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Characterization and phylogeny of the pfp gene of Amycolatopsis methanolica encoding PPi-dependent
A M Alves1, W G Meijer, J W Vrijbloed
1Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute (GBB), University of Groningen, Haren, The Netherlands.
Abstract:
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.
Insights
Amycolatopsis methanolica possesses a unique pyrophosphate-dependent phosphofructokinase (PPi-PFK) enzyme. Gene cloning and expression confirmed its identity, revealing evolutionary links to ATP-dependent forms.
Area of Science:
- Biochemistry
- Molecular Biology
- Enzymology
Background:
- Amycolatopsis methanolica utilizes a pyrophosphate-dependent phosphofructokinase (PPi-PFK) with characteristics of both ATP- and PPi-dependent enzymes.
- Understanding the genetic basis of this enzyme is crucial for metabolic pathway analysis.
Purpose of the Study:
- To isolate and characterize the gene encoding the PPi-PFK from Amycolatopsis methanolica.
- To investigate the evolutionary relationship between PPi-PFK and other phosphofructokinase enzymes.
Main Methods:
- Isolation of a gene fragment from a genomic library using oligonucleotide probes.
- Nucleotide sequencing of the identified fragment.
- Heterologous expression of the gene in Escherichia coli.
- Phylogenetic analysis of PFK enzymes.
Main Results:
- A 2.3-kb fragment containing an open reading frame (pfp) encoding a 340-amino acid protein was identified.
- Heterologous expression yielded a 45-kDa protein with high PPi-PFK activity.
- Adjacent genes (aroA and chiA) with similarity to synthase and chitinase genes were found.
- Phylogenetic analysis indicated that ATP- and PPi-PFK enzymes evolved from a common ancestor.
Conclusions:
- The gene pfp encodes the PPi-PFK of A. methanolica.
- PPi-PFK enzymes form a monophyletic group, suggesting a shared evolutionary origin with ATP-dependent PFKs.