Search

5‐Nitrosalicylate 1,2‐dioxygenase (5NSDO) is an iron(II)‐dependent dioxygenase involved in the aerobic degradation of 5‐nitroanthranilic acid by the bacterium Bradyrhizobium sp. It catalyzes the opening of the 5‐nitrosalicylate aromatic ring, a key step in the degradation pathway. Besides 5‐nitrosalicylate, the enzyme is also active towards 5‐chlorosalicylate. The X‐ray crystallographic structure of the enzyme was solved at 2.1 Å resolution by molecular replacement using a model from the AI program AlphaFold. The enzyme crystallized in the monoclinic space group P21, with unit‐cell parameters a = 50.42, b = 143.17, c = 60.07 Å, β = 107.3°. 5NSDO belongs to the third class of ring‐cleaving dioxygenases. Members of this family convert para‐diols or hydroxylated aromatic carboxylic acids and belong to the cupin superfamily, which is one of the most functionally diverse protein classes and is named on the basis of a conserved β‐barrel fold. 5NSDO is a tetramer composed of four identical subunits, each folded as a monocupin domain. The iron(II) ion in the enzyme active site is coordinated by His96, His98 and His136 and three water molecules with a distorted octahedral geometry. The residues in the active site are poorly conserved compared with other dioxygenases of the third class, such as gentisate 1,2‐dioxygenase and salicylate 1,2‐dioxygenase. Comparison with these other representatives of the same class and docking of the substrate into the active site of 5NSDO allowed the identification of residues which are crucial for the catalytic mechanism and enzyme selectivity. [ABSTRACT FROM AUTHOR]

White-rot fungi (WRF) are the most efficient lignin-degrading organisms in nature. However, their capacity to use lignin-related aromatic compounds, such as 4-hydroxybenzoate, as carbon sources has only been described recently. Previously, the hydroxyquinol pathway was proposed for the bioconversion of these compounds in fungi, but gene- and structure-function relationships of the full enzymatic pathway remain uncharacterized in any single fungal species. Here, we characterize seven enzymes from two WRF, Trametes versicolor and Gelatoporia subvermispora , which constitute a four-enzyme cascade from 4-hydroxybenzoate to β-ketoadipate via the hydroxyquinol pathway. Furthermore, we solve the crystal structure of four of these enzymes and identify mechanistic differences with the closest bacterial and fungal structural homologs. Overall, this research expands our understanding of aromatic catabolism by WRF and establishes an alternative strategy for the conversion of lignin-related compounds to the valuable molecule β-ketoadipate, contributing to the development of biological processes for lignin valorization. [Display omitted] • We characterize enzymes in the 4-hydroxybenzoate catabolic pathway of white-rot fungi • We identify mechanistic differences between bacterial and fungal structural homologs • We expand our understanding of monomeric aromatic compound catabolism by white-rot fungi • We establish an enzymatic strategy to convert lignin-related compounds to β-ketoadipate Kuatsjah et al. biochemically and structurally characterize enzymes in the 4-hydroxybenzoate catabolic pathway of white-rot fungi. Their findings advance the understanding of aromatic catabolism in fungi and reveal pathways and enzymes that convert lignin-derived compounds into valuable biochemicals, such as β-ketoadipate. [ABSTRACT FROM AUTHOR]

Pink-pigmented facultative methylotrophs have long been studied for their ability to grow on reduced single-carbon (C1) compounds. The C1 groups that support methylotrophic growth may come from a variety of sources. Here, we describe a group of Methylobacterium strains that can engage in methoxydotrophy: they can metabolize the methoxy groups from several aromatic compounds that are commonly the product of lignin depolymerization. Furthermore, these organisms can utilize the full aromatic ring as a growth substrate, a phenotype that has rarely been described in Methylobacterium. We demonstrated growth on p -hydroxybenzoate, protocatechuate, vanillate, and ferulate in laboratory culture conditions. We also used comparative genomics to explore the evolutionary history of this trait, finding that the capacity for aromatic catabolism is likely ancestral to two clades of Methylobacterium , but has also been acquired horizontally by closely related organisms. In addition, we surveyed the published metagenome data to find that the most abundant group of aromatic-degrading Methylobacterium in the environment is likely the group related to Methylobacterium nodulans , and they are especially common in soil and root environments. The demethoxylation of lignin-derived aromatic monomers in aerobic environments releases formaldehyde, a metabolite that is a potent cellular toxin but that is also a growth substrate for methylotrophs. We found that, whereas some known lignin-degrading organisms excrete formaldehyde as a byproduct during growth on vanillate, Methylobacterium do not. This observation is especially relevant to our understanding of the ecology and the bioengineering of lignin degradation. [ABSTRACT FROM AUTHOR]

