Thermostable enzymes and thermophilic cell factories may afford economic advantages inFurthermore, we present evidence suggesting that aside from representing a potential 9 reservoir of thermostable enzymes, thermophilic fungi are amenable to manipulation using 10 classical and molecular genetics. 11Rapid, efficient and robust enzymatic degradation of biomass-derived polysaccharides is 12 currently a major challenge for biofuel production. A prerequisite is the availability of enzymes 13 that hydrolyze cellulose, hemicellulose and other polysaccharides into fermentable sugars at 14 conditions suitable for industrial use. The best studied and most widely used cellulases and to overcome these obstacles is to raise the reaction temperature, thereby increasing hydrolytic 20 rates and reducing contamination risks. AT-rich repetitive regions (Fig. 1) To examine the strategy used by these thermophiles for decomposition of plant cell wall 9 polysaccharides, we used RNA-Seq to compare transcript profiles during growth on barley straw 10 or alfalfa straw to growth on glucose. Alfalfa was chosen to represent dicotyledonous plants, 11 whereas barley was used to represent monocotyledon plants. The major difference between these 12 materials is that the carbohydrates from barley cell wall are mainly cellulose and hemicellulose 13 with a negligible amount of pectin 11 , whereas alfalfa cell wall contains pectin and xylan in 14 roughly similar proportions, each consisting of 15-20% of total carbohydrates 12, . 15 We observed notable differences between the transcriptional profiles of genes encoding conditions. For example, the orthologs in Clades A, B, E, G and P of GH61 are upregulated 8 under growth in complex substrates for both thermophiles (Fig. 2b). An even more striking 9 correlation between transcript levels and orthologs is evident for the GH6 and GH7 cellulases 10 ( Supplementary Fig. 7) where the transcript profiles for the orthologs of the two organisms are Table 7). Thermophilic fungi are major components of the microflora in self-heating composts. They 9 break down cellulose at a faster rate than prodigious, mesophilic cellulase producers such as T. Tables 11-14). On the basis of 24 our comparative analyses of the genomes from two thermophilic fungi, we conclude that their 25 nucleotide and protein features are different from those observed in thermophilic prokaryotes. 26 We also investigated the possibility that thermophilic fungi possess major differences in 27 processes mediating thermophily including heat shock, oxidative stress, membrane biosynthesis, 28 chromatin structure and modification, and fungal cell wall metabolism. We compared the 29 proteins predicted to be involved in these processes in C. globosum, M. thermophila and T. 30 terrestris, but were unable to find differences that can convincingly be interpreted as the Fig. 9). Within the Sordiariales, thermophily 6 is restricted to subgroups of the family Chaetomiaceae. Among fungi more broadly, thermophily 7 also exists in the Zygomycota, but it ...

The meta-cleavage pathway for catechol is one of the major routes for the microbial degradation of aromatic compounds. Pseudomonas sp. strain CF600 grows efficiently on phenol, cresols, and 3,4-dimethylphenol via a plasmid-encoded multicomponent phenol hydroxylase and a subsequent meta-cleavage pathway. The genes for the entire pathway were previously found to be clustered, and the nucleotide sequences of dmpKLMNOPBC and D, which encode the first four biochemical steps of the pathway, were determined. By using a combination of deletion mapping, nucleotide sequence determinations, and polypeptide analysis, we identified the remaining six genes of the pathway. The fifteen genes, encoded in the order dmpKLMNOPQBCDEFGHI, lie in a single operon structure with intergenic spacing that varies between 0 to 70 nucleotides. Homologies found between the newly determined gene sequences and known genes are reported. Enzyme activity assays of deletion derivatives of the operon expressed in Escherichia coli were used to correlate dmpE, G, H, and I with known meta-cleavage enzymes. Although the function of the dmpQ gene product remains unknown, dmpF was found to encode acetaldehyde dehydrogenase (acylating) activity (acetaldehyde:NAD+ oxidoreductase [coenzyme A acylating]; E.C.1.2.1.10). The role of this previously unknown meta-cleavage pathway enzyme is discussed.The central role of catecholic intermediates in aerobic microbial degradation of aromatic compounds is well established. Catechol (1,2-dihydroxybenzene) itself is an intermediate in the degradation of compounds such as benzoate, naphthalene, salicylate, and phenol, and substituted catechols are intermediates in the catabolism of methylated and chlorinated derivatives of these compounds (13,34). A diverse array of enzymes can be elaborated to convert aromatic compounds to central catecholic intermediates. However, the reactions used for oxygenative ring fission of the catechol and the subsequent conversion to Krebs cycle intermediates are limited to one of two metabolic alternatives: those of the ortho-and meta-cleavage pathways. The ortho-cleavage pathways involve ring cleavage between the two hydroxyl groups followed by a well-defined series of reactions leading to P-ketoadipate (reviewed in reference 13). The alternative meta-cleavage pathway involves ring cleavage adjacent to the two catechol hydroxyls, followed by degradation of the ring cleavage product to pyruvate and a short-chain aldehyde (Fig. 1). The use of one pathway or the other is dependent upon the microbial species and/or the nature of the growth substrate.The meta-cleavage pathway was first studied in Pseudomonas strains that can grow at the expense of phenol and cresols (14,29). Since then, the role of the meta-cleavage pathway in aromatic biodegradation by bacteria of many genera, including species of Azotobacter and Alcaligenes and numerous species of Pseudomonas, has been demonstrated (2, 13, 23, 36). In addition, reactions of the lower part of the pathway are involved in the degradation of phenylp...

