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ENZYMES AND PROTEINS

Experimental Evolution of Enzyme Temperature Activity Profile: Selection In Vivo and Characterization of Low-Temperature-Adapted Mutants of Pyrococcus furiosus Ornithine Carbamoyltransferase

Martine Roovers, Rony Sanchez, Christianne Legrain, Nicolas Glansdorff
Martine Roovers
Department of Microbiology, Flanders Interuniversity Institute for Biotechnology (VIB),
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Rony Sanchez
Department of Microbiology, Flanders Interuniversity Institute for Biotechnology (VIB),
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Christianne Legrain
Research Institute J. M. Wiame,B-1070 Brussels, Belgium
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Nicolas Glansdorff
Department of Microbiology, Flanders Interuniversity Institute for Biotechnology (VIB),
Research Institute J. M. Wiame,B-1070 Brussels, Belgium
Department of Microbiology, Free University of Brussels (VUB), and
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DOI: 10.1128/JB.183.3.1101-1105.2001
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ABSTRACT

We have obtained mutants of Pyrococcus furiosusornithine carbamoyltransferase active at low temperatures by selecting for complementation of an appropriate yeast mutant after in vivo mutagenesis. The mutants were double ones, still complementing at 15°C, a temperature already in the psychrophilic range. Their kinetic analysis is reported.

Thermodynamics predicts that substantial increases in the thermostability of any enzyme may require only a small number of structural changes (9). However, in most cases it remains difficult to predict which modification will increase stability while keeping the enzyme functional, since no general strategy for thermophilic adaptation appears to exist (10, 22). The same uncertainty prevails for the converse adaptation, psychrophily. Data about cold-active enzymes are still limited but it already appears that efficient catalysis at low temperature may rely on different strategies which are not simply the mirror image of adaptation to heat (4, 7, 21). From the biological point of view the most relevant approach to bypass the problems raised by engineering alterations of the enzyme temperature profile in vitro is experimental evolution, where selection of phenotypes (and not screening) is combined with natural mechanisms of mutagenesis.

Few examples of in vivo selection for a modified enzyme temperature profile have been reported. In most cases, thermostable variants of a mesophilic enzyme were selected in a thermophilic host (1, 8, 16, 23). The selection in a mesophilic host (Escherichia coli) for variants of a thermophilic enzyme becoming more active at lower temperatures was described only recently (17). In vivo selection of such mutants may become useful to modify enzymes but mainly, from an evolutionary perspective, to understand how readily and in which ways existing structures can adapt to lower temperatures.

We report the selection of modified ornithine carbamoyltransferase (OTCase) from Pyrococcus furiosus (an archaeon with a maximal growth rate at 102°C), which complements a null OTCase mutant of the Saccharomyces cerevisiae strain 12S16 (αura3 Δarg3 leu2) at 30 and even 15°C, achieving in one step the largest temperature jump presently obtained by experimental evolution.

OTCase catalyzes the conversion of ornithine and carbamoylphosphate into citrulline and inorganic phosphate in arginine biosynthesis.P. furiosus catalyzes citrulline synthesis about 35 times faster at 100 than at 30°C (14). The enzyme is a homododecamer, a key feature of its thermostability (24). The corresponding gene, argF (20), was amplified by PCR using primers (5′-GCGAATTCATGGTAGTTAGCTTGGC-3′ and 5′-GCGAATTCCTAAAGAATAGAGGGTG-3′) with extensions carrying EcoRI and SalI recognition sites, respectively, and was inserted in two monocopy E. coli/S. cerevisiae shuttle expression vectors (R&D Systems): pYX112 (with the strong constitutive triose phosphate isomerase promoter) and pYX111 (with the weak constitutive promoter 786). The constructs were made inE. coli and transformed into yeast strain 12S16. Complementation of the Δarg3 mutation was observed at 40, 30, and 15°C with pYX112 but not with pYX111, which remained negative at all three temperatures, thus providing a suitable material for selection of cold-active P. furiosus OTCase mutants.

