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Gene mining, codon optimization and analysis of binding mechanism of an aldo-keto reductase with high activity, better substrate specificity and excellent solvent tolerance.

ABSTRACT: The biosynthesis of chiral alcohols has important value and high attention. Aldo-keto reductases (AKRs) mediated reduction of prochiral carbonyl compounds is an interesting way of synthesizing single enantiomers of chiral alcohols due to the high enantio-, chemo- and regioselectivity of the enzymes. However, relatively little research has been done on characterization and apply of AKRs to asymmetric synthesis of chiral alcohols. In this study, the AKR from Candida tropicalis MYA-3404 (C. tropicalis MYA-3404), was mined and characterized. The AKR shown wider optimum temperature and pH. The AKR exhibited varying degrees of catalytic activity for different substrates, suggesting that the AKR can catalyze a variety of substrates. It is worth mentioning that the AKR could catalytic reduction of keto compounds with benzene rings, such as cetophenone and phenoxyacetone. The AKR exhibited activity on N,N-dimethyl-3-keto-3-(2-thienyl)-1-propanamine (DKTP), a key intermediate for biosynthesis of the antidepressant drug duloxetine. Besides, the AKR still has high activity whether in a reaction system containing 10%-30% V/V organic solvent. What's more, the AKR showed the strongest stability in six common organic solvents, DMSO, acetonitrile, ethyl acetate, isopropanol, ethanol, and methanol. And, it retains more that 70% enzyme activity after 6 hours, suggesting that the AKR has strong solvent tolerance. Furthermore, the protein sequences of the AKR and its homology were compared, and a 3D model of the AKR docking with coenzyme NADPH were constructed. And the important catalytic and binding sites were identified to explore the binding mechanism of the enzyme and its coenzyme. These properties, predominant organic solvents resistance and extensive substrate spectrum, of the AKR making it has potential applications in the pharmaceutical field.

SUBMITTER: Jiang W

PROVIDER: S-EPMC8638942 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Aldo-keto reductases (AKRs) are phase I metabolising enzymes that catalyse the reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H)-dependent reduction of carbonyl groups to yield primary and secondary alcohols on a wide range of substrates, including aliphatic and aromatic aldehydes and ketones, ketoprostaglandins, ketosteroids and xenobiotics. In so doing they functionalise the carbonyl group for conjugation (phase II enzyme reactions). Although functionally diverse, AKRs form a protein superfamily based on their high sequence identity and common protein fold, the (alpha/beta) 8 -barrel structure. Well over 150 AKR enzymes, from diverse organisms, have been annotated so far and given systematic names according to a nomenclature that is based on multiple protein sequence alignment and degree of identity. Annotation of non-vertebrate AKRs at the National Center for Biotechnology Information or Vertebrate Genome Annotation (vega) database does not often include the systematic nomenclature name, so the most comprehensive overview of all annotated AKRs is found on the AKR website (http://www.med.upenn.edu/akr/). This site also hosts links to more detailed and specialised information (eg on crystal structures, gene expression and single nucleotide polymorphisms [SNPs]). The protein-based AKR nomenclature allows unambiguous identification of a given enzyme but does not reflect the wealth of genomic and transcriptomic variation that exists in the various databases. In this context, identification of putative new AKRs and their distinction from pseudogenes are challenging. This review provides a short summary of the characteristic features of AKR biochemistry and structure that have been reviewed in great detail elsewhere, and focuses mainly on nomenclature and database entries of human AKRs that so far have not been subject to systematic annotation. Recent developments in the annotation of SNP and transcript variance in AKRs are also summarised.

Project description:The aldo-keto reductases metabolize a wide range of substrates and are potential drug targets. This protein superfamily includes aldose reductases, aldehyde reductases, hydroxysteroid dehydrogenases and dihydrodiol dehydrogenases. By combining multiple sequence alignments with known three-dimensional structures and the results of site-directed mutagenesis studies, we have developed a structure/function analysis of this superfamily. Our studies suggest that the (alpha/beta)8-barrel fold provides a common scaffold for an NAD(P)(H)-dependent catalytic activity, with substrate specificity determined by variation of loops on the C-terminal side of the barrel. All the aldo-keto reductases are dependent on nicotinamide cofactors for catalysis and retain a similar cofactor binding site, even among proteins with less than 30% amino acid sequence identity. Likewise, the aldo-keto reductase active site is highly conserved. However, our alignments indicate that variation ofa single residue in the active site may alter the reaction mechanism from carbonyl oxidoreduction to carbon-carbon double-bond reduction, as in the 3-oxo-5beta-steroid 4-dehydrogenases (Delta4-3-ketosteroid 5beta-reductases) of the superfamily. Comparison of the proposed substrate binding pocket suggests residues 54 and 118, near the active site, as possible discriminators between sugar and steroid substrates. In addition, sequence alignment and subsequent homology modelling of mouse liver 17beta-hydroxysteroid dehydrogenase and rat ovary 20alpha-hydroxysteroid dehydrogenase indicate that three loops on the C-terminal side of the barrel play potential roles in determining the positional and stereo-specificity of the hydroxysteroid dehydrogenases. Finally, we propose that the aldo-keto reductase superfamily may represent an example of divergent evolution from an ancestral multifunctional oxidoreductase and an example of convergent evolution to the same active-site constellation as the short-chain dehydrogenase/reductase superfamily.

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Gene mining, codon optimization and analysis of binding mechanism of an aldo-keto reductase with high activity, better substrate specificity and excellent solvent tolerance.

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