Characterization and phylogeny of the pfp gene of Amycolatopsis methanolica encoding PPi-dependent phosphofructokinase.
The actinomycete Amycolatopsis methanolica employs a PPi-dependent phosphofructokinase (PPi-PFK) (EC 2.7.1.90) with biochemical characteristics similar to those of both ATP- and PPi-dependent enzymes during growth on glucose. A 2.3-kb PvuII fragment hybridizing to two oligonucleotides based on the amino-terminal amino acid sequence of PPi-PFK was isolated from a genomic library of A. methanolica. Nucleotide sequence analysis of this fragment revealed the presence of an open reading frame encoding a protein of 340 amino acids with a high degree of similarity to PFK proteins. Heterologous expression of this open reading frame in Escherichia coli gave rise to a unique 45-kDa protein displaying a high level of PPi-PFK activity. The open reading frame was therefore designated pfp, encoding the PPi-PFK of A. methanolica. Upstream and transcribed divergently from pfp, a partial open reading frame (aroA) similar to 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase-encoding genes was identified. The partial open reading frame (chiA) downstream from pfp was similar to chitinase genes from Streptomyces species. A phylogenetic analysis of the ATP- and PPi-dependent proteins showed that PPi-PFK enzymes are monophyletic, suggesting that the two types of PFK evolved from a common ancestor.
Alves
A M
AM
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Haren, The Netherlands.
Meijer
W G
WG
Vrijbloed
J W
JW
Dijkhuizen
L
L
D039903:Actinobacteria / Q000201:enzymology / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D015342:DNA Probes; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames / Q000235:genetics; D010770:Phosphotransferases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D010802:Phylogeny; D015183:Restriction Mapping; D017422:Sequence Analysis, DNA
D015342:DNA Probes; D010770:Phosphotransferases; C022502:pyrophosphate-fructose 6-phosphate 1-phosphotransferase