Constraints on the evolution of azole resistance in plant pathogenic fungi

The durability of azole fungicides in controlling agriculturally important pathogenic fungi is unique amongst modern single site fungicides. Today, azoles are still relied on to control pathogens of many crops including cereals, fruits and vegetables, canola and soybeans. Significantly, this widespread use continues despite many reports of azole-resistant fungal strains. In this review, recent reports of azole resistance and the mechanisms associated with resistant phenotypes are discussed. The example of the complex evolution of the azole target sterol 14-demethylase (CYP51) enzyme in modern European populations of the wheat pathogen Mycosphaerella graminicola is used to describe the quantitative and epistatic effects on fungicide sensitivity and enzyme function of target site mutations, and to explore the hypothesis that constraints on CYP51 evolution have ensured the longevity of azoles. In addition, the threats posed by alternative resistance mechanisms causing cross-resistance to all azoles or even unrelated fungicides are discussed, and postulations are made on how using new genomic technologies to gain a greater understanding of azole resistance evolution should enhance the ability to control azole-resistant strains of plant pathogenic fungi in the future

Expanding the set of rhodococcal Baeyer–Villiger monooxygenases by high-throughput cloning, expression and substrate screening

To expand the available set of Baeyer–Villiger monooxygenases (BVMOs), we have created expression constructs for producing 22 Type I BVMOs that are present in the genome of Rhodococcus jostii RHA1. Each BVMO has been probed with a large panel of potential substrates. Except for testing their substrate acceptance, also the enantioselectivity of some selected BVMOs was studied. The results provide insight into the biocatalytic potential of this collection of BVMOs and expand the biocatalytic repertoire known for BVMOs. This study also sheds light on the catalytic capacity of this large set of BVMOs that is present in this specific actinomycete. Furthermore, a comparative sequence analysis revealed a new BVMO-typifying sequence motif. This motif represents a useful tool for effective future genome mining efforts.

Substrate binding tunes the reactivity of hispidin 3-hydroxylase, a flavoprotein monooxygenase involved in fungal bioluminescence

Fungal bioluminescence was recently shown to depend on a unique oxygen-dependent system of several enzymes. However, the identities of the enzymes did not reveal the full biochemical details of this process, as the enzymes do not bear resemblance to those of other luminescence systems, and thus the properties of the enzymes involved in this fascinating process are still unknown. Here, we describe the characterization of the penultimate enzyme in the pathway, hispidin 3-hydroxylase, from the luminescent fungus Mycena chlorophos (McH3H), which catalyzes the conversion of hispidin to 3-hydroxyhispidin. 3-Hydroxyhispidin acts as a luciferin substrate in luminescent fungi. McH3H was heterologously expressed in Escherichia coli and purified by affinity chromatography with a yield of 100 mg/liter. McH3H was found to be a single component monomeric NAD(P)H-dependent FAD-containing monooxygenase having a preference for NADPH. Through site-directed mutagenesis, based on a modeled structure, mutant enzymes were created that are more efficient with NADH. Except for identifying the residues that tune cofactor specificity, these engineered variants may also help in developing new hispidin-based bioluminescence applications. We confirmed that addition of hispidin to McH3H led to the formation of 3-hydroxyhispidin as sole aromatic product. Rapid kinetic analysis revealed that reduction of the flavin cofactor by NADPH is boosted by hispidin binding by nearly 100-fold. Similar to other class A flavoprotein hydroxylases, McH3H did not form a stable hydroperoxyflavin intermediate. These data suggest a mechanism by which the hydroxylase is tuned for converting hispidin into the fungal luciferin.</p

Mesozoic-Cenozoic crustaceans preserved within echinoid tests and bivalve shells

Associations of crustaceans with echinoids (Echinodermata) and bivalves (Mollusca) are not uncommon in modern oceans. Here we record the occurrence of anomurans, brachyurans and isopods within echinoid tests and bivalve shells from the Middle Jurassic of France, the Upper Jurassic of the Czech Republic, the Eocene of Croatia and the Miocene of Austria. Additionally a new genus and species of fossil cirolanid isopod from the Middle Jurassic of France is described. The present examples are interpreted as crustacean sheltering, probably for safe and undisturbed moulting (ecdysis), within a vacant host test or shell. However, accidental association (washed in) or even food remains cannot be ruled out entirelyWeb of Science90361160

