title: Substrate recognition by the hetero-octameric ATP phosphoribosyltransferase from Lactococcus lactis.
abstract: Two families of ATP phosphoribosyl transferases (ATP-PRT) join ATP and 5-phosphoribosyl-1 pyrophosphate (PRPP) in the first reaction of histidine biosynthesis. These consist of a homohexameric form found in all three kingdoms and a hetero-octameric form largely restricted to bacteria. Hetero-octameric ATP-PRTs consist of four HisGS catalytic subunits related to periplasmic binding proteins and four HisZ regulatory subunits that resemble histidyl-tRNA synthetases. To clarify the relationship between the two families of ATP-PRTs and among phosphoribosyltransferases in general, we determined the steady state kinetics for the hetero-octameric form and characterized the active site by mutagenesis. The KmPRPP (18.4 +/- 3.5 microM) and kcat (2.7 +/- 0.3 s-1) values for the PRPP substrate are similar to those of hexameric ATP-PRTs, but the Km for ATP (2.7 +/- 0.3 mM) is 4-fold higher, suggestive of tighter regulation by energy charge. Histidine and AMP were determined to be noncompetitive (Ki = 81.1 microM) and competitive (Ki = 1.44 mM) inhibitors, respectively, with values that approximate their intracellular concentrations. Mutagenesis experiments aimed at investigating the side chains recognizing PRPP showed that 5'-phosphate contacts (T159A and T162A) had the largest (25- and 155-fold, respectively) decreases in kcat/Km, while smaller decreases were seen with mutants making cross subunit contacts (K50A and K8A) to the pyrophosphate moiety or contacts to the 2'-OH group. Despite their markedly different quaternary structures, hexameric and hetero-octameric ATRP-PRTs exhibit similar functional parameters and employ mechanistic strategies reminiscent of the broader PRT superfamily.
authors.affiliation: Department of Microbiology and Molecular Genetics, University of Vermont, B403 Given Building, 89 Beaumont Avenue, Burlington, Vermont 05405, USA.
authors.forename: Karen S
authors.initials: KS
authors.lastname: Champagne
authors.forename: Elise
authors.initials: E
authors.lastname: Piscitelli
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Francklyn
mesh_terms: D001276:ATP Phosphoribosyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D020134:Catalytic Domain / Q000235:genetics; D006639:Histidine / Q000096:biosynthesis / Q000737:chemistry; D013294:Lactococcus lactis / Q000201:enzymology* / Q000235:genetics; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D010754:Phosphoribosyl Pyrophosphate / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding / Q000235:genetics; D020836:Protein Structure, Quaternary / Q000235:genetics; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D046912:Multiprotein Complexes; D021122:Protein Subunits; D000249:Adenosine Monophosphate; D006639:Histidine; D010754:Phosphoribosyl Pyrophosphate; D001276:ATP Phosphoribosyltransferase