Structural and functional analysis of PUR2,5 gene encoding bifunctional enzyme of de novo purine biosynthesis in Ogataea (Hansenula) polymorpha CBS 4732T

• Published in: Microbiological research 2014-05, Vol.169 (5-6), p.378-387
• Main Authors: Stoyanov, Anton, Petrova, Penka, Lyutskanova, Dimitrinka, Lahtchev, Kantcho
• Format: Article
• Language: English
• Subjects: Bifunctional enzyme; Binding Sites; Carbon-Nitrogen Ligases - chemistry; Carbon-Nitrogen Ligases - genetics; Carbon-Nitrogen Ligases - metabolism; Cloning, Molecular; DNA, Fungal - chemistry; DNA, Fungal - genetics; Escherichia coli - genetics; Gene Knockout Techniques; Genetic Complementation Test; Humans; Molecular Sequence Data; Molecular Weight; Ogataea (Hansenula) polymorpha; Pichia - enzymology; Pichia - genetics; Promoter Regions, Genetic; Purine biosynthesis; Purines - biosynthesis; Sequence Analysis, DNA; Sequence Homology, Amino Acid; TATA Box
• ISSN: 0944-5013; 1618-0623
• DOI: 10.1016/j.micres.2013.08.008

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 (2369bp) was cloned by genetic complementation of adenine requiring mutation. It encodes a bifunctional enzyme of 789 amino acids (85kDa) 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.