Fusarium head blight (FHB) is a plant disease with serious economic and health impacts. It is caused by fungal species belonging to the genus Fusarium and the mycotoxins they produce. Although it has proved difficult to combat this disease, one strategy that has been examined is the introduction of an indigenous fungal protective gene into cereals such as wheat barley and rice. Thus far the gene of choice has been tri101 whose gene product catalyzes the transfer of an acetyl group from acetyl coenzyme A to the C3 hydroxyl moiety of several trichothecene mycotoxins. In vitro this has been shown to reduce the toxicity of the toxins by ϳ100-fold but has demonstrated limited resistance to FHB in transgenic cereal. To understand the molecular basis for the differences between in vitro and in vivo resistance the three-dimensional structures and kinetic properties of two TRI101 orthologs isolated from Fusarium sporotrichioides and Fusarium graminearum have been determined. The kinetic results reveal important differences in activity of these enzymes toward B-type trichothecenes such as deoxynivalenol. These differences in activity can be explained in part by the three-dimensional structures for the ternary complexes for both of these enzymes with coenzyme A and trichothecene mycotoxins. The structural and kinetic results together emphasize that the choice of an enzymatic resistance gene in transgenic crop protection strategies must take into account the kinetic profile of the selected protein.

Background: LaeA, a putative methyltransferase in Aspergillus nidulans, is a master regulator of secondary metabolism. Results: LaeA automethylates at a methionine residue near the AdoMet-binding site. This modification is not required for in vivo function. Conclusion: Automethylation of LaeA reveals a novel protein methionine methyltransferase activity. Significance: Elucidating the substrate(s) of LaeA will provide insights into the physiological function of LaeA in modulating gene expression.

In bacteria, the dehydration of 2-methylcitrate to yield 2-methylaconitate in the 2-methylcitric acid cycle is catalyzed by a cofactor-less (PrpD) enzyme or by an aconitase-like (AcnD) enzyme. Bacteria that use AcnD also require the function of the PrpF protein, whose function was previously unknown. To gain insights into the function of PrpF, the three-dimensional crystal structure of the PrpF protein from the bacterium Shewanella oneidensis was solved at 2.0 Å resolution. The protein fold of PrpF is strikingly similar to those of the non-PLP-dependent diaminopimelate epimerase from Haemophilus influenzae, a putative proline racemase from Brucella melitensis, and to a recently deposited structure of a hypothetical protein from Pseudomonas aeruginosa. Results from in vitro studies show that PrpF isomerizes trans-aconitate to cis-aconitate. It is proposed that PrpF catalysis of the cis-trans isomerization proceeds through a base-catalyzed proton abstraction coupled with a rotation about C2-C3 bond of 2-methylaconitate, and that residue Lys73 is critical for PrpF function. The newly identified function of PrpF as a non-PLP-dependent isomerase, together with the fact that PrpD-containing bacteria do not require PrpF, suggest that the isomer of 2-methylaconitate that serves as a substrate of aconitase must have the same stereochemistry as that synthesized by PrpD. From this, it follows that the 2-methylaconitate isomer generated by AcnD is not a substrate of aconitase, and that PrpF is required to generate the correct isomer. As a consequence, the isomerase activity of PrpF may now be viewed as an integral part of the 2-methylcitric acid cycle.Keywords: cis-trans isomerases; non-PLP-dependent isomerases protein fold; propionate catabolism; 2-methylcitric acid cycle; 2-methylcitrate dehydratase; 2-methylaconitate isomerase; aconitase; Shewanella physiology In nature, short-chain fatty acids (e.g., acetate, propionate, butyrate) are very abundant (>0.1 M) in environments such as soil and the human intestine, and are of great relevance to agriculture and human health and nutrition (Cummings et al. 1987;Cummings 1995;Buckel 1999 Abbreviations: 2-MC, 2-methylcitrate; 2-MCC, 2-methylcitric acid cycle; AMP, adenosine monophosphate; DMSO, dimethylsulfoxide; dNTP, deoxynucleoside triphosphate; DTT, dithiothreitol; TCEP, tris(2-carboxyethyl)phosphine hydrochloride; HEPES, N-(2-hydroxyethyl)-piperazine-N9-2-ethanesulfonic acid; EDTA, ethylenediaminetetraacetic acid; RMS, root mean square; rTEV, recombinant tobacco etch virus; OD 600 , optical density measured at 600 nm; PEI, polyethyleneimine; MAD phasing, multiple wavelength anomalous dispersion phasing.Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi

Fusarium head blight is a devastating disease of cereal crops whose worldwide incidence is increasing and at present there is no satisfactory way of combating this pathogen or its associated toxins. There is a wide variety of trichothecene mycotoxins and they all contain a 12,13-epoxytrichothecene skeleton but differ in their substitutions. Indeed, there is considerable variation in the toxin profile across the numerous Fusarium species that has been ascribed to differences in the presence or absence of biosynthetic enzymes and their relative activity. This article addresses the source of differences in acetylation at the C15 position of the trichothecene molecule. Here, we present the in vitro structural and biochemical characterization of TRI3, a 15-O-trichothecene acetyltransferase isolated from F. sporotrichioides and the ''in vivo'' characterization of Dtri3 mutants of deoxynivalenol (DON) producing F. graminearum strains. A kinetic analysis shows that TRI3 is an efficient enzyme with the native substrate, 15-decalonectrin, but is inactive with either DON or nivalenol. The structure of TRI3 complexed with 15-decalonectrin provides an explanation for this specificity and shows that Tri3 and Tri101 (3-O-trichothecene acetyltransferase) are evolutionarily related. The active site residues are conserved across all sequences for TRI3 orthologs, suggesting that differences in acetylation at C15 are not due to differences in Tri3. The tri3 deletion mutant shows that acetylation at C15 is required for DON biosynthesis even though DON lacks a C15 acetyl group. The enzyme(s) responsible for deacetylation at the 15 position of the trichothecene mycotoxins have not been identified.

BackgroundChemical mutagenesis screens are useful to identify mutants involved in biological processes of interest. Identifying the mutation from such screens, however, often fails when using methodologies involving transformation of the mutant to wild type phenotype with DNA libraries.ResultsHere we analyzed Illumina sequence of a chemically derived mutant of Aspergillus nidulans and identified a gene encoding a C2H2 transcription factor termed RsrA for regulator of stress response. RsrA is conserved in filamentous fungal genomes, and upon deleting the gene in three Aspergillus species (A. nidulans, A. flavus and A. fumigatus), we found two conserved phenotypes: enhanced resistance to oxidative stress and reduction in sporulation processes. For all species, rsrA deletion mutants were more resistant to hydrogen peroxide treatment. In depth examination of this latter characteristic in A. nidulans showed that upon exposure to hydrogen peroxide, RsrA loss resulted in global up-regulation of several components of the oxidative stress metabolome including the expression of napA and atfA, the two bZIP transcription factors mediating resistance to reactive oxygen species (ROS) as well as NapA targets in thioredoxin and glutathione systems. Coupling transcriptional data with examination of ΔrsrAΔatfA and ΔrsrAΔnapA double mutants indicate that RsrA primarily operates through NapA-mediated stress response pathways. A model of RsrA regulation of ROS response in Aspergillus is presented.ConclusionRsrA, found in a highly syntenic region in Aspergillus genomes, coordinates a NapA mediated oxidative response in Aspergillus fungi.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1011) contains supplementary material, which is available to authorized users.