title: Understanding functional divergence in proteins by studying intragenomic homologues.
abstract: Studies of intragenomic homologues in bacterial genomes can provide valuable insights into functional divergence. Three GTP cyclohydrolase II homologues in the Streptomyces coelicolor genome have been shown to catalyze two related but distinct reactions [Spoonamore, J. E., Dahlgran, A. L., Jacobsen, N. E., and Bandarian, V. (2006) Biochemistry 45, 12144-12155]. Two of the homologues, SCO 1441 and 2687, convert GTP to 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (APy); one of the homologues (SCO 6655) produces 2-amino-5-formylamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate (FAPy). We show herein that the differences in the fate of GTP in SCO 6655 relative to SCO 1441 and 2687 results from a single amino acid substitution in the active site of the protein: a Tyr residue in the active sites of SCO 1441 and SCO 2687 is replaced with a Met in SCO 6655. Site-directed interchange of this residue in the three S. coelicolor intragenomic homologues is necessary and sufficient for interconversion of catalytic function which, except for SCO 1441, occurs with little loss of catalytic efficiency. Furthermore, we show that of 14 additional site-directed variants at this position of SCO 6655, His confers catalytic efficiency within 1 order of magnitude of that of the wild type and supports conversion of GTP to both FAPy and APy. The results demonstrate a clear set of mutational events that permit GCH II to produce either FAPy or APy. These results highlight a mechanism whereby functional divergence can be achieved in enzymes that catalyze multistep transformations.
authors.affiliation: Department of Biochemistry and Molecular Biophysics, The University of Arizona, Tucson, Arizona 85721, USA.
authors.forename: James E
authors.initials: JE
authors.lastname: Spoonamore
authors.forename: Vahe
authors.initials: V
authors.lastname: Bandarian
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000502:physiology; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D017124:Conserved Sequence; D006136:GTP Cyclohydrolase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D016680:Genome, Bacterial; D006868:Hydrolysis; D053858:Metabolic Networks and Pathways; D008954:Models, Biological; D008958:Models, Molecular; D011110:Polymorphism, Genetic / Q000502:physiology; D017422:Sequence Analysis, DNA; D012689:Sequence Homology, Nucleic Acid; D048372:Streptomyces coelicolor / Q000201:enzymology / Q000235:genetics*; D013329:Structure-Activity Relationship
chemical_list: D001426:Bacterial Proteins; D006136:GTP Cyclohydrolase