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.

Thermobia domestica belongs to an ancient group of insects and has a remarkable ability to digest crystalline cellulose without microbial assistance. By investigating the digestive proteome of Thermobia, we have identified over 20 members of an uncharacterized family of lytic polysaccharide monooxygenases (LPMOs). We show that this LPMO family spans across several clades of the Tree of Life, is of ancient origin, and was recruited by early arthropods with possible roles in remodeling endogenous chitin scaffolds during development and metamorphosis. Based on our in-depth characterization of Thermobia’s LPMOs, we propose that diversification of these enzymes toward cellulose digestion might have endowed ancestral insects with an effective biochemical apparatus for biomass degradation, allowing the early colonization of land during the Paleozoic Era. The vital role of LPMOs in modern agricultural pests and disease vectors offers new opportunities to help tackle global challenges in food security and the control of infectious diseases.

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Several independent studies of bacterial degradation of nitrate ester explosives have demonstrated the involvement of flavin-dependent oxidoreductases related to the old yellow enzyme (OYE) of yeast. Some of these enzymes also transform the nitroaromatic explosive 2,4,6-trinitrotoluene (TNT). In this work, catalytic capabilities of five members of the OYE family were compared, with a view to correlating structure and function. The activity profiles of the five enzymes differed substantially; no one compound proved to be a good substrate for all five enzymes. TNT is reduced, albeit slowly, by all five enzymes. The nature of the transformation products differed, with three of the five enzymes yielding products indicative of reduction of the aromatic ring. Our findings suggest two distinct pathways of TNT transformation, with the initial reduction of TNT being the key point of difference between the enzymes. Characterization of an active site mutant of one of the enzymes suggests a structural basis for this difference.Large expanses of land and groundwater have been polluted through the manufacture, deployment, and disposal of explosives. Energetic compounds such as glycerol trinitrate (GTN), pentaerythritol tetranitrate (PETN), and 2,4,6-trinitrotoluene (TNT) are unique to nature, and their recalcitrance to biodegradation means they tend to persist in the environment. It is of significant interest to understand how certain microorganisms have evolved and adapted to utilize these toxic compounds as nitrogen sources for growth. Not surprisingly the enzymes mediating the microbial degradation of explosives have been the focus of much research for the remediation of contaminated land and groundwater.A number of bacterial strains able to degrade the nitrate ester explosives GTN and PETN have been isolated from contaminated soil (5,7,34,36). In each of these strains, a nicotinamide cofactor-dependent oxidoreductase catalyzes the reductive cleavage of the nitrate ester group to yield an alcohol and nitrite (6,15,29). The enzymes have similar amino acid sequences, and all contain flavin mononucleotide as a noncovalently bound cofactor. It has become apparent that some of these enzymes are also able to reduce TNT. Many flavoenzymes catalyze the reduction of nitro groups (8), and the products of TNT reduction by nitrate ester reductases include hydroxylamino-dinitrotoluenes (HADNTs) and amino-dinitrotoluenes (ADNTs). However, in addition, both PETN reductase from Enterobacter cloacae PB2 (14) and xenobiotic reductase B from Pseudomonas fluorescens I-C (25) catalyze reduction of the TNT aromatic ring by hydride addition, yielding hydride-and dihydride-Meisenheimer complexes (compounds 2 to 6, Fig. 1) and nitrite. Most current attempts at TNT bioremediation focus on nitro group reduction, where some of the resulting products bind tightly to soil (1, 2); unfortunately, it is difficult to demonstrate the long-term fate of such immobilized TNT derivatives. Nonaromatic transformation products, such as hydride-and dihydride-Meisen...

There is major international concern over the wide-scale contamination of soil and associated ground water by persistent explosives residues. 2,4,6-Trinitrotoluene (TNT) is one of the most recalcitrant and toxic of all the military explosives. The lack of affordable and effective cleanup technologies for explosives contamination requires the development of better processes. Significant effort has recently been directed toward the use of plants to extract and detoxify TNT. To explore the possibility of overcoming the high phytotoxic effects of TNT, we expressed bacterial nitroreductase in tobacco plants. Nitroreductase catalyzes the reduction of TNT to hydroxyaminodinitrotoluene (HADNT), which is subsequently reduced to aminodinitrotoluene derivatives (ADNTs). Transgenic plants expressing nitroreductase show a striking increase in ability to tolerate, take up, and detoxify TNT. Our work suggests that expression of nitroreductase (NR) in plants suitable for phytoremediation could facilitate the effective cleanup of sites contaminated with high levels of explosives.

Here we report the first structure of a cocaine-degrading enzyme. The bacterial esterase, cocE, hydrolyzes pharmacologically active (-)-cocaine to a non-psychoactive metabolite with a rate faster than any other reported cocaine esterase (kcat = 7.8 s-1 and KM = 640 nM). Because of the high catalytic proficiency of cocE, it is an attractive candidate for novel protein-based therapies for cocaine overdose. The crystal structure of cocE, solved by multiple anomalous dispersion (MAD) methods, reveals that cocE is a serine esterase composed of three domains: (i) a canonical alpha/beta hydrolase fold (ii) an alpha-helical domain that caps the active site and (iii) a jelly-roll-like beta-domain that interacts extensively with the other two domains. The active site was identified within the interface of all three domains by analysis of the crystal structures of transition state analog adduct and product complexes, which were refined at 1.58 A and 1.63 A resolution, respectively. These structural studies suggest that substrate recognition arises partly from interactions between the benzoyl moiety of cocaine and a highly evolved specificity pocket.

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