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Characterization of enzymatic properties of two novel enzymes, 3,4-dihydroxyphenylacetate dioxygenase and 4-hydroxyphenylacetate 3-hydroxylase, from Sulfobacillus acidophilus TPY.

ABSTRACT: BACKGROUND:As an environmental pollutant, 4-hydroxyphenylacetate (4-HPA) was a product of softwood lignin decomposition and was found in industrial effluents from olive oil production. Sulfobacillus acidophilus TPY was a moderately thermoacidophilic bacterium capable of degrading aromatic compounds including 4-HPA. The enzymes involved in the degradation of 4-HPA and the role of this strain in the bioremediation of marine pollutants need to be illustrated. RESULTS:3,4-dihydroxyphenylacetate dioxygenase (DHPAO) encoded by mhpB2 and two components of 4-hydroxydroxyphenylacetate (4-HPA) 3-hydroxylase encoded by hpaB and hpaC from S. acidophilus TPY, a moderately thermoacidophilic bacterium, involved in the degradation of 4-HPA possessed quite low amino acid sequence identity (22-53%) with other ever reported corresponding enzymes, which suggest their novelty. These two enzymes were expressed in E. coli and purified to homogeneity. DHPAO activity in E. coli was revealed by spraying with catechol or 3,4-dihydroxyphenylacetate (3,4-DHPA) on the colonies to make them turn brilliant yellow color. DHPAO possessed total activity of 7.81?U and 185.95?U/mg specific activity at the first minute when 3,4-DHPA was served as substrate. DHPAO was a thermophilic enzyme with optimum temperature of 50?°C and optimum substrate of 3,4-DHPA. The small component (HpaC) was a flavoprotein, and both HpaB and HpaC of 4-HPA 3-hydroxylase were NADH-dependent and essential in the conversion of 4-HPA to 3,4-DHPA. 4-HPA 3-hydroxylase possessed 3.59?U total activity and 27.37?U/mg specific activity at the first minute when enzymatic coupled assay with DHPAO was applied in the enzymatic determination. CONCLUSIONS:The ability of this extreme environmental marine strain to degrade catechol and substituted catechols suggest its applications in the bioremediation of catechol and substituted catechols polluted marine environments.

SUBMITTER: Guo W

PROVIDER: S-EPMC6375179 | biostudies-literature | 2019 Feb

REPOSITORIES: biostudies-literature

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Characterization of enzymatic properties of two novel enzymes, 3,4-dihydroxyphenylacetate dioxygenase and 4-hydroxyphenylacetate 3-hydroxylase, from Sulfobacillus acidophilus TPY.

Guo Wenbin W Zhou Wengen W Zhou Hongbo H Chen Xinhua X

BMC microbiology 20190213 1

<h4>Background</h4>As an environmental pollutant, 4-hydroxyphenylacetate (4-HPA) was a product of softwood lignin decomposition and was found in industrial effluents from olive oil production. Sulfobacillus acidophilus TPY was a moderately thermoacidophilic bacterium capable of degrading aromatic compounds including 4-HPA. The enzymes involved in the degradation of 4-HPA and the role of this strain in the bioremediation of marine pollutants need to be illustrated.<h4>Results</h4>3,4-dihydroxyphe ...[more]

