Kenneth E. Hammel

Preparation of human drug metabolites using fungal peroxygenases.

The synthesis of hydroxylated and O- or N-dealkylated human drug metabolites (HDMs) via selective monooxygenation remains a challenging task for synthetic organic chemists. Here we report that aromatic peroxygenases (APOs; EC 1.11.2.1) secreted by the agaric fungi Agrocybe aegerita and Coprinellus radians catalyzed the H₂O₂-dependent selective monooxygenation of diverse drugs, including acetanilide, dextrorphan, ibuprofen, naproxen, phenacetin, sildenafil and tolbutamide. Reactions included the hydroxylation of aromatic rings and aliphatic side chains, as well as O- and N-dealkylations and exhibited different regioselectivities depending on the particular APO used. At best, desired HDMs were obtained in yields greater than 80% and with isomeric purities up to 99%. Oxidations of tolbutamide, acetanilide and carbamazepine in the presence of H₂¹⁸O₂ resulted in almost complete incorporation of ¹⁸O into the corresponding products, thus establishing that these reactions are peroxygenations. The deethylation of phenacetin-d₁ showed an observed intramolecular deuterium isotope effect [(k(H)/k(D))(obs)] of 3.1±0.2, which is consistent with the existence of a cytochrome P450-like intermediate in the reaction cycle of APOs. Our results indicate that fungal peroxygenases may be useful biocatalytic tools to prepare pharmacologically relevant drug metabolites.

Proteomic and Functional Analysis of the Cellulase System Expressed by Postia placenta during Brown Rot of Solid Wood

ABSTRACT Brown rot basidiomycetes have an important ecological role in lignocellulose recycling and are notable for their rapid degradation of wood polymers via oxidative and hydrolytic mechanisms. However, most of these fungi apparently lack processive (exo-acting) cellulases, such as cellobiohydrolases, which are generally required for efficient cellulolysis. The recent sequencing of the Postia placenta genome now permits a proteomic approach to this longstanding conundrum. We grew P. placenta on solid aspen wood, extracted proteins from the biodegrading substrate, and analyzed tryptic digests by shotgun liquid chromatography-tandem mass spectrometry. Comparison of the data with the predicted P. placenta proteome revealed the presence of 34 likely glycoside hydrolases, but only four of these—two in glycoside hydrolase family 5, one in family 10, and one in family 12—have sequences that suggested possible activity on cellulose. We expressed these enzymes heterologously and determined that they all exhibited endoglucanase activity on phosphoric acid-swollen cellulose. They also slowly hydrolyzed filter paper, a more crystalline substrate, but the soluble/insoluble reducing sugar ratios they produced classify them as nonprocessive. Computer simulations indicated that these enzymes produced soluble/insoluble ratios on reduced phosphoric acid-swollen cellulose that were higher than expected for random hydrolysis, which suggests that they could possess limited exo activity, but they are at best 10-fold less processive than cellobiohydrolases. It appears likely that P. placenta employs a combination of oxidative mechanisms and endo-acting cellulases to degrade cellulose efficiently in the absence of a significant processive component.

Localizing gene regulation reveals a staggered wood decay mechanism for the brown rot fungus Postia placenta

Significance Wood-decomposing fungi are key players in the carbon cycle and are models for making energy from lignocellulose, sustainably. Our study focuses on brown rot fungi that selectively remove carbohydrates, leaving most lignin behind. These fungi often decompose wood faster than their lignin-degrading white rot ancestors, despite losses in genes involved in plant cell wall hydrolysis. To explain brown rot, many have implicated reactive oxygen species (ROS) in facilitating hydrolysis, with microenvironmental gradients partitioning ROS from enzymes. By spatially colocalizing gene expression and enzyme activities as Postia placenta colonizes wood, we provide evidence of an oxidative-hydrolytic two-step mechanism controlled by differential expression, not microenvironments, and we highlight 549 genes (∼4% of the genome) that are upregulated during this unique pretreatment. Wood-degrading brown rot fungi are essential recyclers of plant biomass in forest ecosystems. Their efficient cellulolytic systems, which have potential biotechnological applications, apparently depend on a combination of two mechanisms: lignocellulose oxidation (LOX) by reactive oxygen species (ROS) and polysaccharide hydrolysis by a limited set of glycoside hydrolases (GHs). Given that ROS are strongly oxidizing and nonselective, these two steps are likely segregated. A common hypothesis has been that brown rot fungi use a concentration gradient of chelated metal ions to confine ROS generation inside wood cell walls before enzymes can infiltrate. We examined an alternative: that LOX components involved in ROS production are differentially expressed by brown rot fungi ahead of GH components. We used spatial mapping to resolve a temporal sequence in Postia placenta, sectioning thin wood wafers colonized directionally. Among sections, we measured gene expression by whole-transcriptome shotgun sequencing (RNA-seq) and assayed relevant enzyme activities. We found a marked pattern of LOX up-regulation in a narrow (5-mm, 48-h) zone at the hyphal front, which included many genes likely involved in ROS generation. Up-regulation of GH5 endoglucanases and many other GHs clearly occurred later, behind the hyphal front, with the notable exceptions of two likely expansins and a GH28 pectinase. Our results support a staggered mechanism for brown rot that is controlled by differential expression rather than microenvironmental gradients. This mechanism likely results in an oxidative pretreatment of lignocellulose, possibly facilitated by expansin- and pectinase-assisted cell wall swelling, before cellulases and hemicellulases are deployed for polysaccharide depolymerization.