Mutational and structural analysis of an ancestral fungal dye decolorizing peroxidase

Dye-decolorizing peroxidases (DyPs) constitute a superfamily of heme-containing peroxidases that are related neither to animal nor to plant peroxidase families. These are divided into four classes (types A, B, C, and D) based on sequence features. The active site of DyPs contains two highly conserved distal ligands, an aspartate and an arginine, the roles of which are still controversial. These ligands have mainly been studied in class A-C bacterial DyPs, largely because no effective recombinant expression systems have been developed for the fungal (D-type) DyPs. In this work, we employ ancestral sequence reconstruction (ASR) to resurrect a D-type DyP ancestor, AncDyPD-b1. Expression of AncDyPD-b1 in Escherichia coli results in large amounts of a heme-containing soluble protein and allows for the first mutagenesis study on the two distal ligands of a fungal DyP. UV-Vis and resonance Raman (RR) spectroscopic analyses, in combination with steady-state kinetics and the crystal structure, reveal fine pH-dependent details about the heme active site structure and show that both the aspartate (D222) and the arginine (R390) are crucial for hydrogen peroxide reduction. Moreover, the data indicate that these two residues play important but mechanistically different roles on the intraprotein long-range electron transfer process. Database: Structural data are available in the PDB database under the accession number 7ANV.</p

Discovery and characterization of a putrescine oxidase from Rhodococcus erythropolis NCIMB 11540

A gene encoding a putrescine oxidase (PuO(Rh), EC 1.4.3.10) was identified from the genome of Rhodococcus erythropolis NCIMB 11540. The gene was cloned in the pBAD vector and overexpressed at high levels in Escherichia coli. The purified enzyme was shown to be a soluble dimeric flavoprotein consisting of subunits of 50 kDa and contains non-covalently bound flavin adenine dinucleotide as a cofactor. From all substrates, the highest catalytic efficiency was found with putrescine (K(M) = 8.2 μM, k(cat) = 26 s(−1)). PuO(Rh) accepts longer polyamines, while short diamines and monoamines strongly inhibit activity. PuO(Rh) is a reasonably thermostable enzyme with t(1/2) at 50°C of 2 h. Based on the crystal structure of human monoamine oxidase B, we constructed a model structure of PuO(Rh), which hinted to a crucial role of Glu324 for substrate binding. Mutation of this residue resulted in a drastic drop (five orders of magnitude) in catalytic efficiency. Interestingly, the mutant enzyme showed activity with monoamines, which are not accepted by wt-PuO(Rh). ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00253-007-1310-4) contains supplementary material, which is available to authorized users

Orientational phase transitions in the hexagonal phase of a diblock copolymer melt under shear flow

We generalize the earlier theory by Fredrickson [J. Rheol. v.38, 1045 (1994)] to study the orientational behaviour of the hexagonal phase of diblock copolymer melt subjected to steady shear flow. We use symmetry arguments to show that the orientational ordering in the hexagonal phase is a much weaker effect than in the lamellae. We predict the parallel orientation to be stable at low and the perpendicular orientation at high shear rates. Our analysis reproduces the experimental results by Tepe et al. [Macromolecules v.28, 3008 (1995)] and explains the difficulties in experimental observation of the different orientations in the hexagonal phase.Comment: 21 pages, 6 eps figures, submitted to Physical Review

Editorial: Actinobacteria, a Source of Biocatalytic Tools

Actinobacteria (Actinomycetes) represent one of the largest and most diverse phyla among the Bacteria. The characteristics and phylogeny of actinobacteria have been well-described throughout the years (Anteneh and Franco; Embley et al., 1994; Stackebrandt et al., 1997a,b; Stach and Bull, 2005; Stackebrandt and Schumann, 2006; Ventura et al., 2007; Gao and Gupta, 2012; Goodfellow, 2012a,b; Schrempf, 2013; Lawson, 2018; Lewin et al., 2016). Still actinobacteria are hotspots for discovery of new biomolecules and enzyme activities, fueling an active field of research. The remarkable diversity is displayed by various lifestyles, distinct morphologies, a wide spectrum of physiological and metabolic activities, as well as genetics