PMID: 30760216

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Project description:Sulfobacillus acidophilus TPY, isolated from a hydrothermal vent in the Pacific Ocean, is a moderately thermoacidophilic Gram-positive bacterium that can oxidize ferrous iron or sulfur compounds to obtain energy. In this study, comparative transcriptomic analyses of S. acidophilus TPY were performed under different redox conditions. Based on these results, pathways involved in sulfur metabolism were proposed. Additional evidence was obtained by analyzing mRNA abundance of selected genes involved in the sulfur metabolism of sulfur oxygenase reductase (SOR)-overexpressed S. acidophilus TPY recombinant under different redox conditions. Comparative transcriptomic analyses of S. acidophilus TPY cultured in the presence of ferrous sulfate (FeSO4) or elemental sulfur (S0) were employed to detect differentially transcribed genes and operons involved in sulfur metabolism. The mRNA abundances of genes involved in sulfur metabolism decreased in cultures containing elemental sulfur, as opposed to cultures in which FeSO4 was present where an increase in the expression of sulfur metabolism genes, particularly sulfite reductase (SiR) involved in the dissimilatory sulfate reduction, was observed. SOR, whose mRNA abundance increased in S0 culture, may play an important role in the initial sulfur oxidation. In order to confirm the pathways, SOR overexpression in S. acidophilus TPY and subsequent mRNA abundance analysis of sulfur metabolism-related genes were carried out. Conjugation-based transformation of pTrc99A derived plasmid from heterotrophic E. coli to facultative autotrophic S. acidophilus TPY was developed in this study. Transconjugation between E. coli and S. acidophilus was performed on modified solid 2:2 medium at pH 4.8 and 37°C for 72 h. The SOR-overexpressed recombinant S. acidophilus TPY-SOR had a [Formula: see text]-accumulation increase, higher oxidation/ reduction potentials (ORPs) and lower pH compared with the wild type strain in the late growth stage of S0 culture condition. The transcript level of sor gene in the recombinant strain increased in both S0 and FeSO4 culture conditions, which influenced the transcription of other genes in the proposed sulfur metabolism pathways. Overall, these results expand our understanding of sulfur metabolism within the Sulfobacillus genus and provide a successful gene-manipulation method.

Project description:β-Hydroxy-α-amino acids are useful compounds for pharmaceutical development. Enzymatic synthesis of β-hydroxy-α-amino acids has attracted considerable interest as a selective, sustainable, and environmentally benign process. In this study, we identified a novel amino acid hydroxylase, AEP14369, from Sulfobacillus thermotolerans Y0017, which is included in a previously constructed CAS-like superfamily protein library, to widen the variety of amino acid hydroxylases. The detailed structures determined by nuclear magnetic resonance and X-ray crystallography analysis of the enzymatically produced compounds revealed that AEP14369 catalyzed threo-β-selective hydroxylation of l-His and l-Gln in a 2-oxoglutarate-dependent manner. Furthermore, the production of l-threo-β-hydroxy-His and l-threo-β-hydroxy-Gln was achieved using Escherichia coli expressing the gene encoding AEP14369 as a whole-cell biocatalyst. Under optimized reaction conditions, 137 mM (23.4 g liter-1) l-threo-β-hydroxy-His and 150 mM l-threo-β-hydroxy-Gln (24.3 g liter-1) were obtained, indicating that the enzyme is applicable for preparative-scale production. AEP14369, an l-His/l-Gln threo-β-hydroxylase, increases the availability of 2-oxoglutarate-dependent hydroxylase and opens the way for the practical production of β-hydroxy-α-amino acids in the future. The amino acids produced in this study would also contribute to the structural diversification of pharmaceuticals that affect important bioactivities. IMPORTANCE Owing to an increasing concern for sustainability, enzymatic approaches for producing industrially useful compounds have attracted considerable attention as a powerful complement to chemical synthesis for environment-friendly synthesis. In this study, we developed a bioproduction method for β-hydroxy-α-amino acid synthesis using a newly discovered enzyme. AEP14369 from the moderate thermophilic bacterium Sulfobacillus thermotolerans Y0017 catalyzed the hydroxylation of l-His and l-Gln in a regioselective and stereoselective fashion. Furthermore, we biotechnologically synthesized both l-threo-β-hydroxy-His and l-threo-β-hydroxy-Gln with a titer of over 20 g liter-1 through whole-cell bioconversion using recombinant Escherichia coli cells. As β-hydroxy-α-amino acids are important compounds for pharmaceutical development, this achievement would facilitate future sustainable and economical industrial applications.

Dataset Information

Characterization of enzymatic properties of two novel enzymes, 3,4-dihydroxyphenylacetate dioxygenase and 4-hydroxyphenylacetate 3-hydroxylase, from Sulfobacillus acidophilus TPY.

Publications

Characterization of enzymatic properties of two novel enzymes, 3,4-dihydroxyphenylacetate dioxygenase and 4-hydroxyphenylacetate 3-hydroxylase, from Sulfobacillus acidophilus TPY.

Similar Datasets

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