Evidence from Serpula lacrymans that 2,5-Dimethoxyhydroquinone Is a Lignocellulolytic Agent of Divergent Brown Rot Basidiomycetes

ABSTRACT Basidiomycetes that cause brown rot of wood are essential biomass recyclers in coniferous forest ecosystems and a major cause of failure in wooden structures. Recent work indicates that distinct lineages of brown rot fungi have arisen independently from ligninolytic white rot ancestors via loss of lignocellulolytic enzymes. Brown rot thus proceeds without significant lignin removal, apparently beginning instead with oxidative attack on wood polymers by Fenton reagent produced when fungal hydroquinones or catechols reduce Fe3+ in colonized wood. Since there is little evidence that white rot fungi produce these metabolites, one question is the extent to which independent lineages of brown rot fungi may have evolved different Fe3+ reductants. Recently, the catechol variegatic acid was proposed to drive Fenton chemistry in Serpula lacrymans, a brown rot member of the Boletales (D. C. Eastwood et al., Science 333:762-765, 2011). We found no variegatic acid in wood undergoing decay by S. lacrymans. We found also that variegatic acid failed to reduce in vitro the Fe3+ oxalate chelates that predominate in brown-rotting wood and that it did not drive Fenton chemistry in vitro under physiological conditions. Instead, the decaying wood contained physiologically significant levels of 2,5-dimethoxyhydroquinone, a reductant with a demonstrated biodegradative role when wood is attacked by certain brown rot fungi in two other divergent lineages, the Gloeophyllales and Polyporales. Our results suggest that the pathway for 2,5-dimethoxyhydroquinone biosynthesis may have been present in ancestral white rot basidiomycetes but do not rule out the possibility that it appeared multiple times via convergent evolution.

REGULATION OF GENE EXPRESSION DURING THE ONSET OF LIGNINOLYTIC OXIDATION BY PHANEROCHAETE CHRYSOSPORIUM ON SPRUCE WOOD

ABSTRACT Since uncertainty remains about how white rot fungi oxidize and degrade lignin in wood, it would be useful to monitor changes in fungal gene expression during the onset of ligninolysis on a natural substrate. We grew Phanerochaete chrysosporium on solid spruce wood and included oxidant-sensing beads bearing the fluorometric dye BODIPY 581/591 in the cultures. Confocal fluorescence microscopy of the beads showed that extracellular oxidation commenced 2 to 3 days after inoculation, coincident with cessation of fungal growth. Whole transcriptome shotgun sequencing (RNA-seq) analyses based on the v.2.2 P. chrysosporium genome identified 356 genes whose transcripts accumulated to relatively high levels at 96 h and were at least four times the levels found at 40 h. Transcripts encoding some lignin peroxidases, manganese peroxidases, and auxiliary enzymes thought to support their activity showed marked apparent upregulation. The data were also consistent with the production of ligninolytic extracellular reactive oxygen species by the action of manganese peroxidase-catalyzed lipid peroxidation, cellobiose dehydrogenase-catalyzed Fe3+ reduction, and oxidase-catalyzed H2O2 production, but the data do not support a role for iron-chelating glycopeptides. In addition, transcripts encoding a variety of proteins with possible roles in lignin fragment uptake and processing, including 27 likely transporters and 18 cytochrome P450s, became more abundant after the onset of extracellular oxidation. Genes encoding cellulases showed little apparent upregulation and thus may be expressed constitutively. Transcripts corresponding to 165 genes of unknown function accumulated more than 4-fold after oxidation commenced, and some of them may merit investigation as possible contributors to ligninolysis.

Spatial mapping of extracellular oxidant production by a white rot basidiomycete on wood reveals details of ligninolytic mechanism.

Oxidative cleavage of the recalcitrant plant polymer lignin is a crucial step in global carbon cycling, and is accomplished most efficiently by fungi that cause white rot of wood. These basidiomycetes secrete many enzymes and metabolites with proposed ligninolytic roles, and it is not clear whether all of these agents are physiologically important during attack on natural lignocellulosic substrates. One new approach to this problem is to infer properties of ligninolytic oxidants from their spatial distribution relative to the fungus on the lignocellulose. We grew Phanerochaete chrysosporium on wood sections in the presence of oxidant-sensing beads based on the ratiometric fluorescent dye BODIPY 581/591. The beads, having fixed locations relative to the fungal hyphae, enabled spatial mapping of cumulative extracellular oxidant distributions by confocal fluorescence microscopy. The results showed that oxidation gradients occurred around the hyphae, and data analysis using a mathematical reaction-diffusion model indicated that the dominant oxidant during incipient white rot had a half-life under 0.1 s. The best available hypothesis is that this oxidant is the cation radical of the secreted P. chrysosporium metabolite veratryl alcohol.