Pseudomonas sp. strain CF600 metabolizes phenol and some of its methylated derivatives via a plasmidencoded phenol hydroxylase and meta-cleavage pathway. The genes encoding the multicomponent phenol hydroxylase of this strain are located within a 5.5-kb SacI-NruI fragment. We report the nucleotide sequence and the polypeptide products of this 5.5-kb (14) and into Pseudomonas strain PB2701 by electroporation with a Bio-Rad Gene Pulser. Ampicillin at 100 ,ug/ml and carbenicillin at 1 to 2 mg/ml were used for selection of plasmid-encoded ,-lactamase in E. coli and Pseudomonas strains, respectively.Plasmids expressing phenol hydroxylase genes. Plasmids were constructed by using the broad-host-range tac expression vectors pMMB66HE and pMMB66EH (8), and derivatives thereof, pMMB66HEA and pMMB66EHA (22), that lack expression of the plasmid-encoded lacdq repressor gene. Subfragments of the DNA shown in Fig. 1

The crystal structure of the bifunctional enzyme 4-hydroxy-2-ketovalerate aldolase (DmpG)͞acylating acetaldehyde dehydrogenase (DmpF), which is involved in the bacterial degradation of toxic aromatic compounds, has been determined by multiwavelength anomalous dispersion (MAD) techniques and refined to 1.7-Å resolution. Structures of the two polypeptides represent a previously unrecognized subclass of metal-dependent aldolases, and of a CoA-dependent dehydrogenase. The structure reveals a mixed state of NAD ؉ binding to the DmpF protomer. Domain movements associated with cofactor binding in the DmpF protomer may be correlated with channeling and activity at the DmpG protomer. In the presence of NAD ؉ a 29-Å-long sequestered tunnel links the two active sites. Two barriers are visible along the tunnel and suggest control points for the movement of the reactive and volatile acetaldehyde intermediate between the two active sites. E nzymatic channeling is a process by which intermediates are moved directly between active sites in a sequential reaction pathway without equilibrating with the bulk phase (1, 2). Channeling processes are particularly advantageous over the free diffusion of reaction products through the bulk solvent because they can protect chemically labile intermediates from breakdown, prevent loss of nonpolar intermediates by diffusion across cell membranes, or protect the cell from toxic intermediates. Crystallographic studies on a number of different enzyme systems involved in substrate channeling (3-11) have revealed important structural factors that mediate intersubunit or interdomain communication and facilitate the efficient transfer of intermediates between distant active sites.A bifunctional aldolase-dehydrogenase catalyzes the final two steps of the meta-cleavage pathway for catechol, an intermediate in many bacterial species in the degradation of phenols, toluates, naphthalene, biphenyls and other aromatic compounds (reviewed in ref. 12). Thus, 4-hydroxy-2-ketovalerate aldolase (DmpG; EC 4.1.3.-) and acetaldehyde dehydrogenase (acylating) (DmpF; EC 1.2.1.10) from a methylphenol-degrading pseudomonad convert 4-hydroxy-2-ketovalerate to pyruvate and acetyl-CoA by way of the intermediate acetaldehyde (Scheme 1).The two enzymes are tightly associated with each other (13,14). Whereas the aldolase appears to be inactive when expressed without the dehydrogenase, the dehydrogenase retains some activity when expressed in the absence of aldolase (14), suggesting that at least the dehydrogenase active site is distinct from that of the aldolase. Several lines of evidence are consistent with channeling of acetaldehyde between the active sites. For example, the conversion of 4-hydroxy-2-ketovalerate to acetyl-CoA occurs Ϸ20 times faster than that of acetaldehyde to acetyl-CoA, and the K m for acetaldehyde exceeds 50 mM, a physiologically irrelevant concentration (J.P., unpublished data). In addition, it has been shown that the aldolase activity is stimulated by the addition of the dehydrogenase cofactor to t...