Alcaligenes aquatilis QD168 is a marine, aromatic hydrocarbon-degrading bacterium, isolated from an oil-polluted sediment of Quintero Bay, an industrial-coastal zone that has been chronically impacted by diverse pollutants. The aims of this study were to characterize the phylogenomic positions of Alcaligenes spp. and to characterize the genetic determinants and the physiological response of A. aquatilis QD168 to model environmental stressors (benzene, oxidizing agents, and salt). Phylogenomic analyses, using 35 housekeeping genes, clustered A. aquatilis QD168 with four other strains of Alcaligenes spp. (A. aquatilis BU33N, A. faecalis JQ135, A. faecalis UBA3227, and A. faecalis UBA7629). Genomic sequence analyses of A. aquatilis QD168 with 25 Alcaligenes spp., using ANIb, indicated that A. aquatilis BU33N is the closest related strain, with 96.8% ANIb similarity. Strain QD168 harbors 95 genes encoding proteins of seven central catabolic pathways, as well as sixteen peripheral catabolic pathways/reactions for aromatic compounds. A. aquatilis QD168 was able to grow on 3-hydroxybenzoate, 4-hydroxybenzoate, benzoate, benzene, 3-hydroxycinnamate, cinnamate, anthranilate, benzamide, 4-aminobenzoate, nicotinate, toluene, biphenyl and tryptophan, as sole carbon or nitrogen source. Benzene degradation was further analyzed by growth, metabolite identification and gene expression analyses. Benzene strongly induced the expression of the genes encoding phenol hydroxylase (dmpP) and catechol 1,2-dioxygenase (catA). Additionally, 30 genes encoding transcriptional regulators, scavenging enzymes, oxidative damage repair systems and isozymes involved in oxidative stress response were identified. Oxidative stress response of strain QD168 to hydrogen peroxide and paraquat was characterized, demonstrating that A. aquatilis QD168 is notably more resistant to paraquat than to H2O2. Genetic determinants (47 genes) for osmoprotective responses were identified, correlating with observed high halotolerance by strain QD168. The physiological adaptation of A. aquatilis QD168 to environmental stressors such as pollutants, oxidative stress and salinity may be exploited for bioremediation of oil-polluted saline sites. [ABSTRACT FROM AUTHOR]

Bioconversion of lignin-related aromatic compounds relies on robust catabolic pathways in microbes. Sphingobium sp. SYK-6 (SYK-6) is a well-characterized aromatic catabolic organism that has served as a model for microbial lignin conversion, and its utility as a biocatalyst could potentially be further improved by genome-wide metabolic analyses. To this end, we generate a randomly barcoded transposon insertion mutant (RB-TnSeq) library to study gene function in SYK-6. The library is enriched under dozens of enrichment conditions to quantify gene fitness. Several known aromatic catabolic pathways are confirmed, and RB-TnSeq affords additional detail on the genome-wide effects of each enrichment condition. Selected genes are further examined in SYK-6 or Pseudomonas putida KT2440, leading to the identification of new gene functions. The findings from this study further elucidate the metabolism of SYK-6, while also providing targets for future metabolic engineering in this organism or other hosts for the biological valorization of lignin. [Display omitted] • An RB-TnSeq library is generated in the aromatic catabolic bacterium, Sphingobium sp. SYK-6 • The library is enriched under dozens of growth conditions to determine gene fitness • Fitness profiling identifies details of lignin-related aromatic catabolism and tolerance • This RB-TnSeq library is a resource for future fitness profiling in additional conditions Bleem et al. develop a randomly barcoded transposon insertion (RB-TnSeq) library in Sphingobium sp. SYK-6 and then apply it to quantify gene fitness in aromatic catabolism and stress tolerance conditions. The library identifies details of SYK-6 metabolism, provides metabolic engineering targets for biological lignin valorization, and enables future fitness profiling. [ABSTRACT FROM AUTHOR]

