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The Catalytic Plasticity of Enzymes in Higher Plants

The Catalytic Plasticity of Enzymes in Higher Plants. Plant Biochemistry Spring 2004. What is Catalytic Plasticity?. Functional “flexibility” or “promiscuity” of an enzyme  Catalyzes its primary reaction  Catalyzes one or more alternative reactions

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The Catalytic Plasticity of Enzymes in Higher Plants

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  1. The Catalytic Plasticity of Enzymes in Higher Plants Plant Biochemistry Spring 2004

  2. What is Catalytic Plasticity? • Functional “flexibility” or “promiscuity” of an enzyme  Catalyzes its primary reaction  Catalyzes one or more alternative reactions • A catalytically plastic enzyme can adopt an entirely different function with minor changes in its primary sequence or conformation.

  3. The Significance of Catalytic Plasticity • Catalytically plastic enzymes can be simply modified to trigger the evolution of enzymes with new functions • Conservation of energy normally used for protein synthesis Molecular Basis of Catalytic Plasticity • Catalytically plastic enzymes may adopt multiple conformations • Theoretically, each conformation could provide a different active site that can accept a different substrate • Therefore, each conformation may be able to catalyze a different reaction

  4. Catalytically Plastic Enzymes Aminoglycoside kinase • Movement of an amino acid loop  Creates 2 different active sites • Active sites capable of catalyzing the phosphorylation of aminoglycosides (ex. Kanamycin, Lividomycin) on different sites

  5. Catalytically Plastic Enzymes Trans-sialidase • Isomerization of a Tyr side chain in the active site  Conformational change Creates 2 different active sites • 1st active site: cleavage of sialic acid from sugar chains • 2nd active site: attachment of sialic acid to another substrate. Tyrosine Isomerization

  6. Catalytically Plastic Antibodies SPE7 (An antibody to dinitrophenol) • Exists in two radically different conformations • Each conformation is capable of binding different antigens • 1st conformation: deep and narrow cleft  binds dinitrophenol • 2nd conformation: flat surface  promiscuously binds a protein antigen Deep and Narrow Cleft: Binds Dinitrophenol Flat Surface: Binds a Protein Antigen

  7. Catalytic Plasticity of Fatty Acid Modification Enzymes • Enzymes that modify fatty acids have a high degree of structural similarity • Desaturases: Saturated FA  Monounsaturated FA Monounsaturated FA  Diunsaturated FA • Hydroxylases: FA  Hydroxy FA • Enzymes can be inter-converted by just a few amino acid substitutions • Higher plants: extensive diversity in the composition of seed-storage fatty acids

  8. Fatty Acid Diversity in Higher Plants

  9. Desaturases and Hydroxylases 9 9 12 12 Oleate 12-desaturase Linoleic acid (18:2Δ9,12) Oleic acid (18:1Δ9) 7 Amino Acid Substitutions 9 12 Oleate 12-hydroxylase Oleic acid (18:1Δ9) Ricinoleic acid (18:1Δ9-OH) 12 9

  10. Desaturases and Hydroxylases Oleate 12-desaturase and Oleate 12-hydroxylase • Integral membrane proteins • Non-heme-iron containing enzymes • Catalyze reactions via a di-iron cluster • 3 Histidine clusters essential for catalysis • Belong to a large family of functionally diverse enzymes Other Members of This Family • Alkane hydroxylase • Xylene monooxygenase • Carotene ketolase • Sterol methyl oxidase

  11. Oleate Desaturases and Hydroxylases • Oleic acid hydroxylase from Lesquerella Oleic acid desaturase from Arabidopsis • Oleic acid hydroxylase from Lesquerella Oleic acid hydroxylase from Ricinus 81% Sequence Identity 71% Sequence Identity • The deaturase and hydroxylase are more similar than both hydroxylases • Oleate hydroxylation arose independently by the genetic conversion of desaturase to hydroxylase • Only 7 residues strictly conserved in the desaturases but divergent in the hydroxylases • These 7 residues must be important for catalytic specificity

