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Pseudomonas mendocina KR1 grows on toluene as a sole carbon and energy source. A multicomponent oxygenase was partially purified from toluene-grown cells and separated into three protein components. The reconstituted enzyme system, in the presence of NADH and Fe2 , oxidized toluene to p-cresol as the first detectable product. Experiments with p-deuterotoluene led to the isolation of p-cresol which retained 68% of the deuterium initially present in the parent molecule. When the reconstituted enzyme system was incubated with toluene in the presence of 1802, the oxygen in p-cresol was shown to be derived from molecular oxygen. The results demonstrate that P. mendocina KR1 initiates degradation of toluene by a multicomponent enzyme system which has been designated toluene-4-monooxygenase.Several small aromatic hydrocarbons can be utilized by bacteria as sole carbon and energy sources for growth. Degradation of these hydrocarbons is initiated by either oxidation of an alkyl side group (26,36) or direct attack on the aromatic ring (1,9,11,12,20). Toluene has been used by a number of laboratories as a model compound to study the metabolism of aromatic hydrocarbons. When utilized as a sole carbon and energy source, toluene can be metabolized via oxidation of the methyl group to yield benzyl alcohol as the first metabolite (30,36). This type of oxidation is catalyzed by enzymes of the TOL pathway. Toluene may also be degraded by enzymes of the TOD pathway, in which initial attack is by dioxygenation of the aromatic nucleus at the 2,3 position (11,25,38). In this case, the first detectable product is (+)-cis-l(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene (cis-toluene dihydrodiol). Recently, toluene has been shown to be oxidized by whole cells of organisms which grow on toluene to o-cresol (33) and p-cresol (35a) as initial metabolites.A bacterium which grows on toluene as a sole carbon and energy source has been isolated and identified in our laboratory as Pseudomonas mendocina KR1 (31a). After growth with toluene, whole cells of this organism oxidize p-cresol at a rapid rate. We now report the separation into three protein components of the enzyme system responsible for the conversion of toluene to p-cresol. The reconstituted enzyme system catalyzed oxidation of toluene to p-cresol by an NAD(P)H-dependent monooxygenation reaction in which one atom of molecular oxygen is incorporated into the hydroxyl group of p-cresol. The reaction proceeded with a concomitant NIH shift, which suggests that toluene-3,4-oxide is the first initial oxidation product.( 6,7,8-tetrahydropterine dihydrochloride, DNase I, Bis-Tris propane, Trizma base (Tris), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonate (HEPES), morpholine propane sulfonate (MOPS), dithiothreitol (DTT), acrylamide, and N,N,N',N'-tetramethylethylenediamine were from Sigma Chemical Co., Rockford, Ill.; 2-(N-morpholine)ethanesulfate was from U.S. Biochemical Corp., Cleveland, Ohio; sodium dodecyl sulfate (SDS) was from Fisher Scientific, Fairlawn, N.J.; 2-amino-6,7 -dimethyl -...

The unusual architecture of the enzyme (MsAcT) isolated from Mycobacterium smegmatis forms the mechanistic basis for favoring alcoholysis over hydrolysis in water. Unlike hydrolases that perform alcoholysis only under anhydrous conditions, MsAcT demonstrates alcoholysis in substantially aqueous media and, in the presence of hydrogen peroxide, has a perhydrolysis:hydrolysis ratio 50-fold greater than that of the best lipase tested. The crystal structures of the apoenzyme and an inhibitor-bound form have been determined to 1.5 A resolution. MsAcT is an octamer in the asymmetric unit and forms a tightly associated aggregate in solution. Relative to other structurally similar monomers, MsAcT contains several insertions that contribute to the oligomerization and greatly restrict the shape of the active site, thereby limiting its accessibility. These properties create an environment by which MsAcT can catalyze transesterification reactions in an aqueous medium and suggests how a serine hydrolase can be engineered to be an efficient acyltransferase.

SummaryEmissions of biogenic volatile organic compounds (VOCs) form an important part of the global carbon cycle, comprising a significant proportion of net ecosystem productivity. They impact atmospheric chemistry and contribute directly and indirectly to greenhouse gases. Isoprene, emitted largely from plants, comprises one third of total VOCs, yet in contrast to methane, which is released in similar quantities, we know little of its biodegradation. Here, we report the genome of an isoprene degrading isolate, Rhodococcus sp. AD45, and, using mutagenesis shows that a plasmid-encoded soluble di-iron centre isoprene monooxygenase (IsoMO) is essential for isoprene metabolism. Using RNA sequencing (RNAseq) to analyse cells exposed to isoprene or epoxyisoprene in a substrate-switch time-course experiment, we show that transcripts from 22 contiguous genes, including those encoding IsoMO, were highly upregulated, becoming among the most abundant in the cell and comprising over 25% of the entire transcriptome. Analysis of gene transcription in the wild type and an IsoMO-disrupted mutant strain showed that epoxyisoprene, or a subsequent product of isoprene metabolism, rather than isoprene itself, was the inducing molecule. We provide a foundation of molecular data for future research on the environmental biological consumption of this important, climate-active compound.

Perhydrolase S54V (AcT) effectively catalyzes the perhydrolysis of propylene glycol diacetate (PGD) to generate peracetic acid (PAA). PAA is a potent oxidant used for sanitization and disinfection, with broad effectiveness against bacteria, yeasts, fungi, and spores. In this study, active and stable composites are developed by incorporating AcT–carbon nanotube conjugates into polymer and latex‐based paint. At a conjugate loading of 0.16% (w/v), the composite generated 11 mM PAA in 20 min, capable of killing more than 99% spores initially charged at 106 colony‐forming units per milliliter.