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Extensive lysine methylation in hyperthermophilic crenarchaea: potential implications for protein stability and recombinant enzymes.

ABSTRACT: In eukarya and bacteria, lysine methylation is relatively rare and is catalysed by sequence-specific lysine methyltransferases that typically have only a single-protein target. Using RNA polymerase purified from the thermophilic crenarchaeum Sulfolobus solfataricus, we identified 21 methyllysines distributed across 9 subunits of the enzyme. The modified lysines were predominantly in alpha-helices and showed no conserved sequence context. A limited survey of the Thermoproteus tenax proteome revealed widespread modification with 52 methyllysines in 30 different proteins. These observations suggest the presence of an unusual lysine methyltransferase with relaxed specificity in the crenarchaea. Since lysine methylation is known to enhance protein thermostability, this may be an adaptation to a thermophilic lifestyle. The implications of this modification for studies and applications of recombinant crenarchaeal enzymes are discussed.

SUBMITTER: Botting CH

PROVIDER: S-EPMC2929605 | biostudies-literature | 2010 Aug

REPOSITORIES: biostudies-literature

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Extensive lysine methylation in hyperthermophilic crenarchaea: potential implications for protein stability and recombinant enzymes.

Botting Catherine H CH Talbot Paul P Paytubi Sonia S White Malcolm F MF

Archaea (Vancouver, B.C.) 20100805

In eukarya and bacteria, lysine methylation is relatively rare and is catalysed by sequence-specific lysine methyltransferases that typically have only a single-protein target. Using RNA polymerase purified from the thermophilic crenarchaeum Sulfolobus solfataricus, we identified 21 methyllysines distributed across 9 subunits of the enzyme. The modified lysines were predominantly in alpha-helices and showed no conserved sequence context. A limited survey of the Thermoproteus tenax proteome revea ...[more]

PMID: 20811616

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Project description:BackgroundHorseradish Peroxidase (HRP) plays important roles in many biotechnological fields, including diagnostics, biosensors and biocatalysis. Often, it is used in immobilised form. With conventional immobilisation techniques, the enzyme adheres in random orientation: the active site may face the solid phase rather than bulk medium, impeding substrate access and leading to sub-optimal catalytic performance. The ability to immobilise HRP in a directional manner, such that the active site would always face outwards from the insoluble matrix, would maximise the immobilised enzyme's catalytic potential and could increase HRP's range of actual and potential applications.ResultsWe have replaced arginine residues on the face of glycan-free recombinant HRP opposite to the active site by lysines. Our strategy differs from previous reports of specific HRP immobilisation via an engineered affinity tag or single reactive residue. These conservative Arg-to-Lys substitutions provide a means of multipoint covalent immobilisation such that the active site will always face away from the immobilisation matrix. One triple and one pentuple mutant were generated by substitution of solvent-exposed arginines on the "back" of the polypeptide (R118, R159 and R283) and of residues known to influence stability (K232 and K241). Orientated HRP immobilisation was demonstrated using a modified polyethersulfone (PES) membrane; the protein was forced to orientate its active site away from the membrane and towards the bulk solution phase. Mutant properties and bioinformatic analysis suggested the reversion of K283R to improve stability, thus generating two additional mutants (K118/R159K and R118K/K232N/K241F/R283K). While most mutants were less stable in free solution than wild type rHRP, the quadruple revertant regained some stability over its mutant counterparts. A greater degree of immobilisation on CNBr-activated Sepharosetrade mark was noted with increased lysine content; however, only marginal gains in solvent stability resulted from immobilisation on this latter matrix.ConclusionDirectional, orientated, immobilisation of rHRP mutants onto an activated, modified polyethersulfone membrane has been achieved with excellent retention of catalytic activity; however, re-engineering of acceptable stability characteristics into the "immobilisation mutants" will determine their applicability in diagnosis and biosensor development.

Dataset Information

Extensive lysine methylation in hyperthermophilic crenarchaea: potential implications for protein stability and recombinant enzymes.

Publications

Extensive lysine methylation in hyperthermophilic crenarchaea: potential implications for protein stability and recombinant enzymes.

Similar Datasets

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