The Catalytic Plasticity of Enzymes in Higher Plants

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!