The advent of biotechnology-derived, herbicide-resistant crops has revolutionized farming practices in many countries. Facile, highly effective, environmentally sound, and profitable weed control methods have been rapidly adopted by crop producers who value the benefits associated with biotechnology-derived weed management traits. But a rapid rise in the populations of several troublesome weeds that are tolerant or resistant to herbicides currently used in conjunction with herbicide-resistant crops may signify that the useful lifetime of these economically important weed management traits will be cut short. We describe the development of soybean and other broadleaf plant species resistant to dicamba, a widely used, inexpensive, and environmentally safe herbicide. The dicamba resistance technology will augment current herbicide resistance technologies and extend their effective lifetime. Attributes of both nuclear- and chloroplast-encoded dicamba resistance genes that affect the potency and expected durability of the herbicide resistance trait are examined.

Candida albicans cells of opposite mating types are thought to conjugate during infection in mammalian hosts, but paradoxically, the mating-competent opaque state is not stable at mammalian body temperatures. We found that anaerobic conditions stabilize the opaque state at 37°C, block production of farnesol, and permit in vitro mating at 37°C at efficiencies of up to 84%. Aerobically, farnesol prevents mating because it kills the opaque cells necessary for mating, and as a corollary, farnesol production is turned off in opaque cells. These in vitro observations suggest that naturally anaerobic sites, such as the efficiently colonized gastrointestinal (GI) tract, could serve as niches for C. albicans mating. In a direct test of mating in the mouse GI tract, prototrophic cells were obtained from auxotrophic parent cells, confirming that mating will occur in this organ. These cells were true mating products because they were tetraploid, mononuclear, and prototrophic, and they contained the heterologous hisG marker from one of the parental strains.

Dicamba (3,6-dichloro-2-methoxybenzoic acid) is a widely used herbicide that is efficiently degraded by soil microbes. These microbes use a novel Rieske non-heme oxygenase, dicamba monooxygenase (DMO), to catalyze the oxidative demethylation of dicamba to 3,6-dichlorosalicylic acid (DCSA) and formaldehyde. We have determined the crystal structures of DMO in the free state, bound to its substrate dicamba, and bound to the product DCSA at 2.10−1.75 Å resolution. The structures show that the DMO active site uses a combination of extensive hydrogen bonding and steric interactions to correctly orient chlorinated, ortho-substituted benzoic acid-like substrates for catalysis. Unlike other Rieske aromatic oxygenases, DMO oxygenates the exocyclic methyl group, rather than the aromatic ring, of its substrate. This first crystal structure of a Rieske demethylase shows that the Rieske oxygenase structural scaffold can be co-opted to perform varied types of reactions on xenobiotic substrates.

The first committed step in methanopterin biosynthesis is catalyzed by 4-(␤-D-ribofuranosyl)aminobenzene 5-phosphate (RFA-P) synthase. Unlike all known phosphoribosyltransferases, ␤-RFA-P synthase catalyzes the unique formation of a C-riboside instead of an N-riboside in the condensation of p-aminobenzoic acid (pABA) and 5-phospho-␣-D-ribosyl-1-pyrophosphate (PRPP) to produce 4-(␤-D-ribofuranosyl)aminobenzene 5-phosphate (␤-RFA-P), CO 2 , and inorganic pyrophosphate (PP i ). Here we report the successful cloning, active overexpression in Escherichia coli, and purification of this homodimeric enzyme containing two 36.2-kDa subunits from the methanogen Methanococcus jannaschii. Steady-state initial velocity and product inhibition kinetic studies indicate an ordered Bi-Ter mechanism involving binding of PRPP, then pABA, followed by release of the products CO 2 , then ␤-RFA-P, and finally PP i . The Michaelis parameters are as follows: K m pABA, 0.15 mM; K m PRPP, 1.50 mM; V max , 375 nmol/min/mg; k cat , 0.23 s ؊1 . CO 2 showed uncompetitive inhibition, K i ‫؍‬ 0.990 mM, under varied PRPP and saturated pABA, and a mixed type of inhibition, K 1 ‫؍‬ 1.40 mM and K 2 ‫؍‬ 3.800 mM, under varied pABA and saturated PRPP. RFA-P showed uncompetitive inhibition, K i ‫؍‬ 0.210 mM, under varied PRPP and saturated pABA, and again uncompetitive, K i ‫؍‬ 0.300 mM, under saturated PRPP and varied pABA. PP i exhibits competitive inhibition, K i ‫؍‬ 0.320 mM, under varied PRPP and saturated pABA, and a mixed type of inhibition, K 1 ‫؍‬ 0.60 mM and K 2 ‫؍‬ 1.900 mM, under saturated PRPP and varied pABA. Synthase lacks any chromogenic cofactor, and the presence of pyridoxal phosphate and the mechanistically related pyruvoyl cofactors has been strictly excluded.The first step in methanopterin biosynthesis is catalyzed by 4-(␤-D-ribofuranosyl)aminobenzene 5Ј-phosphate (␤-RFA-P) 1 synthase. This enzyme catalyzes the condensation between para-aminobenzoic acid (pABA) and 5-phospho-␣-D-ribosyl-1-pyrophosphate (PRPP) with concomitant formation of ␤-RFA-P, CO 2 , and inorganic pyrophosphate (PP i ) (1). This enzyme is a phosphoribosyltransferase and a decarboxylase and forms a C-riboside, which is unique among phosphoribosyltransferases and pABA-dependent enzymes. For example, in an early step in tetrahydrofolate biosynthesis, dihydropteroate synthase catalyzes a condensation between the amino group of pABA and dihydropterin pyrophosphate to generate dihydropteroate, eliminating PP i . Thus, ␤-RFA-P synthase and dihydropteroate synthase both use pABA as a substrate and produce PP i as product; however, the amino group is the nucleophile in dihydropteroate synthase, whereas the aromatic ring carbon 4 (C-4) is the nucleophile in ␤-RFA-P synthase (2, 3).How does RFA-P synthase generate an electrophilic center at C-1 of PRPP? How does this enzyme poise ring carbon-4 of pABA for nucleophilic attack on the C-1 of PRPP and activate this position for decarboxylation? The mechanism shown in Scheme 1 is our working hypothesis. When PRPP binds, C-1 is ...

