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Identification and functional analysis of a new type of Z,E-mixed prenyl reductase from mycobacteria
Tohru Abe1, Mariko Hakamata2, Akihito Nishiyama2
1Department of Agriculture, Faculty of Agriculture and Graduate School of Science and Technology, Niigata University, Japan.
Abstract:
Isoprenoids with reduced Z,E-mixed prenyl groups are found in various organisms. To date, only polyprenol reductases (PR-Dol) involved in dolichol biosynthesis have been identified as enzymes capable of reducing Z,E-mixed prenyl groups. Although C35 -isoprenoids with reduced Z,E-mixed prenyl groups are found in mycobacteria, Z,E-mixed heptaprenyl reductase (HepR) remains unidentified. In the present study, the identification and functional analysis of HepR was performed. No PR-Dol homolog gene was detected in the genome of Mycolicibacterium vanbaalenii. However, a homolog of geranylgeranyl reductase (GGR), which reacts with an all-E prenyl group as a substrate, was encoded in the genome; thus, we analyzed it as a HepR candidate. In vitro enzymatic assay and in vivo gene suppression analysis identified the GGR homolog as HepR and revealed that HepR catalyzes the reduction of ω- and E- prenyl units in Z,E-mixed heptaprenyl diphosphates, and C35 -isoprenoids are mainly biosynthesized using E,E,E-geranylgeranyl diphosphate as a precursor. Thus, it was demonstrated that the Z,E-mixed prenyl reductase family exists in the GGR homologs. To the best of our knowledge, this is the first identification of a new type of Z,E-mixed prenyl reductase with no sequence homology to PR-Dol. The substrate specificity of HepR significantly differed from that of GGR, suggesting that it is a new enzyme. HepR homologs are widely distributed in mycobacterial genomes, and lipid analysis suggests that many strains, including pathogenic species, produce HepR metabolites. The discovery of this new enzyme will promote further research on Z,E-mixed isoprenoids.
Insights
Researchers identified a new enzyme, heptaprenyl reductase (HepR), in mycobacteria that reduces mixed prenyl groups. This discovery expands our understanding of isoprenoid biosynthesis and identifies a new enzyme family within geranylgeranyl reductase homologs.
Area of Science:
- Biochemistry
- Molecular Biology
- Microbiology
Background:
- Isoprenoids with reduced Z,E-mixed prenyl groups are biologically significant but their synthesis pathways are not fully understood.
- Polyprenol reductases (PR-Dol) are the only known enzymes capable of reducing these mixed prenyl groups.
- Mycobacteria synthesize C35-isoprenoids with reduced Z,E-mixed prenyl groups, yet the specific reductase remained unidentified.
Purpose of the Study:
- To identify and functionally characterize the enzyme responsible for reducing Z,E-mixed prenyl groups in *Mycolicibacterium vanbaalenii*.
- To determine if known reductases, like geranylgeranyl reductase (GGR), could perform this function.
- To investigate the distribution and significance of this newly identified reductase in mycobacteria.
Main Methods:
- Bioinformatic analysis to search for PR-Dol homologs in *M. vanbaalenii* genome.
- Identification and analysis of a GGR homolog as a candidate enzyme.
- In vitro enzymatic assays to confirm substrate specificity and catalytic activity.
- In vivo gene suppression studies to assess the enzyme's function in the organism.
- Lipid analysis of various mycobacterial strains.
Main Results:
- No PR-Dol homolog was found in *M. vanbaalenii*; however, a GGR homolog was identified as a potential heptaprenyl reductase (HepR).
- HepR was confirmed to catalyze the reduction of ω- and E- prenyl units in Z,E-mixed heptaprenyl diphosphates.
- C35-isoprenoids are primarily synthesized using E,E,E-geranylgeranyl diphosphate, with HepR playing a crucial role.
- HepR represents a novel class of Z,E-mixed prenyl reductase, distinct from PR-Dol and exhibiting different substrate specificity compared to GGR.
- HepR homologs are widespread in mycobacteria, including pathogenic species, suggesting broad biological relevance.
Conclusions:
- The GGR homolog in *M. vanbaalenii* functions as HepR, a novel Z,E-mixed prenyl reductase.
- This discovery establishes the existence of a new prenyl reductase family within GGR homologs.
- HepR's unique substrate specificity highlights it as a distinct enzyme, opening new avenues for isoprenoid research.