The pYX111 argF plasmid was transformed into the E. coli XL1-Red mutator strain (mutS mutT mutD), deficient in three primary DNA repair pathways (Stratagene). Plasmid DNA was recovered from a pool of about 30,000 colonies. Fragments bearing only the argF gene were separated from mutations in vector DNA by recloning in pYX111, and the resulting library was used to select for 12S16 transformants growing at 30°C in the absence of arginine. Two double mutants (dm1 and dm2) were found to complement the yeast Δarg3 strain: TAC to TGC plus GAG to GGG for dm1 (i.e., Y227C plus E277G) and GCT to GAT plus GAG to GGG for dm2 (i.e., A240D plus E277G). They thus share the E277G substitution. All three mutations affect the ornithine binding domain of the OTCase monomer (Fig. 1). The three single mutants still complemented 12S16, though to a lesser extent (see serial dilution tests displayed in Fig. 2). Interestingly, complementation still occurred at 15°C. In liquid minimal medium without arginine, generation times at 30°C were about 6 h for the double mutants and 10 h for the single mutants, compared with 2 h for strain 12S16 in the presence of arginine.

Fig. 1.
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Fig. 1.

Structural representation of the P. furiosusOTCase monomer. The amino acids that have been replaced in the low-temperature-active mutants are indicated.

Fig. 2.
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Fig. 2.

Effects of mutations on cell growth. Serial tenfold dilutions of yeast cells were plated on minimal medium with or without arginine and grown at 30°C for 3 days or at 15°C for 7 days.

High levels of enzyme were achieved by inserting the cognate genes into pYX213, which contained the inducible gal1 promoter. OTCase was purified (using successively a monoQ and an arginine-Sepharose column [Pharmacia] [14]) from 2-liter cultures grown in the presence of 2% galactose. From a calibrated Superose P12 HR 10/30 column (Pharmacia), the double mutant enzymes eluted at the same position as the wild-type (WT) OTCase, corresponding to a molecular mass of 400 ± 20 kDa.

The effect of temperature on citrulline synthesis was studied with purified enzyme between 22 and 55°C (Fig.3), with CP thermolability (13) preventing assays at higher temperatures. Activation energies were, respectively, 42 kJ/mol for mutant Y227C + E277G, 57 kJ/mol for mutant A240D + E277G, and 48 kJ/mol for WT P. furiosus OTCase (as for OTCase purified from P. furiosus cells [15]).

Fig. 3.
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Fig. 3.

Temperature dependence of P. furiosus OTCase dm1 and dm2 compared to the WT. Open square (□), WT; full square (■), dm1; triangle (▴), dm2.

The Kmapp for carbamoylphosphate was similar in WT and mutated P. furiosus OTCases (about 0.1 mM). For ornithine, however, the affinity varied considerably among mutant OTCases at 55°C (Table1). Mutation Y227C had only a marginal influence, whereas mutations A240D and E277G resulted in a 10- to 14-fold increase in Kmapp compared to WT OTCase. In dm2, where both mutations are present, the effect was much more pronounced.

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Table 1.

Kinetic behavior and stability of WT and mutant OTCases

At 30°C, the turnover number (kcat) of the double mutants was about six times higher than for the wild type (eight times higher at 55°C) and for the single mutants, 1.5-fold higher (two to three times higher at 55°C). This explains why only the double mutants were initially selected. Catalytic efficiencies (kcat/Km) at 55°C decreased 2- to 15-fold, mainly due to the increase in the respectiveKm values. Interestingly, higher catalytic efficiencies were observed at 30 than at 55°C for both double mutants (Table 1).

Efficient catalysis at low temperature would appear to require enzymes with a higher kcat and/orkcat/Km ratio than their mesophilic or thermophilic homologues in order to compensate for the reduction of reaction rates at low temperature (4, 7). However, it may be difficult to both increase thekcat and decrease the Kmof an enzyme. Moreover, an increase in Km may actually lower the activation energy of the reaction (4). One may thus expect some enzymes to have adopted this strategy when it was compatible with the requirements of metabolism, as in the present case, when forced accumulation of the metabolic precursor ornithine allows the selection to play mainly on the kcat.

An increase in enzyme activity at low temperature is expected to result from an increase of flexibility, at least at the active site if not in the whole structure. Indeed, the frequent (but not universal [3]) thermolability of cold-adapted enzymes is regarded as a consequence of their flexibility. Double mutants dm1 and dm2 show a dramatic increase in OTCase thermolability (Table 1). The substitutions reported here are located in the ornithine-binding domain of the OTCase monomer (Fig. 1) (15, 24). Glutamic acid 277, which is substituted for in both double mutants, is highly conserved among OTCases (11). This residue interacts with the peptide backbone of histidine 273 by a hydrogen bond. H273 itself lies in the close vicinity of the tetrad H268CLP, which together with the triad SMG defines the ornithine-binding domain of OTCases (25). The absence of this H bond in the E277G mutant may increase the flexibility of the HCLP configuration. This could be the basis of the observed increase inKmapp Orn and an important feature of adaptation to lower temperatures.