The salicylate 1,2-dioxygenase ( SDO) from the bacterium Pseudaminobacter salicylatoxidans BN12 is a versatile gentisate 1,2-dioxygenase ( GDO) that converts both gentisate (2,5-dihydroxybenzoate) and various monohydroxylated substrates. Several variants of this enzyme were rationally designed based on the previously determined enzyme structure and sequence differences between the SDO and the 'conventional' GDO from Corynebacterium glutamicum. This was undertaken in order to define the structural elements that give the SDO its unique ability to dioxygenolytically cleave (substituted) salicylates. SDO variants M103 L, G106 A, G111 A, R113 G, S147 R and F159 Y were constructed and it was found that G106 A oxidized only gentisate; 1-hydroxy-2-naphthoate and salicylate were not converted. This indicated that this enzyme variant behaves like previously known 'conventional' GDOs. Crystals of the G106 A SDO variant and its complexes with salicylate and gentisate were obtained under anaerobic conditions, and the structures were solved and analyzed. The amino acid residue Gly106 is located inside the SDO active site cavity but does not directly interact with the substrates. Crystal structures of G106 A SDO complexes with gentisate and salicylate showed a different binding mode for salicylate when compared with the wild-type enzyme. Thus, salicylate coordinated in the G106 A variant with the catalytically active Fe(II) ion in an unusual and unproductive manner because of the inability of salicylate to displace a hydrogen bond that was formed between Trp104 and Asp174 in the G106 A variant. It is proposed that this type of unproductive substrate binding might generally limit the substrate spectrum of 'conventional' GDOs. Database Structural data are available in the Protein Data Bank databases under the accession numbers 3NST, 3NWA, 3NVC [ABSTRACT FROM AUTHOR]

The major utilization pathway for lignin-derived aromatic compounds in microorganisms is the β-ketoadipate pathway. Through this pathway, the aromatic compounds protocatechuate and catechol are converted to acetyl coenzyme A and succinyl coenzyme A. The enzymes of the protocatechuate branch of this pathway are encoded by the pca genes. Here, we describe a gene cluster in Streptomyces coelicolor containing the pca structural genes and a regulatory gene required for the catabolism of protocatechuate. We found that transcription of the structural genes in S. coelicolor is induced by protocatechuate and p-hydroxybenzoate. We also observed inducible transcription of pca structural genes in the ligninolytic strain Streptomyces viridosporus ATCC 39115. Disruption of a gene encoding a putative MarR family transcription factor that is divergently transcribed from the pca structural genes resulted in constitutive transcription of the structural genes. Thus, the transcription factor encoded by this gene is an apparent negative regulator of pca gene transcription in S. coelicolor. Our findings suggest how Streptomyces bacteria could be engineered for and used in biotechnology for the utilization and degradation of lignin and lignin-derived aromatic compounds. [ABSTRACT FROM AUTHOR]

Heavy metal co-contamination in crude oil-polluted environments may inhibit microbial bioremediation of hydrocarbons. The model heavy metal-resistant bacterium Cupriavidus metallidurans CH34 possesses cadmium and mercury resistance, as well as genes related to the catabolism of hazardous BTEX aromatic hydrocarbons. The aims of this study were to analyze the aromatic catabolic potential of C. metallidurans CH34 and to determine the functionality of the predicted benzene catabolic pathway and the influence of cadmium and mercury on benzene degradation. Three chromosome-encoded bacterial multicomponent monooxygenases (BMMs) are involved in benzene catabolic pathways. Growth assessment, intermediates identification, and gene expression analysis indicate the functionality of the benzene catabolic pathway. Strain CH34 degraded benzene via phenol and 2-hydroxymuconic semialdehyde. Transcriptional analyses revealed a transition from the expression of catechol 2,3-dioxygenase (tomB) in the early exponential phase to catechol 1,2-dioxygenase (catA1 and catA2) in the late exponential phase. The minimum inhibitory concentration to Hg (II) and Cd (II) was significantly lower in the presence of benzene, demonstrating the effect of co-contamination on bacterial growth. Notably, this study showed that C. metallidurans CH34 degraded benzene in the presence of Hg (II) or Cd (II). [ABSTRACT FROM AUTHOR]