Lignin decomposition is sustained under fluctuating redox conditions in humid tropical forest soils

Lignin mineralization represents a critical flux in the terrestrial carbon (C) cycle, yet little is known about mechanisms and environmental factors controlling lignin breakdown in mineral soils. Hypoxia is thought to suppress lignin decomposition, yet potential effects of oxygen (O2 ) variability in surface soils have not been explored. Here, we tested the impact of redox fluctuations on lignin breakdown in humid tropical forest soils during ten-week laboratory incubations. We used synthetic lignins labeled with 13 C in either of two positions (aromatic methoxyl or propyl side chain Cβ ) to provide highly sensitive and specific measures of lignin mineralization seldom employed in soils. Four-day redox fluctuations increased the percent contribution of methoxyl C to soil respiration relative to static aerobic conditions, and cumulative methoxyl-C mineralization was statistically equivalent under static aerobic and fluctuating redox conditions despite lower soil respiration in the latter treatment. Contributions of the less labile lignin Cβ to soil respiration were equivalent in the static aerobic and fluctuating redox treatments during periods of O2 exposure, and tended to decline during periods of O2 limitation, resulting in lower cumulative Cβ mineralization in the fluctuating treatment relative to the static aerobic treatment. However, cumulative mineralization of both the Cβ - and methoxyl-labeled lignins nearly doubled in the fluctuating treatment relative to the static aerobic treatment when total lignin mineralization was normalized to total O2 exposure. Oxygen fluctuations are thought to be suboptimal for canonical lignin-degrading microorganisms. However, O2 fluctuations drove substantial Fe reduction and oxidation, and reactive oxygen species generated during abiotic Fe oxidation might explain the elevated contribution of lignin to C mineralization. Iron redox cycling provides a potential mechanism for lignin depletion in soil organic matter. Couplings between soil moisture, redox fluctuations, and lignin breakdown provide a potential link between climate variability and the biochemical composition of soil organic matter.

A Highly Diastereoselective Oxidant Contributes to Ligninolysis by the White Rot Basidiomycete Ceriporiopsis subvermispora

ABSTRACT The white rot basidiomycete Ceriporiopsis subvermispora delignifies wood selectively and has potential biotechnological applications. Its ability to remove lignin before the substrate porosity has increased enough to admit enzymes suggests that small diffusible oxidants contribute to delignification. A key question is whether these unidentified oxidants attack lignin via single-electron transfer (SET), in which case they are expected to cleave its propyl side chains between Cα and Cβ and to oxidize the threo-diastereomer of its predominating β-O-4-linked structures more extensively than the corresponding erythro-diastereomer. We used two-dimensional solution-state nuclear magnetic resonance (NMR) techniques to look for changes in partially biodegraded lignin extracted from spruce wood after white rot caused by C. subvermispora. The results showed that (i) benzoic acid residues indicative of Cα—Cβ cleavage were the major identifiable truncated structures in lignin after decay and (ii) depletion of β-O-4-linked units was markedly diastereoselective with a threo preference. The less selective delignifier Phanerochaete chrysosporium also exhibited this diastereoselectivity on spruce, and a P. chrysosporium lignin peroxidase operating in conjunction with the P. chrysosporium metabolite veratryl alcohol did likewise when cleaving synthetic lignin in vitro. However, C. subvermispora was significantly more diastereoselective than P. chrysosporium or lignin peroxidase-veratryl alcohol. Our results show that the ligninolytic oxidants of C. subvermispora are collectively more diastereoselective than currently known fungal ligninolytic oxidants and suggest that SET oxidation is one of the chemical mechanisms involved.

Acridine Orange Indicates Early Oxidation of Wood Cell Walls by Fungi

Colonization of wood blocks by brown and white rot fungi rapidly resulted in detectable wood oxidation, as shown by a reduced phloroglucinol response, a loss of autofluorescence, and acridine orange (AO) staining. This last approach is shown to provide a novel method for identifying wood oxidation. When lignin was mildly oxidized, the association between AO and lignin was reduced such that stained wood sections emitted less green light during fluorescence microscopy. This change was detectable after less than a week, an interval that past work has shown to be too short for significant delignification of wood. Although fungal hyphae were observed in only a few wood lumina, oxidation was widespread, appearing relatively uniform over regions several hundred micrometers from the hyphae. This observation suggests that both classes of fungi release low molecular weight mild oxidants during the first few days of colonization.