Kirk Matthew Schnorr

Plectasin is a peptide antibiotic with therapeutic potential from a saprophytic fungus

Animals and higher plants express endogenous peptide antibiotics called defensins. These small cysteine-rich peptides are active against bacteria, fungi and viruses. Here we describe plectasin—the first defensin to be isolated from a fungus, the saprophytic ascomycete Pseudoplectania nigrella. Plectasin has primary, secondary and tertiary structures that closely resemble those of defensins found in spiders, scorpions, dragonflies and mussels. Recombinant plectasin was produced at a very high, and commercially viable, yield and purity. In vitro, the recombinant peptide was especially active against Streptococcus pneumoniae, including strains resistant to conventional antibiotics. Plectasin showed extremely low toxicity in mice, and cured them of experimental peritonitis and pneumonia caused by S. pneumoniae as efficaciously as vancomycin and penicillin. These findings identify fungi as a novel source of antimicrobial defensins, and show the therapeutic potential of plectasin. They also suggest that the defensins of insects, molluscs and fungi arose from a common ancestral gene.

Lignocellulose degradation mechanisms across the Tree of Life

Organisms use diverse mechanisms involving multiple complementary enzymes, particularly glycoside hydrolases (GHs), to deconstruct lignocellulose. Lytic polysaccharide monooxygenases (LPMOs) produced by bacteria and fungi facilitate deconstruction as does the Fenton chemistry of brown-rot fungi. Lignin depolymerisation is achieved by white-rot fungi and certain bacteria, using peroxidases and laccases. Meta-omics is now revealing the complexity of prokaryotic degradative activity in lignocellulose-rich environments. Protists from termite guts and some oomycetes produce multiple lignocellulolytic enzymes. Lignocellulose-consuming animals secrete some GHs, but most harbour a diverse enzyme-secreting gut microflora in a mutualism that is particularly complex in termites. Shipworms however, house GH-secreting and LPMO-secreting bacteria separate from the site of digestion and the isopod Limnoria relies on endogenous enzymes alone. The omics revolution is identifying many novel enzymes and paradigms for biomass deconstruction, but more emphasis on function is required, particularly for enzyme cocktails, in which LPMOs may play an important role.

A novel approach to the antimicrobial activity of maggot debridement therapy

Objectives Commercially produced sterile green bottle fly Lucilia sericata maggots are successfully employed by practitioners worldwide to clean a multitude of chronic necrotic wounds and reduce wound bacterial burdens during maggot debridement therapy (MDT). Secretions from the maggots exhibit antimicrobial activity along with other activities beneficial for wound healing. With the rise of multidrug-resistant bacteria, new approaches to identifying the active compounds responsible for the antimicrobial activity within this treatment are imperative. Therefore, the aim of this study was to use a novel approach to investigate the output of secreted proteins from the maggots under conditions mimicking clinical treatments. Methods cDNA libraries constructed from microdissected salivary glands and whole maggots, respectively, were treated with transposon-assisted signal trapping (TAST), a technique selecting for the identification of secreted proteins. Several putative secreted components of insect immunity were identified, including a defensin named lucifensin, which was produced recombinantly as a Trx-fusion protein in Escherichia coli, purified using immobilized metal affinity chromatography and reverse-phase HPLC, and tested in vitro against Gram-positive and Gram-negative bacterial strains. Results Lucifensin was active against Staphylococcus carnosus, Streptococcus pyogenes and Streptococcus pneumoniae (MIC 2 mg/L), as well as Staphylococcus aureus (MIC 16 mg/L). The peptide did not show antimicrobial activity towards Gram-negative bacteria. The MIC of lucifensin for the methicillin-resistant S. aureus and glycopeptide-intermediate S. aureus isolates tested ranged from 8 to >128 mg/L. Conclusions The TAST results did not reveal any highly secreted compounds with putative antimicrobial activity, implying an alternative antimicrobial activity of MDT. Lucifensin showed antimicrobial activities comparable to other defensins and could have potential as a future drug candidate scaffold, for redesign for other applications besides the topical treatment of infected wounds.

Structural characterization of a unique marine animal family 7 cellobiohydrolase suggests a mechanism of cellulase salt tolerance

Nature uses a diversity of glycoside hydrolase (GH) enzymes to convert polysaccharides to sugars. As lignocellulosic biomass deconstruction for biofuel production remains costly, natural GH diversity offers a starting point for developing industrial enzymes, and fungal GH family 7 (GH7) cellobiohydrolases, in particular, provide significant hydrolytic potential in industrial mixtures. Recently, GH7 enzymes have been found in other kingdoms of life besides fungi, including in animals and protists. Here, we describe the in vivo spatial expression distribution, properties, and structure of a unique endogenous GH7 cellulase from an animal, the marine wood borer Limnoria quadripunctata (LqCel7B). RT-quantitative PCR and Western blot studies show that LqCel7B is expressed in the hepatopancreas and secreted into the gut for wood degradation. We produced recombinant LqCel7B, with which we demonstrate that LqCel7B is a cellobiohydrolase and obtained four high-resolution crystal structures. Based on a crystallographic and computational comparison of LqCel7B to the well-characterized Hypocrea jecorina GH7 cellobiohydrolase, LqCel7B exhibits an extended substrate-binding motif at the tunnel entrance, which may aid in substrate acquisition and processivity. Interestingly, LqCel7B exhibits striking surface charges relative to fungal GH7 enzymes, which likely results from evolution in marine environments. We demonstrate that LqCel7B stability and activity remain unchanged, or increase at high salt concentration, and that the L. quadripunctata GH mixture generally contains cellulolytic enzymes with highly acidic surface charge compared with enzymes derived from terrestrial microbes. Overall, this study suggests that marine cellulases offer significant potential for utilization in high-solids industrial biomass conversion processes.

