Computational Thermostabilization of an Enzyme

Thermostabilizing an enzyme while maintaining its activity for industrial or biomedical applications can be difficult with traditional selection methods. We describe a rapid computational approach that identified three mutations within a model enzyme that produced a 10°C increase in apparent melting temperature Tm and a 30-fold increase in half-life at 50°C, with no reduction in catalytic efficiency. The effects of the mutations were synergistic, giving an increase in excess of the sum of their individual effects. The redesigned enzyme induced an increased, temperature-dependent bacterial growth rate under conditions that required its activity, thereby coupling molecular and metabolic engineering.

[1]  Robert Blair Vocci.  Geology , 1882, Nature.

[2]  F. Young.  Biochemistry , 1955, The Indian Medical Gazette.

[3]  Purification and Some Properties of Cytosine Deaminase from Bakers’ Yeast , 1989 .

[4]  D C Rees,et al.  Hyperthermophiles: taking the heat and loving it. , 1995, Structure.

[5]  S. L. Mayo,et al.  De novo protein design: fully automated sequence selection. , 1997, Science.

[6]  Roland L. Dunbrack,et al.  Bayesian statistical analysis of protein side‐chain rotamer preferences , 1997, Protein science : a publication of the Protein Society.

[7]  R. A. Scott,et al.  Dissecting contributions to the thermostability of Pyrococcus furiosus rubredoxin: beta-sheet chimeras. , 1997, Biochemistry.

[8]  Stephen L. Mayo,et al.  Design, structure and stability of a hyperthermophilic protein variant , 1998, Nature Structural Biology.

[9]  P. Engel,et al.  Protein thermostability in extremophiles. , 1998, Biochimie.

[10]  V. Schramm.  Enzymatic transition states and transition state analog design. , 1998, Annual review of biochemistry.

[11]  B. Dahiyat,et al.  In silico design for protein stabilization. , 1999, Current opinion in biotechnology.

[12]  S. A. Marshall,et al.  Energy functions for protein design. , 1999, Current opinion in structural biology.

[13]  M. Karplus,et al.  Effective energy function for proteins in solution , 1999, Proteins.

[14]  S. L. Mayo,et al.  Computational protein design. , 1999, Structure.

[15]  A. Rehemtulla,et al.  Superiority of yeast over bacterial cytosine deaminase for enzyme/prodrug gene therapy in colon cancer xenografts. , 1999, Cancer research.

[16]  D. Baker,et al.  Native protein sequences are close to optimal for their structures. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[17]  J R Desjarlais,et al.  Computational protein design. , 2001, Current opinion in chemical biology.

[18]  G. Dachs,et al.  Gene directed enzyme/prodrug therapy of cancer: Historical appraisal and future prospectives , 2001, Journal of cellular physiology.

[19]  R. Sterner,et al.  Thermophilic Adaptation of Proteins , 2001, Critical reviews in biochemistry and molecular biology.

[20]  Raphael Guerois,et al.  Energy estimation in protein design. , 2002, Current opinion in structural biology.

[21]  D. E. Benson,et al.  Converting a maltose receptor into a nascent binuclear copper oxygenase by computational design. , 2002, Biochemistry.

[22]  Anirban Kundu,et al.  Development of a cytokine analog with enhanced stability using computational ultrahigh throughput screening , 2002, Protein science : a publication of the Protein Society.

[23]  Julia M. Shifman,et al.  Modulating calmodulin binding specificity through computational protein design. , 2002, Journal of molecular biology.

[24]  L. Looger,et al.  Computational design of receptor and sensor proteins with novel functions , 2003, Nature.

[25]  K. Mace,et al.  Engineering the proteolytic specificity of activated protein C improves its pharmacological properties , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[26]  Daniel Herschlag,et al.  Challenges in enzyme mechanism and energetics. , 2003, Annual review of biochemistry.

[27]  D. Baker,et al.  A large scale test of computational protein design: folding and stability of nine completely redesigned globular proteins. , 2003, Journal of molecular biology.

[28]  B. Stoddard,et al.  The 1.14 A crystal structure of yeast cytosine deaminase: evolution of nucleotide salvage enzymes and implications for genetic chemotherapy. , 2003, Structure.

[29]  D. Baker,et al.  Design of a Novel Globular Protein Fold with Atomic-Level Accuracy , 2003, Science.

[30]  Ceslovas Venclovas,et al.  Assessment of progress over the CASP experiments , 2003, Proteins.

[31]  Jeremy C. Smith,et al.  The role of dynamics in enzyme activity. , 2003, Annual review of biophysics and biomolecular structure.

[32]  V. Eijsink,et al.  Rational engineering of enzyme stability. , 2004, Journal of biotechnology.

[33]  Loren L Looger,et al.  Computational Design of a Biologically Active Enzyme , 2004, Science.

[34]  宁北芳,et al.  疟原虫var基因转换速率变化导致抗原变异[英]／Paul H, Robert P, Christodoulou Z, et al//Proc Natl Acad Sci U S A , 2005 .