In wild-type OTCase Y227 probably forms an internal aromatic interaction with Y159 since both phenyl ring planes are perpendicular and separated by 4.5 Å (24). A reduction in aromatic interactions may be related to protein adaptation at low temperatures (2). Therefore, the Y227C substitution could partly destabilize OTCase. Moreover, the shorter side chain of the cysteine residue introduces a cavity within the core of the protein, which could also lower enzyme stability by increasing flexibility (19). Note that a shortening of side chain is also observed in the E277G substitution.

Mutation A240D is less readily interpretable without further studies. Indeed, it slightly increases the thermoresistance of OTCase and, when present in dm2 (A240D plus E277G), enhances the thermoresistance of the enzyme with respect to mutation E277G alone. A240, a nonconserved residue, is situated in a turn exposed to the solvent (24); the substitution gives an additional acidic surface residue. This could improve solvent interactions which, as in class C β-lactamase from Psychrobacter immobilis A5 (5), appear to be a determinant of flexibility. On the other hand, the negative charge of the aspartate residue could result in a stabilizing electrostatic interaction with R244, which in the wild-type enzyme interacts with E276, in the close vicinity of the residues involved in ornithine binding (24). Consequently, ornithine binding could be affected in this mutant and the new electrostatic interaction could increase thermoresistance. Thus in the case of A240D, a possible local gain of flexibility by an increased interaction with the solvent would appear compatible with an increase of thermoresistance. Narinx and coworkers also reported a mutant of a cold-active subtilisin which, besides improved specific activity at low temperatures, displayed increased stability as well (18). A twofold increase in low temperature catalysis of P. furiosus β-glucosidase CelB was also obtained without loss of stability (12). Interestingly, the two single mutants Y227C and A240D differ dramatically in thermostability despite their similar kinetic behavior. Catalytic activity and resistance to thermodenaturation can thus be separated in P. furiosusOTCase, implying that they can involve different regions of the protein. Indeed, the two residues are located in distinct parts of the protein, Y227 being in the interior and A240 at the surface of the enzyme.

The very possibility of modifying so extensively the temperature activity profile of an enzyme by only two mutations is interesting from the point of view of evolution. Moreover, if adverse temperature could, like other kinds of physiological disturbances (6), transiently increase the rate of mutation, it would become conceivable that a succession of moderate temperature stresses could lead to the accumulation of mutations in a number of critical, limiting steps at a rate sufficient to bring global adaptation to another temperature range within closer reach than generally assumed.

ACKNOWLEDGMENTS

This work was supported by a grant from the Flanders Scientific Fund (FWO).

We thank M. Demarez for his help with purification and B. Clantin and V. Villeret for assistance in structural interpretation.

FOOTNOTES

    • Received 26 June 2000.
    • Accepted 25 October 2000.
  • Copyright © 2001 American Society for Microbiology

