cation radical (S′=1/2) in high-valent iron porphyrin complexes of the type ¦X-(TMP)Fe=O¦+(Cl−), X=I, Br2, Br4 were generated by mCPBA oxidation of corresponding Fe(III) porphyrins. The halogen substitution at the peripheral positions of the porphyrin leads to distortion of the planar porphyrin ring of ¦(TMP)Fe=O¦+. The new species have beeen investigated by temperature-dependent EPR and field-dependent Mössbauer spectroscopy; for the evaluation of spectra, we adopted the spin-Hamiltonian formalism including exchange interaction explicitly. As in ¦(TMP)Fe=O¦+, strong ferromagnetic spin coupling was observed with|J0|D=0.9–1 and a zero-field spltting ofD∼32 cm−1. For consistent parametrization of EPR and Mössbauer results, anisotropic coupling had to be introduced. Compared to ¦(TMP)Fe=O¦+ [1], analysis of the spectroscopic data shows that zero-field splitting and spin coupling is only slightly affected by the halogen distortion of the porphyrin structure.

Identity and mechanisms of alkane-oxidizing metalloenzymes from deep-sea hydrothermal vents

Six aerobic alkanotrophs (organism that can metabolize alkanes as their sole carbon source) isolated from deep-sea hydrothermal vents were characterized using the radical clock substrate norcarane to determine the metalloenzyme and reaction mechanism used to oxidize alkanes. The organisms studied were Alcanivorax sp. strains EPR7 and MAR14, Marinobacter sp. strain EPR21, Nocardioides sp. strains EPR26w, EPR28w, and Parvibaculum hydrocarbonoclasticum strain EPR92. Each organism was able to grow on n-alkanes as the sole carbon source and therefore must express genes encoding an alkane-oxidizing enzyme. Results from the oxidation of the radical-clock diagnostic substrate norcarane demonstrated that five of the six organisms (EPR7, MAR14, EPR21, EPR26w, and EPR28w) used an alkane hydroxylase functionally similar to AlkB to catalyze the oxidation of medium-chain alkanes, while the sixth organism (EPR92) used an alkane-oxidizing cytochrome P450 (CYP)-like protein to catalyze the oxidation. DNA sequencing indicated that EPR7 and EPR21 possess genes encoding AlkB proteins, while sequencing results from EPR92 confirmed the presence of a gene encoding CYP-like alkane hydroxylase, consistent with the results from the norcarane experiments.