Most plant-microbe interactions do not result in disease; natural products restrict non-host pathogens. We found that sulforaphane (4-methylsulfinylbutyl isothiocyanate), a natural product derived from aliphatic glucosinolates, inhibits growth in Arabidopsis of non-host Pseudomonas bacteria in planta. Multiple sax genes (saxCAB/F/D/G) were identified in Pseudomonas species virulent on Arabidopsis. These sax genes are required to overwhelm isothiocyanate-based defenses and facilitate a disease outcome, especially in the young leaves critical for plant survival. Introduction of saxCAB genes into non-host strains enabled them to overcome these Arabidopsis defenses. Our study shows that aliphatic isothiocyanates, previously shown to limit damage by herbivores, are also crucial, robust, and developmentally regulated defenses that underpin non-host resistance in the Arabidopsis-Pseudomonas pathosystem.

SummaryAn immunological analysis of an Escherichia coli strain unable to synthesize the main pyruvate formate-lyase enzyme Pfl revealed the existence of a weak, crossreacting 85 kDa polypeptide that exhibited the characteristic oxygen-dependent fragmentation typical of a glycyl radical enzyme. Polypeptide fragmentation of this cross-reacting species was shown to be dependent on Pfl activase. Cloning and sequence analysis of the gene encoding this protein revealed that it coded for a new enzyme, termed TdcE, which has 82% identity with Pfl. On the basis of RNA analyses, the tdcE gene was shown to be part of a large operon that included the tdcABC genes, encoding an anaerobic threonine dehydratase, tdcD, coding for a propionate kinase, tdcF, the function of which is unknown, and the tdcG gene, which encodes a L-serine dehydratase. Expression of the tdcABCDEFG operon was strongly catabolite repressed. Enzyme studies showed that TdcE has both pyruvate formate-lyase and 2-ketobutyrate formate-lyase activity, whereas the TdcD protein is a new propionate/acetate kinase. By monitoring culture supernatants from various mutants using 1 H nuclear magnetic resonance (NMR), we followed the anaerobic conversion of L-threonine to propionate. These studies confirmed that 2-ketobutyrate, the product of threonine deamination, is converted in vivo by TdcE to propionyl-CoA. These studies also revealed that Pfl and an as yet unidentified thiamine pyrophosphate-dependent enzyme(s) can perform this reaction. Double null mutants deficient in phosphotransacetylase (Pta) and acetate kinase (AckA) or AckA and TdcD were unable to metabolize threonine to propionate, indicating that propionyl-CoA and propionyl-phosphate are intermediates in the pathway and that ATP is generated during the conversion of propionyl-P to propionate by AckA or TdcD.

Background: GlgE is a maltosyltransferase involved in bacterial ␣-glucan biosynthesis and is a genetically validated antituberculosis target. Results: We have determined the catalytic properties of Streptomyces coelicolor GlgE and solved its structure. Conclusion:The enzyme has the same catalytic properties as Mycobacterium tuberculosis GlgE and the structure reveals how GlgE functions. Significance: The structure will help guide the development of inhibitors with therapeutic potential.

Hydroxycinnamic acid amides are a class of secondary metabolites distributed widely in plants. We have identified two sinapoyl spermidine derivatives, N-((4′-O-glycosyl)-sinapoyl),N′-sinapoylspermidine and N,N′-disinapoylspermidine, which comprise the two major polyamine conjugates that accumulate in Arabidopsis thaliana seed. Using metabolic profiling of knockout mutants to elucidate the functions of members of the BAHD acyltransferase family in Arabidopsis, we have also identified two genes encoding spermidine disinapoyl transferase (SDT) and spermidine dicoumaroyl transferase (SCT) activities. At2g23510, which is expressed mainly in seeds, encodes a spermidine sinapoyl CoA acyltransferase (SDT) that is required for the production of disinapoyl spermidine and its glucoside in Arabidopsis seed. The structurally related BAHD enzyme encoded by At2g25150 is expressed specifically in roots and has spermidine coumaroyl CoA acyltransferase (SCT) activity both in vitro and in vivo.

