Background: Microbes are rich sources of enzymes and esterases are one of the most important classes of enzymes because of their potential for application in the field of food, agriculture, pharmaceuticals and bioremediation. Due to limitations in their cultivation, only a small fraction of the complex microbial communities can be cultured from natural habitats. Thus to explore the catalytic potential of uncultured organisms, the metagenomic approach has turned out to be an effective alternative method for direct mining of enzymes of interest. Based on activity-based screening method, an esterase-positive clone was obtained from metagenomic libraries. Results: Functional screening of a soil metagenomic fosmid library, followed by transposon mutagenesis led to the identification of a 1179 bp esterase gene, estM2, that encodes a 392 amino acids long protein (EstM2) with a translated molecular weight of 43.12 kDa. Overproduction, purification and biochemical characterization of the recombinant protein demonstrated carboxylesterase activity towards short-chain fatty acyl esters with optimal activity for p-nitrophenyl butyrate at pH 8.0 and 37 °C. Amino acid sequence analysis and subsequent phylogenetic analysis suggested that EstM2 belongs to the family VIII esterases that bear modest similarities to class C β-lactamases. EstM2 possessed the conserved S-x-x-K motif of class C β-lactamases but did not exhibit β-lactamase activity. Guided by molecular docking analysis, EstM2 was shown to hydrolyze a wide range of di-and monoesters of alkyl-, aryl-and benzyl-substituted phthalates. Thus, EstM2 displays an atypical hydrolytic potential of biotechnological significance within family VIII esterases. Conclusions: This study has led to the discovery of a new member of family VIII esterases. To the best of our knowledge, this is the first phthalate hydrolase (EstM2), isolated from a soil metagenomic library that belongs to a family possessing β-lactamase like catalytic triad. Based on its catalytic potential towards hydrolysis of both phthalate diesters and phthalate monoesters, this enzyme may find use to counter the growing pollution caused by phthalatebased plasticizers in diverse geological environment and in other aspects of biotechnological applications.

No abstract

Strain ST-14, characterized as a member of the genus Cupriavidus, was capable of utilizing 2-and 4-nitrobenzoates individually as sole sources of carbon and energy. Biochemical studies revealed the assimilation of 2-and 4-nitrobenzoates via 3-hydroxyanthranilate and protocatechuate, respectively. Screening of a genomic fosmid library of strain ST-14 constructed in Escherichia coli identified two gene clusters, onb and pob-pca, to be responsible for the complete degradation of 2-nitrobenzoate and protocatechuate, respectively. Additionally, a gene segment (pnb) harboring the genes for the conversion of 4-nitrobenzoate to protocatechuate was unveiled by transposome mutagenesis. Reverse transcription-PCR analysis showed the polycistronic nature of the gene clusters, and their importance in the degradation of 2-and 4-nitrobenzoates was ascertained by gene knockout analysis. Cloning and expression of the relevant pathway genes revealed the transformation of 2-nitrobenzoate to 3-hydroxyanthranilate and of 4-nitrobenzoate to protocatechuate. Finally, incorporation of functional 3-nitrobenzoate dioxygenase into strain ST-14 allowed the recombinant strain to utilize 3-nitrobenzoate via the existing protocatechuate metabolic pathway, thereby allowing the degradation of all three isomers of mononitrobenzoate by a single bacterial strain. IMPORTANCEMononitrobenzoates are toxic chemicals largely used for the production of various value-added products and enter the ecosystem through industrial wastes. Bacteria capable of degrading mononitrobenzoates are relatively limited. Unlike other contaminants, these man-made chemicals have entered the environment since the last century, and it is believed that bacteria in nature evolved not quite efficiently to assimilate these compounds; as a consequence, to date, there are only a few reports on the bacterial degradation of one or more isomers of mononitrobenzoate. In the present study, fortunately, we have been able to isolate a Cupriavidus sp. strain capable of assimilating both 2-and 4-nitrobenzoates as the sole carbon source. Results of the biochemical and molecular characterization of catabolic genes responsible for the degradation of mononitrobenzoates led us to manipulate a single enzymatic step, allowing the recombinant host organism to expand its catabolic potential to assimilate 3-nitrobenzoate.

