Abstract
The methionine salvage pathway is widely distributed among some eubacteria, yeast, plants and animals and recycles the sulfur-containing metabolite 5-methylthioadenosine (MTA) to methionine. In eukaryotic cells, the methionine salvage pathway takes place in the cytosol and usually involves six enzymatic activities: MTA phosphorylase (MTAP, EC 2.4.2.28), 5′-methylthioribose-1-phosphate isomerase (mtnA, EC 5.3.1.23), 5′-methylthioribulose-1-phosphate dehydratase (mtnB, EC: 4.2.1.109), 2,3-dioxomethiopentane-1-phosphate enolase/phosphatase (mtnC, EC 3.1.3.77), aci-reductone dioxygenase (mtnD, EC 1.13.11.54) and 4-methylthio-2-oxo-butanoate (MTOB) transaminase (EC 2.6.1.-). The aim of this study was to complete the available information on the methionine salvage pathway in human by identifying the enzyme responsible for the dehydratase step. Using a bioinformatics approach, we propose that a protein called APIP could perform this role. The involvement of this protein in the methionine salvage pathway was investigated directly in HeLa cells by transient and stable short hairpin RNA interference. We show that APIP depletion specifically impaired the capacity of cells to grow in media where methionine is replaced by MTA. Using a Shigella mutant auxotroph for methionine, we confirm that the knockdown of APIP specifically affects the recycling of methionine. We also show that mutation of three potential phosphorylation sites does not affect APIP activity whereas mutation of the potential zinc binding site completely abrogates it. Finally, we show that the N-terminal region of APIP that is missing in the short isoform is required for activity. Together, these results confirm the involvement of APIP in the methionine salvage pathway, which plays a key role in many biological functions like cancer, apoptosis, microbial proliferation and inflammation.
Highlights
Methionine is an essential amino acid involved in major functions such as protein synthesis, formation of polyamines, DNA and protein methylation and protection against reactive oxygen species though the generation of glutathione [1]
It has been shown that the level and activity of ornithine decarboxylase, the ratecontrolling enzyme in polyamine synthesis, can be modulated by the first and last metabolites of the methionine salvage pathway: MTA and 4-methhylthio-2-oxo-butanoate (MTOB) [5,6,7]
To confirm that APIP is the most likely human protein capable of performing mtnB activity, we performed a BLAST search on UniProtKB using the sequences of the Bacillus subtilis and Saccharomyces cerevisiae enzymes
Summary
Methionine is an essential amino acid involved in major functions such as protein synthesis, formation of polyamines, DNA and protein methylation and protection against reactive oxygen species though the generation of glutathione [1]. The methionine that is not used for protein synthesis is converted into S-adenosylmethionine (SAM), the principal methyl donor (Figure 1A). The methionine salvage pathway and the polyamine synthesis seem to be tightly coupled, probably in order to maintain intracellular levels of SAM. It has been shown that the level and activity of ornithine decarboxylase, the ratecontrolling enzyme in polyamine synthesis, can be modulated by the first and last metabolites of the methionine salvage pathway: MTA and 4-methhylthio-2-oxo-butanoate (MTOB) [5,6,7]. The methionine salvage pathway takes place in the cytosol and involves six enzymatic activities: MTA phosphorylase (MTAP, EC 2.4.2.28), 59methylthioribose-1-phosphate isomerase (mtnA, EC 5.3.1.23), 59methylthioribulose-1-phosphate dehydratase (mtnB, EC 4.2.1.109), 2,3-dioxomethiopentane-1-phosphate enolase/phosphatase (mtnC, EC 3.1.3.77), aci-reductone dioxygenase (mtnD, EC 1.13.11.54) and MTOB transaminase (EC 2.6.1.-) [4]. The transamination step can be catalyzed by a range of transaminases, which preferentially use aromatic and branched chain amino group donors [9]
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