Pseudomonas sp. strain CF600 is an efficient degrader of phenol and methylsubstituted phenols. These compounds are degraded by the set of enzymes encoded by the plasmid located dmpoperon. The sequences of all the fifteen structural genes required to encode the nine enzymes of the catabolic pathway have been determined and the corresponding proteins have been purified. In this review the interplay between the genetic analysis and biochemical characterisation of the catabolic pathway is emphasised. The first step in the pathway, the conversion of phenol to catechol, is catalysed by a novel multicomponent phenol hydroxylase. Here we summarise similarities of this enzyme with other multicomponent oxygenases, particularly methane monooxygenase (EC 1.14.13.25). The other enzymes encoded by the operon are those of the well-known meta-cleavage pathway for catechol, and include the recently discovered meta-pathway enzyme aldehyde dehydrogenase (acylating) (EC 1.2.1.10). The known properties of these meta-pathway enzymes, and isofunctional enzymes from other aromatic degraders, are summarised. Analysis of the sequences of the pathway proteins, many of which are unique to the meta-pathway, suggests new approaches to the study of these generally little-characterised enzymes. Furthermore, biochemical studies of some of these enzymes suggest that physical associations between meta-pathway enzymes play an important role. In addition to the pathway enzymes, the specific regulator of phenol catabolism, DmpR, and its relationship to the XylR regulator of toluene and xylene catabolism is discussed.

Thermostable enzymes and thermophilic cell factories may afford economic advantages inFurthermore, we present evidence suggesting that aside from representing a potential 9 reservoir of thermostable enzymes, thermophilic fungi are amenable to manipulation using 10 classical and molecular genetics. 11Rapid, efficient and robust enzymatic degradation of biomass-derived polysaccharides is 12 currently a major challenge for biofuel production. A prerequisite is the availability of enzymes 13 that hydrolyze cellulose, hemicellulose and other polysaccharides into fermentable sugars at 14 conditions suitable for industrial use. The best studied and most widely used cellulases and to overcome these obstacles is to raise the reaction temperature, thereby increasing hydrolytic 20 rates and reducing contamination risks. AT-rich repetitive regions (Fig. 1). one PL3 and two GH28). Pectin lyases are most active at neutral to alkaline pH whereas GH28 To examine the strategy used by these thermophiles for decomposition of plant cell wall 9 polysaccharides, we used RNA-Seq to compare transcript profiles during growth on barley straw 10 or alfalfa straw to growth on glucose. Alfalfa was chosen to represent dicotyledonous plants, 11 whereas barley was used to represent monocotyledon plants. The conditions. For example, the orthologs in Clades A, B, E, G and P of GH61 are upregulated 8 under growth in complex substrates for both thermophiles (Fig. 2b). An even more striking 9 correlation between transcript levels and orthologs is evident for the GH6 and GH7 cellulases Table 7). 14 Secretomes and exo-proteomes 15In addition to extracellular CAZymes involved in digestion of polysaccharide nutrients, the Thermophilic fungi are major components of the microflora in self-heating composts. They 9 break down cellulose at a faster rate than prodigious, mesophilic cellulase producers such as T. Fig. 8 We also investigated the possibility that thermophilic fungi possess major differences in 27 processes mediating thermophily including heat shock, oxidative stress, membrane biosynthesis, 28 chromatin structure and modification, and fungal cell wall metabolism. We compared the 29 proteins predicted to be involved in these processes in C. globosum, M. thermophila and T. 30terrestris, but were unable to find differences that can convincingly be interpreted as the Fig. 9) Thermophilic fungi are ubiquitous organisms commonly found in decomposing organic matter. 25The biotechnological utility of these fungi has been recognized for many years. enzymes from the thermophiles exhibit higher hydrolytic capacity than their counterparts from 6 mesophiles at temperatures ranging from 30 °C to 60 °C (Fig. 3). One explanation is that the 7 enzymes from the thermophiles possess higher specific activity toward lignocellulosic biomass.8

