title: The HI0073/HI0074 protein pair from Haemophilus influenzae is a member of a new nucleotidyltransferase family: structure, sequence analyses, and solution studies.
abstract: The crystal structure of HI0074 from Haemophilus influenzae, a protein of unknown function, has been determined at a resolution of 2.4 A. The molecules form an up-down, four-helix bundle, and associate into homodimers. The fold is most closely related to the substrate-binding domain of KNTase, yet the amino acid sequences of the two proteins exhibit no significant homology. Sequence analyses of completely and incompletely sequenced genomes reveal that the two adjacent genes, HI0074 and HI0073, and their close relatives comprise a new family of nucleotidyltransferases, with 15 members at the time of writing. The analyses also indicate that this is one of eight families of a large nucleotidyltransferase superfamily, whose members were identified based on the proximity of the nucleotide- and substrate-binding domains on the respective genomes. Both HI0073 and HI0074 were annotated "hypothetical" in the original genome sequencing publication. HI0073 was cloned, expressed, and purified, and was shown to form a complex with HI0074 by polyacrylamide gel electrophoresis under nondenaturing conditions, analytic size exclusion chromatography, and dynamic light scattering. Double- and single-stranded DNA binding assays showed no evidence of DNA binding to HI0074 or to HI0073/HI0074 complex despite the suggestive shape of the putative binding cleft formed by the HI0074 dimer.
authors.affiliation: Center for Advanced Research in Biotechnology, University of Maryland Biotechnology Institute, Rockville, Maryland 20850, USA.
authors.forename: Christopher
authors.initials: C
authors.lastname: Lehmann
authors.forename: Kap
authors.initials: K
authors.lastname: Lim
authors.forename: Vani Rao
authors.initials: VR
authors.lastname: Chalamasetty
authors.forename: Wojciech
authors.initials: W
authors.lastname: Krajewski
authors.forename: Eugene
authors.initials: E
authors.lastname: Melamud
authors.forename: Andrey
authors.initials: A
authors.lastname: Galkin
authors.forename: Andrew
authors.initials: A
authors.lastname: Howard
authors.forename: Zvi
authors.initials: Z
authors.lastname: Kelman
authors.forename: Prasad T
authors.initials: PT
authors.lastname: Reddy
authors.forename: Alexey G
authors.initials: AG
authors.lastname: Murzin
authors.forename: Osnat
authors.initials: O
authors.lastname: Herzberg
mesh_terms: D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D018360:Crystallography, X-Ray; D004247:DNA / Q000235:genetics / Q000378:metabolism; D019281:Dimerization; D016680:Genome, Bacterial; D006193:Haemophilus influenzae / Q000201:enzymology* / Q000235:genetics*; D046911:Macromolecular Substances; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009713:Nucleotidyltransferases / Q000737:chemistry* / Q000235:genetics*; D011485:Protein Binding; D011487:Protein Conformation; D011544:Pseudogenes / Q000235:genetics; D012645:Selenomethionine / Q000032:analysis / Q000378:metabolism; D016415:Sequence Alignment; D017385:Sequence Homology; D012996:Solutions
chemical_list: D001426:Bacterial Proteins; D046911:Macromolecular Substances; D012996:Solutions; D004247:DNA; D012645:Selenomethionine; D009713:Nucleotidyltransferases