  12. Experimental Evidence for Catalytic Plasticity The Role of the Seven Conserved Residues in Desaturases Site-directed Mutagenesis N C Lesquerella hydroxylase (LFAH12) 7 Collective Mutations N C Arabidopsis FAD2desaturase (FAD2) • Normal (FAD2, LFAH12) and mutant (m7FAD2, m7LFAH12) genes expressed in yeast using GAL1 promoter • Cells harvested • Analysis of fatty acid composition by GC

  13. Desaturases and Hydroxylases 9 9 12 12 Oleate 12-desaturase Linoleic acid (18:2Δ9,12) Oleic acid (18:1Δ9) 9 12 Oleate 12-hydroxylase Oleic acid (18:1Δ9) Ricinoleic acid (18:1Δ9-OH) 12 9

  14. Experimental Evidence for Catalytic Plasticity The Role of the Seven Residues • FAD2: Converted from a strict desaturase into a bifunctional desaturase/hydroxylase • LFAH12: Converted from a hydroxylase/desaturase into a strict desaturase (Linoleic acid)

  15. Experimental Evidence for Catalytic Plasticity Amino Acids Required for Desaturase vs. Hydroxylase Activities Site-directed Mutagenesis N C Lesquerella hydroxylase (LFAH12) 6 Collective Mutations (All Combinations) 7 Individual Mutations N C Arabidopsis FAD2desaturase (FAD2) Individual Mutations • Hydroxylase activities were very similar to WT hydroxylase (LFAH12) • No single amino acid is solely responsible for hydroxylase activity

  16. Experimental Evidence for Catalytic Plasticity Amino Acids Required for Desaturase and Hydroxylase Activities All seven constructs: • Ratio of diunsaturated to hydroxylated fatty acids similar to m7LFAH12 • As few as 6 AAs determine the desaturase/hydroxylase activities • Reduced desaturase activity F218Y-G105A construct: • Similar activity to F218Y and G105A • F218Y and G105A are redundant • Changes in catalytic activity due to combined effects of several AAs

  17. Experimental Evidence for Catalytic Plasticity Amino Acids Required for Desaturase vs. Hydroxylase Activities Site-directed Mutagenesis N H H H C Arabidopsis FAD2desaturase (FAD2) 4 Collective Mutations 4 Collective Mutations 4 Individual Mutations N H H H C Lesquerella hydroxylase (LFAH12) Castorhydroxylase (CFAH) Collective Mutations  m4FAD2: 90-fold increase in accumulation of hydroxylated fatty acids Single Amino Acid Subsitutions • T148I: 15-fold increase in accumulation of hydroxylated fatty acids • M324I: 50-fold increase in accumulation of hydroxylated fatty acids

  18. Catalytic Plasticity of Oleate Desaturase and Oleate Hydroxylase Substitution of Seven Amino Acids • Oleate 12-desaturase: From strict desaturase to bifunctional desaturase/hydroxylase • Oleate 12-hydroxylase: From desaturase/hydroxylase to strict desaturase Amino Acids that Determine Catalytic Specificity • As few as 6 amino acids determine desaturation vs. hydroxylation • Only 4 amino acid changes sufficient to convert a strict desaturase into a bifunctional desaturase/hydroxylase • Catalytic specificity due to combined effects of several amino acids • T148 and M324: Critical determinants of hydroxylase activity

  19. Practical Applications Catalytic Plasticity in Plants • Natural Plant Products: Spearmint vs. Peppermint • High throughput screening: finding more efficient enzymes • Creating enzymes with different inducible catalytic activities  A desaturase/hydroxylase/monooxygenase  Desired catalysis can be achieved by the addition of a molecule that triggers a conformational change Desaturase Oleic acid Oxo oleate Ricinoleic acid Oleic acid Monooxygenase Hydroxylase Oleic acid Linoleic acid

  20. THE END GO LAKERS! DOWN WITH THE SPURS!

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