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Catalytic Plasticity of the Aspartate/Glutamate Racemase Superfamily

By Florian A Fisch

Abstract

The bacterial and archaeal Asp/Glu racemase enzyme superfamily contains a variety of catalytic functions that have great potential for use in industrial biocatalysis. Members of this superfamily include aspartate racemases (AspRs), glutamate racemases (GluRs), hydantoin racemases (HydRs), arylmalonate decarboxylases (AMDs) and maleate cis-trans isomerases (MIs). Despite their catalytic diversity, all characterised members share the same protein fold, catalytic cysteine residues and reaction intermediate. Attempts to exploit this evolutionary flexibility for new processes have had limited success so far, showing that the employed mechanisms are not yet fully understood. For example, the well-characterised Bordetella bronchiseptica AMD (BbAMD) enantiospecifically decarboxylates a range of arylmalonates was but is not able to decarboxylate alkylmalonates despite considerable efforts made by site directed mutagenesis.\ud \ud \ud In this work an investigation of the sequence diversity of the superfamily was undertaken and a range of BbAMD sequence homologues was tested for both aryl- and alkylmalonate decarboxylation (Chapter 3). However, none of the homologues exhibited decarboxylation activity. Targeted mutation of active site residues in an attempt to introduce decarboxylase activity was also unsuccessful. In an alternative approach to identify new alkylmalonate decarboxylating enzymes, a range of bacterial strains capable of processing alkylmalonates was isolated using selective enrichment from soil samples (Appendix D).\ud \ud \ud The only characterised superfamily enzymes without a described three dimensional protein structure are MIs. In order to illuminate the distinct mechanism of MIs, the activity of the superfamily member Nocardia farcinica MI (NfMI) was characterised (Chapter 4) and its structure was determined by X-ray crystallography (Chapter 5). A potent inhibitor and substrate analogue bromomaleate was found. Mutagenesis of the active site cysteine dyad confirmed its catalytic role and Cys76 was found to be more important than Cys194. The data support a mechanism initiated by nucleophilic attack by Cys76 on the double bond of maleate. Although alternative mechanisms cannot be excluded at present, these findings indicate that the mechanistic chemistry in the superfamily is more adaptable than previously thought.\u

Publisher: Biology (York)
Year: 2009
OAI identifier: oai:etheses.whiterose.ac.uk:799

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Citations

  1. (2004). Acta Cryst. doi
  2. (1972). Cis-Trans Isomerisation. doi
  3. (2007). Cofaktor-unabhängige Decarboxylasen. 13133-79 Ed., Deutsche Bundesstiftung Umwelt (DBU)
  4. (2009). Crystal structure of glutamate racemase from Listeria monocytogenes in complex with acetate ion. Center for Structural Genomics of Infectious Diseases (CSGID)
  5. (2009). Crystal structure of glutamate racemase from Listeria monocytogenes. Center for Structural Genomics of Infectious Diseases (CSGID)
  6. (2007). Crystal structure of hydantoin racemase from Pyrococcus horikoshii OT3. doi
  7. (2007). Crystal structure of phenolic acid decarboxylase (2635953) from Bacillus subtilis at 1.36 A resolution (PDB:
  8. (2007). Crystal structure of ST0656, a function unknown protein from Sulfolobus tokodaii. doi
  9. (2005). Enzymatic asymmetric decarboxylation of disubstituted malonic acids. in European Patent Office,
  10. (1971). Fumarase and crotonase. doi
  11. (1961). Fumarase. doi
  12. (1961). Isomerisation Reactions (Survey).
  13. (2001). Maleic Anhydride, Maleic Acid, and Fumaric Acid. doi
  14. (2003). Methods for identifying racemases.
  15. (2009). New Malonate Decarboxylases for Industiral Applications. doi
  16. (1987). Organic Chemistry, A Short Course, 7th ed.,
  17. (2009). Practical Biotransformations. A Beginner's Guide, doi
  18. (2003). Protein Struct. doi
  19. (2009). Structural Basis of the Enantioselective Decarboxylation by Arylmalonate Decarboxylase.
  20. (1999). Structure and Mechanism doi

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