Picking on someone smaller. Cytochromes P450 catalyze the hydroxylation of thousands of substrates, including alkanes. No naturally occurring P450, however, is known to oxidize the smallest alkanes, ethane and methane. Here we report the direct and selective oxidation of ethane to ethanol using dioxygen, catalyzed by a cytochrome P450 BM‐3 variant engineered for high activity towards small alkanes (see scheme). Achieving P450‐catalyzed oxidation of ethane is a key step in the pathway to P450‐catalyzed methane oxidation and opens new opportunities for the bioconversion of natural gas to fuels and chemicals.

Testing the toxicities and biological activities of the human metabolites of drugs is important for development of safe and effective pharmaceuticals. Producing these metabolites using human cytochrome P450s is difficult, however, because the human enzymes are costly, poorly stable, and slow. We have used directed evolution to generate variants of P450 BM3 from Bacillus megaterium that function via the "peroxide shunt" pathway, using hydrogen peroxide in place of the reductase domain, oxygen and NADPH. Here, we report further evolution of the P450 BM3 heme domain peroxygenase to enhance production of the authentic human metabolites of propranolol by this biocatalytic route. This system offers a versatile, cost-effective, and scaleable route to the synthesis of drug metabolites.

Genes encoding 2-deoxy-D-ribose-5-phosphate aldolase (DERA) homologues from two hyperthermophiles, the archaeon Pyrobaculum aerophilum and the bacterium Thermotoga maritima, were expressed individually in Escherichia coli, after which the structures and activities of the enzymes produced were characterized and compared with those of E. coli DERA. To our surprise, the two hyperthermophilic DERAs showed much greater catalysis of sequential aldol condensation using three acetaldehydes as substrates than the E. coli enzyme, even at a low temperature (25°C), although both enzymes showed much less 2-deoxy-D-ribose-5-phosphate synthetic activity. Both the enzymes were highly resistant to high concentrations of acetaldehyde and retained about 50% of their initial activities after a 20-h exposure to 300 mM acetaldehyde at 25°C, whereas the E. coli DERA was almost completely inactivated after a 2-h exposure under the same conditions. The structure of the P. aerophilum DERA was determined by X-ray crystallography to a resolution of 2.0 Å. The main chain coordinate of the P. aerophilum enzyme monomer was quite similar to those of the T. maritima and E. coli enzymes, whose crystal structures have already been solved. However, the quaternary structure of the hyperthermophilic enzymes was totally different from that of the E. coli DERA. The areas of the subunit-subunit interface in the dimer of the hyperthermophilic enzymes are much larger than that of the E. coli enzyme. This promotes the formation of the unique dimeric structure and strengthens the hydrophobic intersubunit interactions. These structural features are considered responsible for the extremely high stability of the hyperthermophilic DERAs.Using acetaldehyde and D-glyceraldehyde-3-phosphate as substrates, 2-deoxy-D-ribose-5-phosphate aldolase (DERA; EC 4.1.2.4) catalyzes a reversible aldol reaction that generates 2-deoxy-D-ribose-5-phosphate (DRP) (1,17). DERA is unique in that it catalyzes the aldol reaction between two aldehydes, which serve as both the aldol donor and the acceptor components. In addition, DERA is the only aldolase known to accept three aldehydes in a sequential and stereoselective manner during an aldol condensation reaction, which makes it a particularly interesting potential biocatalyst for synthetic organic chemistry. Gijsen and Wong (6) were the first to observe Escherichia coli DERA (DERA Eco ) catalyze a double aldol condensation of three acetaldehyde molecules (Fig. 1): the reaction started with a stereospecific addition of acetaldehyde to a substituted acetaldehyde to form 3-hydroxy-4-substituted butylaldehyde, which in turn reacted with a third acetaldehyde. After the second condensation, the product (compound 1) largely cyclized to form stable 2,4,6-trideoxy-D-erythro-hexapyranoside, which is a useful chiral synthon of hydroxymethylglutaryl-coenzyme A reductase inhibitors. This has prompted investigation of the feasibility of applying DERA Eco for the synthesis of cholesterol-lowering agents (5-7, 23). The practical application of th...

In this study we have replaced all 13 methionine residues in the cytochrome P450 BM-3 heme domain (463 amino acids) with the isosteric methionine analog norleucine. This experiment has provided a means of testing the functional limits of globally incorporating into an enzyme an unnatural amino acid in place of its natural analog, and also an efficient way to test whether inactivation during peroxide-driven P450 catalysis involves methionine oxidation. Although there was no increase in the stability of the P450 under standard reaction conditions (in 10 mM hydrogen peroxide), complete substitution with norleucine resulted in nearly two-fold-increased peroxygenase activity. Thermostability was significantly reduced. The fact that the enzyme can tolerate such extensive amino acid replacement suggests that we can engineer enzymes with unique chemical properties via incorporation of unnatural amino acids while retaining or improving catalytic properties. This system also provides a platform for directing enzyme evolution using an extended set of protein building blocks.

The effect of bubble discharge in water on the growth rate of plants was investigated experimentally for application to plant cultivation systems. Spinach (Spinacia oleracea), radish (Raphanus sativus var. sativus), and strawberry (Fragaria × ananassa) were used as specimens to clarify the effect of the discharge treatment on edible parts of the plants. The specimens were cultivated in pots filled with artificial soil, which included chicken manure charcoal. Distilled water was sprayed on the artificial soil and drained through a hole in the pots to a water storage tank. The water was circulated from the water storage tank to the cultivation pots after 15 or 30 min discharge treatment on alternate days. A magnetic compression-type pulsed power generator was used to produce the bubble discharge with a repetition rate of 250 pps. The plant height in the growth phase and the dry weight of the harvested plants were improved markedly by the discharge treatment in water. The soil and plant analyzer development (SPAD) value of the plants also improved in the growth phase of the plants. The concentration of nitrate nitrogen, which mainly contributed to the improvement of the growth rate, in the water increased with the discharge treatment. The Brix value of edible parts of Fragaria × ananassa increased with the discharge treatment. The inactivation of bacteria in the water was also confirmed with the discharge treatment.

The radiocarbon ((14)C) of total carbon (TC) in atmospheric fine particles was measured at 6 h or 12 h intervals at two sites, 50 and 100 km downwind from Tokyo, Japan (Kisai and Maebashi) in summer 2007. The percent modern carbon (pMC) showed clear diurnal variations with minimums in the daytime. The mean pMC values at Maebashi were 28 ± 7 in the daytime and 45 ± 16 at night (37 ± 15 for the overall period). Those at Kisai were 26 ± 9 in the daytime and 44 ± 8 at night (37 ± 12 for the overall period). This data indicates that fossil sources were major contributors to the daytime TC, while fossil and modern sources had comparable contributions to nighttime TC in the suburban areas. At both sites, the concentration of fossil carbon as well as O(3) and the estimated secondary organic carbon increased in the daytime. These results suggest that fossil sources around Tokyo contributed significantly to the high daytime concentration of secondary organic aerosols (SOA) at the two suburban sites. A comparison of pMC and the ratio of elemental carbon/TC from our particulate samples with those from three end-member sources corroborates the dominant role of fossil SOA in the daytime.

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