This paper describes the design, synthesis, and successful employment of inhibitors of 4-(␤-D-ribofuranosyl)aminobenzene-5-phosphate (RFA-P) synthase, which catalyzes the first committed step in the biosynthesis of methanopterin, to specifically halt the growth of methane-producing microbes. RFA-P synthase catalyzes the first step in the synthesis of tetrahydromethanopterin, a key cofactor required for methane formation and for one-carbon transformations in methanogens. A number of inhibitors, which are N-substituted derivatives of p-aminobenzoic acid (pABA), have been synthesized and their inhibition constants with RFA-P synthase have been determined. Based on comparisons of the inhibition constants among various inhibitors, we propose that the pABA binding site in RFA-P synthase has a relatively large hydrophobic pocket near the amino group. These enzyme-targeted inhibitors arrest the methanogenesis and growth of pure cultures of methanogens. Supplying pABA to the culture relieves the inhibition, indicating a competitive interaction between pABA and the inhibitor at the cellular target, which is most likely RFAP synthase. The inhibitors do not adversely affect the growth of pure cultures of the bacteria (acetogens) that play a beneficial role in the rumen. Inhibitors added to dense ruminal fluid cultures (artificial rumena) halt methanogenesis; however, they do not inhibit volatile fatty acid (VFA) production and, in some cases, VFA levels are slightly elevated in the methanogenesisinhibited cultures. We suggest that inhibiting methanopterin biosynthesis could be considered in strategies to decrease anthropogenic methane emissions, which could have an environmental benefit since methane is a potent greenhouse gas.Biological methane formation is a microbial process catalyzed by methanogens, which are members of the Archaea domain, the third kingdom of life (23). Methanogens are found in most anaerobic environments, including the rumen of domesticated livestock (24). The beneficial effects of methanogenesis include the removal of H 2 formed during the oxidative metabolism of biomass, thus enhancing the biodegradation process (equations 1 and 2). However, there are several negative aspects of ruminant methanogenesis. Since methane production in the rumen results in a loss of between 3 and 12% of feed gross energy, inhibition of methanogenesis has long been considered as a strategy to improve agricultural productivity (25). Inhibition of ruminal methanogenesis can enhance production of the volatile fatty acids (VFAs) that are useful to the host (10). Furthermore, methane is a potent greenhouse gas and thus contributes to the problem of global warming (4). Ruminal methanogenesis produces about 80 million tons of methane per year (11), second only to the mining, processing, and use of coal, oil, and natural gas (100 million tons).Biomass 3 CH 3 COOH ϩ H 2 ϩ CO 2 ϩ NH 3(1)The objective of the research described here is to specifically inhibit a key methanogenic enzyme that is not present in the animal or in ruminal b...