Protocatechuate 4,5-dioxygenase has been purified 100-fold from 4-hydroxybenzoate grown cells of Rhizobium leguminosarum biovar viceae. The purification yielded a homogeneous preparation with specific activity of 321 Units · mg protein. The molecular weight of the homodimeric native protein was 120,000, with subunit molecular weight of 62,000. The optimum pH for catalytic activity was 9.5 and the K for protocatechuate was 20 μM. Physical and catalytic properties of the R. leguminosarum protocatechuate 4,5-dioxygenase were different from the published characteristics of isofunctional enzymes from Pseudomonas paucimobilis and Comamonas testosteroni. [ABSTRACT FROM AUTHOR]

Six species of free-living nitrogen fixing bacteria, Azomonas agilis, Azospirillum brasilense, Azospirillum lipoferum, Azotobacter chroococcum, Azotobacter vinelandii, and Beijerinckia mobilis, were surveyed for their ability to grow and fix N using aromatic compounds as sole carbon and energy source. All six species grew and expressed nitrogenase activity on benzoate, catechol, 4-hydroxybenzoate, naphthalene, protocatechuate, and 4-toluate. In many cases, growth rates on one or more aromatic compounds were comparable to or greater than those on the non-aromatic substrates routinely used for cultivation of the organisms. Specific activity of nitrogenase in extracts of aromatic-grown cells often exceeded that in cells grown on non-aromatic substrates. All six species growing on substrates typically converted to catechol expressed inducible catechol 1,2-dioxygenase and/or catechol 2,3-dioxygenase. When grown on substrates typically converted to protocatechuate, inducible protocatechuate 3,4-dioxygenase and/or protocatechuate 4,5-dioxygenase was expressed. A. chroococcum expressed only ortho cleavage dioxygenases during growth on naphthalene and 4-toluate and only meta cleavage dioxygenases on the other aromatics. B. mobilis expressed only ortho cleavage dioxygenases. The other four species examined expressed both ortho and meta cleavage enzymes. [ABSTRACT FROM AUTHOR]

Pseudomonas fluorescens biovar I was isolated from a pulp mill effluent based on its ability to grow on synthetic media containing 1,2-diarylethane structures as the sole carbon and energy source. Analysis of samples taken from cultures of this strain in benzoin or 4,4′-dimethoxybenzoin (anisoin), showed that cleavage between the two aliphatic carbons takes place prior to ring fission. Intermonomeric cleavage was also obtained with crude extracts. Substrates of this reaction were only those 1,2-diarylethane compounds that supported growth of the bacterium. The purification and partial characterization of an enzyme that catalyzes the NADH-dependent reduction of the carbonyl group of benzoin and anisoin is also reported. [ABSTRACT FROM AUTHOR]

Escherichia coli mutants, unable to grown on 4-hydroxyphenylacetate, have been isolated and found to be defective in the NAD-dependent succinate semialdehyde dehydrogenase. When the mutants are grown with 4-aminobutyrate as sole nitrogen source an NAD-dependent succinate semialdehyde dehydrogenase seen in the parental strain is absent but, as in the parental strain, an NADP-dependent enzyme is induced. Growth of the mutants is inhibited by 4-hydroxyphenylacetate due to the accumulation of succinate semialdehyde. The mutants are more sensitive to inhibition by exogenous succinate semialdehyde than is the parental strain. Secondary mutants able to grow in the presence of 4-hydroxyphenylacetate but still unable to use it as sole carbon source were defective in early steps of 4-hydroxyphenylacetate catabolism and so did not form succinate semialdehyde from 4-hydroxyphenylacetate. The gene encoding the NAD-dependent succinate semialdehyde dehydrogenase of Escherichia coli K-12 was located at min 34.1 on the genetic map. [ABSTRACT FROM AUTHOR]