Bioconversion of biologically active indole derivatives with indole-3-acetic acid-degrading enzymes from Caballeronia glathei DSM50014

A plant auxin hormone indole-3-acetic acid (IAA) can be assimilated by bacteria as an energy and carbon source, although no degradation has been reported for indole-3-propionic acid and indole-3-butyric acid. While significant efforts have been made to decipher the Iac (indole-3-acetic acid catabolism)-mediated IAA degradation pathway, a lot of questions remain regarding the mechanisms of individual reactions, involvement of specific Iac proteins, and the overall reaction scheme. This work was aimed at providing new experimental evidence regarding the biodegradation of IAA and its derivatives. Here, it was shown that Caballeronia glathei strain DSM50014 possesses a full iac gene cluster and is able to use IAA as a sole source of carbon and energy. Next, IacE was shown to be responsible for the conversion of 2-oxoindole-3-acetic acid (Ox-IAA) intermediate into the central intermediate 3-hydroxy-2-oxindole-3-acetic acid (DOAA) without the requirement for IacB. During this reaction, the oxygen atom incorporated into Ox-IAA was derived from water. Finally, IacA and IacE were shown to convert a wide range of indole derivatives, including indole-3-propionic acid and indole-3-butyric acid, into corresponding DOAA homologs. This work provides novel insights into Iac-mediated IAA degradation and demonstrates the versatility and substrate scope of IacA and IacE enzymes

Characterization of <i>Paenibacillus</i> sp. GKG Endo-β-1, 3-Glucanase, a Member of Family 81 Glycoside Hydrolases

Paenibacillus sp. GKG was isolated based on its ability to produce hydrolysis zones on agar plates containing yeast cell wall substrate as the single carbon source. The extracellular enzymes secreted into the culture medium were identified by LC-MS/MS proteomics. Endo-β-1,3-glucanase PsLam81A containing GH81 catalytic and the CBM56 carbohydrate-binding modules was selected for heterologous expression in Escherichia coli. The identity of the recombinant PsLam81A was confirmed by LC-MS/MS proteomics. The PsLam81A showed the highest activity at 60 °C, and the optimal pH range was between 6.5 and 8.0. The analysis of the full-length PsLam81A and truncated PsLam81AΔCBM56 enzymes showed that the CBM56 module improved the hydrolytic activity towards linear β-1,3-glucans—curdlan and pachyman but had no effect on hydrolysis of β-1,3/β1,6-branched glucans—laminarin and yeast β-glucan. The characterization of PsLam81A enzyme broadens current knowledge on the biochemical properties and substrate specificity of family 81 glycoside hydrolases and allows prediction of the necessity of CBM56 module in the process of designing new truncated or chimeric glycosidases

Biochemical and genetic analysis of 4-hydroxypyridine catabolism in arthrobacter sp. strain IN13

N-Heterocyclic compounds are widely spread in the biosphere, being constituents of alkaloids, cofactors, allelochemicals, and artificial substances. However, the fate of such compounds including a catabolism of hydroxylated pyridines is not yet fully understood. Arthrobacter sp. IN13 is capable of using 4-hydroxypyridine as a sole source of carbon and energy. Three substrate-inducible proteins were detected by comparing protein expression profiles, and peptide mass fingerprinting was performed using MS/MS. After partial sequencing of the genome, we were able to locate genes encoding 4-hydroxypyridine-inducible proteins and identify the kpi gene cluster consisting of 16 open reading frames. The recombinant expression of genes from this locus in Escherichiacoli and Rhodococcus erytropolis SQ1 allowed an elucidation of the biochemical functions of the proteins. We report that in Arthrobacter sp. IN13, the initial hydroxylation of 4-hydroxypyridine is catalyzed by a flavin-dependent monooxygenase (KpiA). A product of the monooxygenase reaction is identified as 3,4-dihydroxypyridine, and a subsequent oxidative opening of the ring is performed by a hypothetical amidohydrolase (KpiC). The 3-(N-formyl)-formiminopyruvate formed in this reaction is further converted by KpiB hydrolase to 3-formylpyruvate. Thus, the degradation of 4-hydroxypyridine in Arthrobacter sp. IN13 was analyzed at genetic and biochemical levels, elucidating this catabolic pathwa

Tailoring a Soluble Diiron Monooxygenase for Synthesis of Aromatic <i>N</i>-oxides

The aromatic N-oxides have received increased attention over the last few years due to their potential application in medicine, agriculture and organic chemistry. As a green alternative in their synthesis, the biocatalytic method employing whole cells of Escherichia coli bearing phenol monooxygenase like protein PmlABCDEF (from here on &#8211; PML monooxygenase) has been introduced. In this work, site-directed mutagenesis was used to study the contributions of active site neighboring residues I106, A113, G109, F181, F200, F209 to the regiospecificity of N-oxidation. Based on chromogenic indole oxidation screening, a collection of PML mutants with altered catalytic properties was created. Among the tested mutants, the A113G variant acquired the most distinguishable N-oxidations capacity. This new variant of PML was able to produce dioxides (quinoxaline-1,4-dioxide, 2,5-dimethylpyrazine-1,4-dioxide) and specific mono-N-oxides (2,3,5-trimethylpyrazine-1-oxide) that were unachievable using the wild type PML. This mutant also featured reshaped regioselectivity as N-oxidation shifted towards quinazoline-1-oxide compared to quinazoline-3-oxide that is produced by the wild type PML

