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Employing non-canonical amino acids towards the immobilization of a hyperthermophilic enzyme to increase protein stability.

ABSTRACT: A carboxylesterase derived from Sulfolobus solfataricus P1 was immobilized onto an epoxy-activated Sepharose resin via non-canonical amino acids. The immobilized enzyme exhibited heightened performance in organic solvents, recyclability, and stability at room temperature for over two years. The incorporation of a non-canonical amino acid afforded a high degree of control over the bioorthogonal immobilization reaction. These results indicate that the specificity conferred by genetic code expansion produces advantages in protein immobilization and broadens the utility of such proteins to non-biological settings.

SUBMITTER: Switzer HJ

PROVIDER: S-EPMC10012417 | biostudies-literature | 2023 Mar

REPOSITORIES: biostudies-literature

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Employing non-canonical amino acids towards the immobilization of a hyperthermophilic enzyme to increase protein stability.

Switzer Hannah J HJ Howard Christina A CA Halonski John F JF Peairs Emily M EM Smith Nolan N Zamecnik Maddy P MP Verma Sanjana S Young Douglas D DD

RSC advances 20230314 13

A carboxylesterase derived from <i>Sulfolobus solfataricus</i> P1 was immobilized onto an epoxy-activated Sepharose resin <i>via</i> non-canonical amino acids. The immobilized enzyme exhibited heightened performance in organic solvents, recyclability, and stability at room temperature for over two years. The incorporation of a non-canonical amino acid afforded a high degree of control over the bioorthogonal immobilization reaction. These results indicate that the specificity conferred by genetic ...[more]

PMID: 36926306

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Project description:We have determined the nucleotide sequences of eight ethyl methanesulphonate-induced mutants in Drosophila alcohol dehydrogenase (ADH), of which six were previously characterized by Hollocher and Place [(1988) Genetics 116, 253-263 and 265-274]. Four of these ADH mutants contain a single amino acid change: glycine-17 to arginine, glycine-93 to glutamic acid, alanine-159 to threonine, and glycine-184 to aspartic acid. Although these mutants are inactive, three mutants (Gly17Arg, Gly93Glu and Gly184Asp) form stable homodimers, as well as heterodimers with wild-type ADH, in which the wild-type ADH subunit retains full enzyme activity [Hollocher and Place (1988) Genetics 116, 265-274]. Interestingly, the Ala159Thr mutant does not form either stable homodimers or heterodimers with wild-type ADH, suggesting that alanine-159 is important in stabilizing ADH dimers. The mutations were analysed in terms of a three-dimensional model of ADH using bacterial 20 beta-hydroxysteroid dehydrogenase and rat dihydropteridine reductase as templates. The model indicates that mutations in glycine-17 and glycine-93 affect the binding of NAD+. It also shows that alanine-159 is part of a hydrophobic anchor on the dimer interface of ADH. Replacement of alanine-159 with threonine, which has a larger side chain and can hydrogen bond with water, is likely to reduce the strength of the hydrophobic interaction. The three-dimensional model shows that glycine-184 is close to the substrate binding site. Replacement of glycine-184 with aspartic acid is likely to alter the position of threonine-186, which we propose hydrogen bonds to the carboxamide moiety of NAD+. Also, the negative charge on the aspartic acid side chain may interact with the substrate and/or residues in the substrate binding site. These mutations provide information about ADH catalysis and the stability of dimers, which may also be useful in understanding homologous dehydrogenases, which include the human 17 beta-hydroxysteroid, 11 beta-hydroxysteroid and 15-hydroxyprostaglandin dehydrogenases.

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Employing non-canonical amino acids towards the immobilization of a hyperthermophilic enzyme to increase protein stability.

Publications

Employing non-canonical amino acids towards the immobilization of a hyperthermophilic enzyme to increase protein stability.

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