Members of the cytochromes P450 superfamily (P450s) catalyze a huge variety of oxidation reactions in microbes and higher organisms. Most P450 families are highly divergent, but in contrast the cytochrome P450 14α-sterol demethylase (CYP51) family is one of the most ancient and conserved, catalyzing sterol 14α-demethylase reactions required for essential sterol synthesis across the fungal, animal, and plant kingdoms. Oats (Avena spp.) produce antimicrobial compounds, avenacins, that provide protection against disease. Avenacins are synthesized from the simple triterpene, β-amyrin. Previously we identified a gene encoding a member of the CYP51 family of cytochromes P450, AsCyp51H10 (also known as Saponin-deficient 2, Sad2), that is required for avenacin synthesis in a forward screen for avenacin-deficient oat mutants. sad2 mutants accumulate β-amyrin, suggesting that they are blocked early in the pathway. Here, using a transient plant expression system, we show that AsCYP51H10 is a multifunctional P450 capable of modifying both the C and D rings of the pentacyclic triterpene scaffold to give 12,13β-epoxy-3β,16β-dihydroxy-oleanane (12,13β-epoxy-16β-hydroxy-β-amyrin). Molecular modeling and docking experiments indicate that C16 hydroxylation is likely to precede C12,13 epoxidation. Our computational modeling, in combination with analysis of a suite of sad2 mutants, provides insights into the unusual catalytic behavior of AsCYP51H10 and its active site mutants. Fungal bioassays show that the C12,13 epoxy group is an important determinant of antifungal activity. Accordingly, the oat AsCYP51H10 enzyme has been recruited from primary metabolism and has acquired a different function compared to other characterized members of the plant CYP51 family-as a multifunctional stereo-and regio-specific hydroxylase in plant specialized metabolism.

YvrK was found to contain between 0.86 and 1.14 atoms of manganese/subunit. EPR spectroscopy showed that the metal ion was predominantly but not exclusively in the Mn(II) oxidation state. The hyperfine coupling constant (A ‫؍‬ 9.5 millitesla) of the main g ‫؍‬ 2 signal was consistent with oxygen and nitrogen ligands with hexacoordinate geometry. The structure of YvrK was modeled on the basis of homology with oxalate oxidase, canavalin, and phaseolin, and its hexameric oligomerization was predicted by analogy with proglycinin and homogentisate 1,2-dioxygenase. Although YvrK possesses two potential active sites, only one could be fully occupied by manganese. The possibility that the C-terminal domain active site has no manganese bound and is buried in an intersubunit interface within the hexameric enzyme is discussed. A mechanism for oxalate decarboxylation is proposed, in which both Mn(II) and O 2 are cofactors that act together as a two-electron sink during catalysis.

SummaryThe Elp3 subunit of the Elongator complex is highly conserved from archaea to humans and contains a well-characterized C-terminal histone acetyltransferase (HAT) domain. The central region of Elp3 shares significant sequence homology to the Radical SAM superfamily. Members of this large family of bacterial proteins contain a FeS cluster and use S-adenosylmethionine (SAM) to catalyse a variety of radical reactions. To biochemically characterize this domain we have expressed and purified the corresponding fragment of the Methanocaldococcus jannaschii Elp3 protein. The presence of a Fe 4 S 4 cluster has been confirmed by UV-visible spectroscopy and electron paramagnetic resonance (EPR) spectroscopy and the Fe content determined by both a colorimetric assay and atomic absorption spectroscopy. The cysteine residues involved in cluster formation have been identified by site-directed mutagenesis. The protein binds SAM and the binding alters the EPR spectrum of the FeS cluster. Our results provide biochemical support to the hypothesis that Elp3 does indeed contain the Fe 4 S 4 cluster which characterizes the Radical SAM superfamily and binds SAM, suggesting that Elp3, in addition to its HAT activity, has a second as yet uncharacterized catalytic function. We also present preliminary data to show that the protein cleaves SAM.

Oxalate decarboxylases and oxalate oxidases are members of the cupin superfamily of proteins that have many common features: a manganese ion with a common ligand set, the substrate oxalate, and dioxygen (as either a unique cofactor or a substrate). We have hypothesized that these enzymes share common catalytic steps that diverge when a carboxylate radical intermediate becomes protonated. The Bacillus subtilis decarboxylase has two manganese binding sites, and we proposed that Glu162 on a flexible lid is the site 1 general acid. We now demonstrate that a decarboxylase can be converted into an oxidase by mutating amino acids of the lid that include Glu162 with specificity switches of 282,000 (SEN161-3DAS), 275,000 (SENS161-4DSSN), and 225,000 (SENS161-4DASN). The structure of the SENS161-4DSSN mutant showed that site 2 was not affected. The requirement for substitutions other than of Glu162 was, at least in part, due to the need to decrease the Km for dioxygen for the oxidase reaction. Reversion of decarboxylase activity could be achieved by reintroducing Glu162 to the SENS161-4DASN mutant to give a relative specificity switch of 25,600. This provides compelling evidence for the crucial role of Glu162 in the decarboxylase reaction consistent with it being the general acid, for the role of the lid in controlling the Km for dioxygen, and for site 1 being the sole catalytically active site. We also report the trapping of carboxylate radicals produced during turnover of the mutant with the highest oxidase activity. Such radicals were also observed with the wild-type decarboxylase.