Thermostable enzymes in the amino acid metabolism of Archaea

This project bridges the gap between genomics and proteomics by comparing the observed biochemical function of gene products with the predicted function assigned to them through analysis of sequence homology. As new sequence data becomes available, more and more genes are annotated based on sequence homology alone. However, some genes may have completely different sequences but perform the same function (nonorthologous) or exhibit sequence homology but perform different functions (orthologous). Recent sequencing of genomes in the third domain of life, the archaea, revealed more of these nonorthologous genes. In this study, the genes of interest are those that are involved in amino acid metabolism and the two archaeal representatives are Pyrobaculum aerophilum and Archaeoglobus fulgidus. Chapters 1 and 2 discuss the identification and characterization of a proline dehydrogenase from P. aerophilum and an alanine dehydrogenase from A. fulgidus. The two amino acid dehydrogenases were detected using zymograms and were isolated from the soluble fractions of the organisms. Amino acid sequence analysis of the enzymes revealed new classes of proline and alanine dehydrogenases. Subsequent chapters describe selected enzymes of the aromatic amino acid biosynthesis pathway in A. fulgidus. Four of the seven enzymes in the common aromatic amino acid biosynthesis pathway, also known as the shikimate pathway, along with other terminal pathway enzymes have been annotated in the genome. Shikimate dehydrogenase was the first enzyme for which activity was confirmed and is described in chapter 3. The terminal pathway in the formation of the aromatic amino acids starts with chorismate, the branch point intermediate. Chorismate mutase is the first enzyme in the committed terminal pathway for the biosynthesis of tyrosine and phenylalanine. Chapter 4 reports the unusual fusion of chorismate mutase, prephenate dehydrogenase, and prephenate dehydratase activities in a single polypeptide. The last chapter discusses the identification of two loci that may code for the biocatalyst for the first step in the shikimate pathway. The gene products were tested for 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase activity, but this activity was not detected.