Structure of a dimeric fungal α-type carbonic anhydrase

AoCA binds to AoCA X‐ray crystallography (View interaction)

Structural and electronic determinants of lytic polysaccharide monooxygenase reactivity on polysaccharide substrates.

Lytic polysaccharide monooxygenases (LPMOs) are industrially important copper-dependent enzymes that oxidatively cleave polysaccharides. Here we present a functional and structural characterization of two closely related AA9-family LPMOs from Lentinus similis (LsAA9A) and Collariella virescens (CvAA9A). LsAA9A and CvAA9A cleave a range of polysaccharides, including cellulose, xyloglucan, mixed-linkage glucan and glucomannan. LsAA9A additionally cleaves isolated xylan substrates. The structures of CvAA9A and of LsAA9A bound to cellulosic and non-cellulosic oligosaccharides provide insight into the molecular determinants of their specificity. Spectroscopic measurements reveal differences in copper co-ordination upon the binding of xylan and glucans. LsAA9A activity is less sensitive to the reducing agent potential when cleaving xylan, suggesting that distinct catalytic mechanisms exist for xylan and glucan cleavage. Overall, these data show that AA9 LPMOs can display different apparent substrate specificities dependent upon both productive protein–carbohydrate interactions across a binding surface and also electronic considerations at the copper active site.Copper-dependent lytic polysaccharide monooxygenases (LPMOs) oxidatively cleave polysaccharides. Here the authors present a structure-function characterization of fungal LPMOs, showing that a particular LPMO cleaves xylan using a mechanism that involves an alternative copper coordination geometry.

Sequence analysis and characterization of a novel pectin acetyl esterase from Bacillus subtilis

A recombinant pectin acetyl esterase (YxiM) from Bacillus subtilis was characterised and the enzymatic properties compared to previously characterised pectin acetyl esterases (PAE’s). The YxiM protein had a pH optimum of 8.0 and was stable above pH 4.O. The enzyme released acetate from pectin isolated from different sources and from various synthetic compounds. In addition, YxiM showed a preference for acetylated tobacco homogalacturonan oligomers. Determination of the kinetic constants of YxiM indicated that the enzyme had a higher affinity to acetylated substrates, but a lower specific activity towards these substrates than PaeY from Erwinia chrysanthemi. Pre-treatment of sugar beet pectin with pectin methyl esterase (PME) from Aspergillus aculeatus increased the ability of YxiM to release acetate and similarly, pre-treatment with YxiM increased the ability of PME to release methanol. This demonstrates that YxiM acts in synergy with PME on the modification of plant cell wall pectin.

A novel cellulase for biofuels production: structure of a marine GH7 cellobiohydrolase

There is strong pressure to diversify feedstocks used for biofuel generation, to avoid competition with food crops. In particular, there is increasing emphasis on the utilisation of woody (lignocellulosic) materials that are recalcitrant to degradation. Prospecting for enzymes capable of overcoming this recalcitrance has focused on the relatively few types of microorganisms and animals that have wood-degrading capability. Of particular interest are the GH7-family enzymes that convert cellulose polymers to cellobiose units, the central step in the production of glucose for downstream fermentation to bioethanol. Currently, fungal GH7 enzymes represent the main hydrolytic component in the majority of industrial cocktails, however, their high cost represents a significant barrier to the commercial viability of large-scale biofuel production. We are therefore exploring the rich resource of endogenous lignocellulose-degrading enzymes in wood boring crustaceans that achieve breakdown without the help of microbial mutualists. Unlike animals such as termites that employ a complex community of microbial flora to produce digestive enzymes, the marine crustacean Limnoria quadripunctata has a sterile gut and produces all the necessary enzymatic machinery to efficiently digest these difficult substrates. A successful collaboration between Portsmouth and York Universities, NREL in Golden, USA and Novozymes in Denmark has resulted in the detailed characterisation of the first animal cellobiohydrolase, LqCel7B. Following a transcriptomic analysis [1], we selected a GH7 family enzyme that was highly expressed in the hepatopancreas digestive gland. Biophysical analysis revealed a stable monomeric protein and extensive crystallisation trials produced well diffracting crystals. Four structures have been solved to date, one apo form and three with various bound ligands occupying the active site tunnel. A complex with the inhibitor thiocellobiose diffracted to 1.1 Å resolution. These structures provided the basis for structural comparisons and molecular dynamic simulations and have revealed a host of novel features with industrial potential [2].

Crystal structure of a dimeric fungal α-type carbonic anhydrase

Crystal structure of a dimeric fungal α-type carbonic anhydrase Jose A. Cuesta-Seijo,a,c Martin Simon Borchert,b Jens-Christian Navarro-Poulsen,a Kirk Matthew Schnorr,b Steen Bennike Mortensen,b Leila Lo Leggio,a aBiophysical Chemistry Group, Department of Chemistry, University of Copenhagen, Denmark. bNovozymes A/S, Bagsværd, Denmark. cCarlsberg Laboratory, Copenhagen, Denmark. E-mail: josea.cuesta.seijo@carlsberglab.dk