REFERENCES

  1. 1.↵
    1. Akanuma S.,
    2. Yamagishi A.,
    3. Tanaka N.,
    4. Oshima T.
    Serial increase in the thermal stability of 3-isopropylmalate dehydrogenase from Bacillus subtilis by experimental evolution.Protein Sci.71998698705
    OpenUrlCrossRefPubMedWeb of Science
  2. 2.↵
    1. Davail S.,
    2. Feller G.,
    3. Narinx E.,
    4. Gerday C.
    Cold adaptation of proteins.J. Biol. Chem.2619941744817453
    OpenUrl
  3. 3.↵
    1. Di Fraia R.,
    2. Wilquet V.,
    3. Ciardiello M. A.,
    4. Carratore V.,
    5. Antignani A.,
    6. Camardella L.,
    7. Glansdorff N.,
    8. di Prisco G.
    NADP+-dependent glutamate dehydrogenase in the Antarctic psychrotolerant bacterium Psychrobacter sp. TAD1. Characterization, protein and DNA sequence, and relationship to other glutamate dehydrogenases.Eur. J. Biochem.2672000121131
    OpenUrlPubMed
  4. 4.↵
    1. Feller G.,
    2. Gerday C.
    Psychrophilic enzymes: molecular basis of cold adaptation.Cell. Mol. Life Sci.531997830841
    OpenUrlCrossRefPubMedWeb of Science
  5. 5.↵
    1. Feller G.,
    2. Zekhnini Z.,
    3. Lamotte-Brasseur J.,
    4. Gerday C.
    Enzymes from cold-adapted microorganisms. The class C β-lactamase from the antarctic psychrophile Psychrobacter immobilis A5.Eur. J. Biochem.2441997186191
    OpenUrlPubMedWeb of Science
  6. 6.↵
    1. Foster P. L.
    Adaptive mutation: the uses of adversity.Annu. Rev. Microbiol.471993467504
    OpenUrlCrossRefPubMedWeb of Science
  7. 7.↵
    1. Gerday C.,
    2. Aittaleb M.,
    3. Arpigny J.,
    4. Baise E.,
    5. Chessa J.,
    6. François J.,
    7. Garsoux G.,
    8. Petrescu I.,
    9. Feller G.
    Cold enzymes: a hot topic in cold-adapted organisms Ecology, physiology, enzymology and molecular biology. Margesin R., Schinner F. 1999 257 275 Springer-Verlag Berlin, Germany
  8. 8.↵
    1. Hoseki J.,
    2. Yano T.,
    3. Koyama Y.,
    4. Kuramitsu S.,
    5. Kagamiyama H.
    Directed evolution of thermostable kanamycin-resistance gene: a convenient selection marker for Thermus thermophilus.J. Biochem.1261999951956
    OpenUrlCrossRefPubMedWeb of Science
  9. 9.↵
    1. Jaenicke R.
    What ultrastable globular proteins teach us about protein stabilization.Biochemistry631998312321
    OpenUrlPubMed
  10. 10.↵
    1. Jaenicke R.,
    2. Böhm G.
    The stability of proteins in extreme environments.Curr. Opin. Struct. Biol.81998738748
    OpenUrlCrossRefPubMedWeb of Science
  11. 11.↵
    1. Labedan B.,
    2. Boyen A.,
    3. Baetens M.,
    4. Charlier D.,
    5. Chen P.,
    6. Cunin R.,
    7. Durbecq V.,
    8. Glansdorff N.,
    9. Hervé G.,
    10. Legrain C.,
    11. Liang Z.,
    12. Purcarea C.,
    13. Roovers M.,
    14. Sanchez R.,
    15. Toong T.-L.,
    16. Van de Casteele M.,
    17. Van Vliet F.,
    18. Xu Y.,
    19. Zhang Y.-F.
    The evolutionary history of carbamoyltransferases: a complex set of paralogous genes was already present in the last universal common ancestor.J. Mol. Evol.491999461473
    OpenUrlCrossRefPubMedWeb of Science
  12. 12.↵
    1. Lebbink J.,
    2. Kaper T.,
    3. Bron P.,
    4. van der Oost J.,
    5. de Vos W.
    Improving low-temperature catalysis in the hyperthermostable Pyrococcus furiosus β-glucosidase CelB by directed evolution.Biochemistry39200036563665
    OpenUrlCrossRefPubMed
  13. 13.↵
    1. Legrain C.,
    2. Demarez M.,
    3. Glansdorff N.,
    4. Piérard A.
    Ammonia-dependent synthesis and metabolic channeling of carbamoylphosphate in the hyperthermophilic archeaon Pyrococcus furiosus.Microbiology141199510931099
    OpenUrlCrossRefWeb of Science
  14. 14.↵
    Legrain, C., V. Villeret, M. Roovers, C. Tricot, B. Clantin, J. Van Beeumen, V. Stalon, and N. Glansdorff. The ornithine carbamoyltransferase of Pyrococcus furiosus. Methods Enzymol., in press.
  15. 15.↵
    1. Legrain C.,
    2. Villeret V.,
    3. Roovers M.,
