title: Structural and functional analysis of PUR2,5 gene encoding bifunctional enzyme of de novo purine biosynthesis in Ogataea (Hansenula) polymorpha CBS 4732T.
abstract: We describe the cloning, sequencing and functional characterization of gene PUR2,5, involved in de novo purine biosynthesis of the yeast Ogataea (Hansenula) polymorpha. This gene (2369 bp) was cloned by genetic complementation of adenine requiring mutation. It encodes a bifunctional enzyme of 789 amino acids (85 kDa) that catalyzes the second and the fifth steps of de novo purine biosynthesis pathway and shows dual enzymatic activity - of glycinamide ribotide synthetase (GARS, EC 6.3.4.13) and of aminoimidazole ribotide synthetase (AIRS, EC 6.3.3.1). Nucleotide sequence analysis revealed the presence of putative regulatory elements located in the adjacent 5' region. Canonical motives that function as binding sites for BAS1 transcription activator were found at positions (-593) and (-389). The putative TAATTA-box was located at (-20) to (-14) and AT-rich heteroduplex was found in the 3'-non-translated region. We compared the amino acid sequence of OpPUR2,5p with those of the corresponding enzymes of other yeast species as well as with distant organisms like bacteria Escherichia coli and human Homo sapiens. A successful disruption of OpPUR2,5 gene was done. It was found that OpPUR2,5::LEU2 replacement affects both mating and sporulation processes. OpPUR2,5 sequence is deposited in the GenBank of NCBI with accession no. JF967633.
authors.affiliation: The Stefan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria.
authors.forename: Anton
authors.initials: A
authors.lastname: Stoyanov
authors.affiliation: The Stefan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria.
authors.forename: Penka
authors.initials: P
authors.lastname: Petrova
authors.affiliation: The Stefan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria.
authors.forename: Dimitrinka
authors.initials: D
authors.lastname: Lyutskanova
authors.affiliation: The Stefan Angeloff Institute of Microbiology, Bulgarian Academy of Sciences, Sofia, Bulgaria. Electronic address: kantcholahtchev@yahoo.com.
authors.forename: Kantcho
authors.initials: K
authors.lastname: Lahtchev
mesh_terms: D001665:Binding Sites; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000737:chemistry / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D055786:Gene Knockout Techniques; D005816:Genetic Complementation Test; D006801:Humans; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010843:Pichia / Q000201:enzymology* / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011687:Purines / Q000096:biosynthesis*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D016385:TATA Box
chemical_list: D004271:DNA, Fungal; D011687:Purines; D019731:Carbon-Nitrogen Ligases; C022380:phosphoribosylaminoimidazole synthase; C022387:phosphoribosylamine-glycine ligase; C030985:purine
keywords: Bifunctional enzyme; Ogataea (Hansenula) polymorpha; Purine biosynthesis