In this report, we describe some of the characteristics of the Comamonas testosteroni B-356 biphenyl (BPH)-chlorobiphenyl dioxygenase system, which includes the terminal oxygenase, an iron-sulfur protein (ISP BPH ) made up of an ␣ subunit (51 kDa) and a ␤ subunit (22 kDa) encoded by bphA and bphE, respectively; a ferredoxin (FER BPH ; 12 kDa) encoded by bphF; and a ferredoxin reductase (RED BPH ; 43 kDa) encoded by bphG. ISP BPH subunits were purified from B-356 cells grown on BPH. Since highly purified FER BPH and RED BPH were difficult to obtain from strain B-356, these two components were purified from recombinant Escherichia coli strains by using the His tag purification system. These His-tagged fusion proteins were shown to support BPH 2,3-dioxygenase activity in vitro when added to preparations of ISP BPH in the presence of NADH. FER BPH and RED BPH are thought to pass electrons from NADH to ISP BPH , which then activates molecular oxygen for insertion into the aromatic substrate. The reductase was found to contain approximately 1 mol of flavin adenine dinucleotide per mol of protein and was specific for NADH as an electron donor. The ferredoxin was found to contain a Rieske-type [2Fe-2S] center (⑀ 460 , 7,455 M ؊1 cm ؊1) which was readily lost from the protein during purification and storage. In the presence of RED BPH and FER BPH , ISP BPH was able to convert BPH into both 2,3-dihydro-2,3-dihydroxybiphenyl and 3,4-dihydro-3,4-dihydroxybiphenyl. The significance of this observation is discussed.The enzymatic steps involved in the conversion of biphenyl (BPH) and chlorobiphenyls (PCBs) into, respectively, benzoate and chlorobenzoates have been elucidated in many bacteria (5,8,11,12,23,24,29,32,43). The first step (shown in Fig. 1) involved BPH 2,3-dioxygenase (dox), which transforms BPH into 2,3-dihydro-2,3-dihydroxybiphenyl. This enzyme is believed to determine the substrate selectivity of BPH-degrading bacteria (17).PCB-degrading bacteria have been divided into four groups based on substrate selectivity patterns (4). Pseudomonas sp. strain LB400 has a unique feature in being able to degrade ortho-substituted PCB congeners preferentially (17). Most other bacteria preferentially transform meta-and para-substituted congeners. In another study (submitted for publication) based on alignments of gene and gene product sequences, we determined the existence of two separate BPH dox lineages among gram-negative bacteria. Comamonas testosteroni B-356 BPH dox belongs to a distinct phylogenetic lineage together with Pseudomonas sp. strain KKS102 (11,24). This group is characterized by the fact that the gene encoding the BPH ferredoxin reductase (RED BPH ) is located outside the bph gene cluster and is phylogenetically unrelated to all other known bacterial RED BPH -encoding genes.The two members of the second lineage are strain LB400 (8) and Pseudomonas pseudoalcaligenes KF707 (40). The BPH dox of these strains has a distinct substrate selectivity pattern, suggesting that only minor factors determine this characteri...

The oxygenase component of biphenyl dioxygenase (BPDO) from Comamonas testosteroni B-356 dihydroxylates biphenyl and some polychlorinated biphenyls (PCBs), thereby initiating their degradation. Overexpressed, anaerobically purified BPDO had a specific activity of 4.9 units/mg, and its oxygenase component appeared to contain a full complement of Fe 2 S 2 center and catalytic iron. Oxygenase crystals in space group R3 were obtained under anaerobic conditions using polyethylene glycol as the precipitant. X-ray diffraction was measured to 1.6 Å. Steady-state kinetics assays demonstrated that BPDO had an apparent k cat /K m for biphenyl of (1. The microbial catabolic activities responsible for the degradation of aromatic compounds constitute an essential link in the global carbon cycle. These activities are of considerable practical interest due to their potential to destroy toxic, persistent pollutants, a strategy known as bioremediation (1). In the case of highly chlorinated, structurally diverse xenobiotics such as PCBs, 1 the development of a practical bioremediation technology has been limited in part by the failure of existing microbial catabolic activities to effectively degrade these compounds (1, 2). This failure may arise because these activities have not yet evolved to degrade compounds that have only recently been introduced into the biosphere. An important aspect of the adaptation of catabolic activities for bioremediation is the study of the structure and function of key catabolic enzymes. Such studies provide insight into the molecular basis of an important biological process, thereby facilitating the modification of enzyme specificity and the design of novel metabolic pathways.2BPDO catalyzes the initial reaction in the aerobic degradation of biphenyl and some PCBs. BPDO is a typical aromatic ring-hydroxylating dioxygenase, utilizing O 2 and electrons originating from NADH to transform biphenyl to cis-(2R,3S)-dihydroxy-1-phenylcyclohexa-4,6-diene ( Fig. 1) (3, 4). This dihydroxylation prepares the ring for subsequent degradation by ring cleavage enzymes. The enzyme comprises an FAD-containing reductase (BphG), a Rieske-type ferredoxin (BphF), and a two-subunit oxygenase of ␣ 3 ␤ 3 constitution that contains a Rieske-type Fe 2 S 2 cluster and a mononuclear iron center. Accordingly, BPDO has been classified as a group IIB aromatic ring-hydroxylating dioxygenase together with benzene and toluene dioxygenases (5). Structural and spectroscopic studies of related dioxygenases indicate that the mononuclear iron center orchestrates substrate transformation (reviewed in Ref. 6). BphG, BphF, and the oxygenase Fe 2 S 2 cluster function to transfer electrons from NADH to this center.BPDO is a major determinant of the PCB-catabolizing capabilities of biphenyl-degrading strains, and the enzymes from different strains possess significantly different congener-transforming abilities. For example, BPDO LB400 from Burkholderia cepacia LB400 transforms a much broader range of congeners than BPDO KF707 from Pseudomonas ps...