The gene encoding a nonoxidative decarboxylase capable of catalyzing the transformation of 2-hydroxy-1-naphthoic acid (2H1NA) to 2-naphthol was identified, recombinantly expressed, and purified to homogeneity. The putative gene sequence of the decarboxylase (hndA) encodes a 316-amino-acid protein (HndA) with a predicted molecular mass of 34 kDa. HndA exhibited high identity with uncharacterized amidohydrolase 2 proteins of various Burkholderia species, whereas it showed a modest 27% identity with ␥-resorcylate decarboxylase, a well-characterized nonoxidative decarboxylase belonging to the amidohydrolase superfamily. Biochemically characterized HndA demonstrated strict substrate specificity toward 2H1NA, whereas inhibition studies with HndA indicated the presence of zinc as the transition metal center, as confirmed by atomic absorption spectroscopy. A three-dimensional structural model of HndA, followed by docking analysis, identified the conserved metal-coordinating and substrate-binding residues, while their importance in catalysis was validated by site-directed mutagenesis. IMPORTANCEMicrobial nonoxidative decarboxylases play a crucial role in the metabolism of a large array of carboxy aromatic chemicals released into the environment from a variety of natural and anthropogenic sources. Among these, hydroxynaphthoic acids are usually encountered as pathway intermediates in the bacterial degradation of polycyclic aromatic hydrocarbons. The present study reveals biochemical and molecular characterization of a 2-hydroxy-1-naphthoic acid nonoxidative decarboxylase involved in an alternative metabolic pathway which can be classified as a member of the small repertoire of nonoxidative decarboxylases belonging to the amidohydrolase 2 family of proteins. The strict substrate specificity and sequence uniqueness make it a novel member of the metallo-dependent hydrolase superfamily. Decarboxylase is one of the most important classes of enzymes involved in a large variety of catabolic and anabolic pathways. The majority of the decarboxylases utilize an organic cofactor or a transition metal coupled with dioxygen to activate their substrates leading to the removal of carbon dioxide (1). However, there is a small group of transition metal-dependent decarboxylases that carry out decarboxylation of various aromatic acids in a nonoxidative manner. These nonoxidative decarboxylases act on various lignin-derived compounds, such as 4-hydroxybenzoic acid (2), (carboxy)vanillic acid (3, 4), protocatechuic acid (5), ferulic acid (6), p-coumaric acid (7), and estrogenic phthalate (8). Likewise, oxygen-independent decarboxylases are also involved in the 2-nitrobenzoic acid degradation pathway (9, 10), the tryptophan catabolic pathway (11), and the thymidine salvage pathway (12).Nonoxidative decarboxylases, in general, can broadly be classified into two major groups depending on their oxygen sensitivity. Oxygen-sensitive decarboxylases, viz., 4-hydroxybenzoate decarboxylase (2), 3,4-dihydroxybenzoate decarboxylase (5), and indol...

Burkholderia sp. strain BC1, a soil bacterium, isolated from a naphthalene balls manufacturing waste disposal site, is capable of utilizing 2-hydroxy-1-naphthoic acid (2H1NA) and naphthalene individually as the sole source of carbon and energy. To deduce the pathway for degradation of 2H1NA, metabolites isolated from resting cell culture were identified by a combination of chromatographic and spectrometric analyses. Characterization of metabolic intermediates, oxygen uptake studies and enzyme activities revealed that strain BC1 degrades 2H1NA via 2-naphthol, 1,2,6-trihydroxy-1,2-dihydronaphthalene and gentisic acid. In addition, naphthalene was found to be degraded via 1,2-dihydroxy-1,2-dihydronaphthalene, salicylic acid and gentisic acid, with the putative involvement of the classical nag pathway. Unlike most other Gram-negative bacteria, metabolism of salicylic acid in strain BC1 involves a dual pathway, via gentisic acid and catechol, with the latter being metabolized by catechol 1,2-dioxygenase. Involvement of a nonoxidative decarboxylase in the enzymic transformation of 2H1NA to 2-naphthol indicates an alternative catabolic pathway for the bacterial degradation of hydroxynaphthoic acid. Furthermore, the biochemical observations on the metabolism of structurally similar compounds, naphthalene and 2-naphthol, by similar but different sets of enzymes in strain BC1 were validated by real-time PCR analyses.