    4. Gigot D.,
    5. Dideberg O.,
    6. Piérard A.,
    7. Glansdorff N.
    Biochemical characterisation of ornithine carbamoyltransferase from Pyrococcus furiosus.Eur. J. Biochem.247199710461055
    OpenUrlPubMedWeb of Science
  16. 16.↵
    1. Liao H.,
    2. McKenzie T.,
    3. Hageman R.
    Isolation of a thermostable enzyme variant by cloning and selection in a thermophile.Proc. Natl. Acad. Sci. USA831986576580
    OpenUrlAbstract/FREE Full Text
  17. 17.↵
    1. Merz A.,
    2. Yee M.-C.,
    3. Szadkowski H.,
    4. Pappenberger G.,
    5. Crameri A.,
    6. Stemmer W.,
    7. Yanofsky C.,
    8. Kirschner K.
    Improving the catalytic activity of a thermophilic enzyme at low temperatures.Biochemistry392000880889
    OpenUrlCrossRefPubMed
  18. 18.↵
    1. Narinx E.,
    2. Baise E.,
    3. Gerday C.
    Subtilisin from psychrophilic Antarctic bacteria: characterization and site-directed mutagenesis of residues possibly involved in the adaptation to cold.Protein Eng.10199712711279
    OpenUrlCrossRefPubMedWeb of Science
  19. 19.↵
    1. Rashin A. A.,
    2. Iofin M.,
    3. Honig B.
    Internal cavities and buried waters in globular proteins.Biochemistry25198636193625
    OpenUrlCrossRefPubMed
  20. 20.↵
    1. Roovers M.,
    2. Hethke C.,
    3. Legrain C.,
    4. Thomm M.,
    5. Glansdorff N.
    Isolation of the gene encoding Pyrococcus furiosus ornithine carbamoyltransferase and study of its expression profile in vivo and in vitro.Eur. J. Biochem.247199710381047
    OpenUrlPubMedWeb of Science
  21. 21.↵
    1. Russell N. J.
    Toward a molecular understanding of cold activity of enzymes from psychrophiles.Extremophiles420008390
    OpenUrlCrossRefPubMed
  22. 22.↵
    1. Scandurra R.,
    2. Consalvi V.,
    3. Chiaraluce R.,
    4. Politi L.,
    5. Engel P.
    Protein thermostability in extremophiles.Biochimie801998933941
    OpenUrlPubMed
  23. 23.↵
    1. Tamakoshi M.,
    2. Yamagishi A.,
    3. Oshima T.
    Screening of stable proteins in an extreme thermophile, Thermus thermophilus.Mol. Microbiol.16199510311036
    OpenUrlCrossRefPubMedWeb of Science
  24. 24.↵
    1. Villeret V.,
    2. Clantin B.,
    3. Tricot C.,
    4. Legrain C.,
    5. Roovers M.,
    6. Stalon V.,
    7. Glansdorff N.,
    8. Van Beeumen J.
    The crystal structure of Pyrococcus furiosus ornithine carbamoyltransferase reveals a key role for oligomerization in enzyme stability at extremely high temperatures.Proc. Natl. Acad. Sci. USA95199828012806
    OpenUrlAbstract/FREE Full Text
  25. 25.↵
    1. Villeret V.,
    2. Tricot C.,
    3. Stalon V.,
    4. Dideberg O.
    Crystal structure of Pseudomonas aeruginosa catabolic ornithine transcarbamoylase at 3.0-A resolution: a different oligomeric organization in the transcarbamoylase family.Proc. Natl. Acad. Sci. USA9219951076210766
    OpenUrlAbstract/FREE Full Text
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Experimental Evolution of Enzyme Temperature Activity Profile: Selection In Vivo and Characterization of Low-Temperature-Adapted Mutants of Pyrococcus furiosus Ornithine Carbamoyltransferase
Martine Roovers, Rony Sanchez, Christianne Legrain, Nicolas Glansdorff
Journal of Bacteriology Feb 2001, 183 (3) 1101-1105; DOI: 10.1128/JB.183.3.1101-1105.2001

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Experimental Evolution of Enzyme Temperature Activity Profile: Selection In Vivo and Characterization of Low-Temperature-Adapted Mutants of Pyrococcus furiosus Ornithine Carbamoyltransferase
Martine Roovers, Rony Sanchez, Christianne Legrain, Nicolas Glansdorff
Journal of Bacteriology Feb 2001, 183 (3) 1101-1105; DOI: 10.1128/JB.183.3.1101-1105.2001
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KEYWORDS

Adaptation, Biological
Evolution, Molecular
mutation
Ornithine Carbamoyltransferase
Pyrococcus furiosus

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