Oxyfunctionalization of pyridine derivatives using whole cells of Burkholderia sp. MAK1

Pyridinols and pyridinamines are important intermediates with many applications in chemical industry. The pyridine derivatives are in great demand as synthons for pharmaceutical products. Moreover, pyridines are used either as biologically active substances or as building blocks for polymers with unique physical properties. Application of enzymes or whole cells is an attractive strategy for preparation of hydroxylated pyridines since the methods for chemical synthesis of pyridinols, particularly aminopyridinols, are usually limited or inefficient. Burkholderia sp. MAK1 (DSM102049), capable of using pyridin-2-ol as the sole carbon and energy source, was isolated from soil. Whole cells of Burkholderia sp. MAK1 were confirmed to possess a good ability to convert different pyridin- 2-amines and pyridin-2-ones into their 5-hydroxy derivatives. Moreover, several methylpyridines as well as methylated pyrazines were converted to appropriate N-oxides. In conclusion, regioselective oxyfunctionalization of pyridine derivatives using whole cells of Burkholderia sp. MAK1 is a promising method for the preparation of various pyridin-5-ols and pyridin-N-oxides

Biocatalytic synthesis of asymmetric water-soluble indirubin derivatives /

A method for the synthesis of asymmetric carboxy-substituted indirubins is presented. It employs indole-5-carboxylic acid or indole-6-carboxylic acid and 2-indolinone derivatives as substrates for bacterial monooxygenase-driven enzymatic bioconversion in different bacterial hosts. This bioconversion system achieved the highest titer of monocarboxyindirubin production of up to 327 mg L−1 for 5-bromoindirubin-6′-carboxylic acid during the 16-h incubation period. The purified monocarboxyindirubins exhibited high solubility in water, up to three orders of magnitude higher than that of indirubin. In addition, several monocarboxyindirubins, namely 1-methylindirubin-5′-carboxylic acid, possess potent antiproliferative activity against different cancer cell lines. Therefore, the synthesis method for monocarboxyindirubins described herein is an efficient and environmentally friendly bioconversion system and the synthesized monocarboxyindirubins show great promise due to their high water solubility and potential antiproliferative activity

Engineering of a chromogenic enzyme screening system based on an auxiliary indole-3-carboxylic acid monooxygenase

Here, we present a proof-of-principle for a new high-throughput functional screening of metagenomic libraries for the selection of enzymes with different activities, predetermined by the substrate being used. By this approach, a total of 21 enzyme-coding genes were selected, including members of xanthine dehydrogenase, aldehyde dehydrogenase (ALDH), and amidohydrolase families. The screening system is based on a pro-chromogenic substrate, which is transformed by the target enzyme to indole-3-carboxylic acid. The later compound is converted to indoxyl by a newly identified indole-3-carboxylate monooxygenase (Icm). Due to the spontaneous oxidation of indoxyl to indigo, the target enzyme-producing colonies turn blue. Two types of pro-chromogenic substrates have been tested. Indole-3-carboxaldehydes and the amides of indole-3-carboxylic acid have been applied as substrates for screening of the ALDHs and amidohydrolases, respectively. Both plate assays described here are rapid, convenient, easy to perform, and adaptable for the screening of a large number of samples both in Escherichia coli and Rhodococcus sp. In addition, the fine-tuning of the pro-chromogenic substrate allows screening enzymes with the desired substrate specificity

Cytidine deaminases catalyze the conversion of N( S,O)4^4 -substituted pyrimidine nucleosides

Cytidine deaminases (CDAs) catalyze the hydrolytic deamination of cytidine and 2′-deoxycytidine to uridine and 2′-deoxyuridine. Here, we report that prokaryotic homo-tetrameric CDAs catalyze the nucleophilic substitution at the fourth position of N$^4$-acyl-cytidines, N$^4$-alkyl-cytidines, and N$^4$-alkyloxycarbonyl-cytidines, and S$^4$-alkylthio-uridines and O$^4$-alkyl-uridines, converting them to uridine and corresponding amide, amine, carbamate, thiol, or alcohol as leaving groups. The x-ray structure of a metagenomic CDA_F14 and the molecular modeling of the CDAs used in this study show a relationship between the bulkiness of a leaving group and the volume of the binding pocket, which is partly determined by the flexible β3α3 loop of CDAs. We propose that CDAs that are active toward a wide range of substrates participate in salvage and/or catabolism of variously modified pyrimidine nucleosides. This identified promiscuity of CDAs expands the knowledge about the cellular turnover of cytidine derivatives, including the pharmacokinetics of pyrimidine-based prodrugs