1+1 = 3: A Fusion of 2 Enzymes in the Methionine Salvage Pathway of Tetrahymena thermophila Creates a Trifunctional Enzyme That Catalyzes 3 Steps in the Pathway

Abstract

The methionine salvage pathway is responsible for regenerating methionine from its derivative, methylthioadenosine. The complete set of enzymes of the methionine pathway has been previously described in bacteria. Despite its importance, the pathway has only been fully described in one eukaryotic organism, yeast. Here we use a computational approach to identify the enzymes of the methionine salvage pathway in another eukaryote, Tetrahymena thermophila. In this organism, the pathway has two fused genes, MTNAK and MTNBD. Each of these fusions involves two different genes whose products catalyze two different single steps of the pathway in other organisms. One of the fusion proteins, mtnBD, is formed by enzymes that catalyze non-consecutive steps in the pathway, mtnB and mtnD. Interestingly the gene that codes for the intervening enzyme in the pathway, mtnC, is missing from the genome of Tetrahymena. We used complementation tests in yeast to show that the fusion of mtnB and mtnD from Tetrahymena is able to do in one step what yeast does in three, since it can rescue yeast knockouts of mtnB, mtnC, or mtnD. Fusion genes have proved to be very useful in aiding phylogenetic reconstructions and in the functional characterization of genes. Our results highlight another characteristic of fusion proteins, namely that these proteins can serve as biochemical shortcuts, allowing organisms to completely bypass steps in biochemical pathways.

MtnBD Is a Multifunctional Fusion Enzyme in the Methionine Salvage Pathway of Tetrahymena thermophila

“…While MtnBD shows no similarity to MtnW (DK-MTP-1-P enolase), MtnX (HK-MTPenyl-1-P phosphatase), or MtnC (DK-MTP-1-P enolase/phosphatase), previous genetic experiments suggested that it catalyzes four sequential reactions; dehydration, enolization, dephosphorylation, and dioxygenation [14]. To analyze whether MtnBD catalyzes these four reactions (steps 4 to 7) in vitro , we designed a recombinant protein of T. thermophila MtnBD fused with a histidine-tag at the N-terminus (Fig.…”

“…Gene fusions of mtnA and mtnK , and mtnB and mtnD , which encode enzymes in the methionine salvage pathway (MSP), are present in the genome of T. thermophila [14]. MSP is a universal pathway in all kingdoms [15]; it recycles reduced sulfur from 5-methylthioadenosine (MTA), a by-product of polyamine synthesis, to methionine (Fig.…”

“…Enzyme names are derived from Ashida et al [19] and Salim et al [14]. The MSP recycles organic sulfur from MTA, a by-product of polyamine synthesis, to methionine.…”

“…1B) [14], [18]–[21], [30], [34]–[36]. Thus, the MSP is catalyzed by different enzyme combinations in different organisms, despite going through the same metabolites.…”

“…In terms of multifunctionality, the most notable enzyme is T. thermophila MtnBD, the fusion protein of MtnB and MtnD. This enzyme probably catalyzes the four reaction steps of MtnB, MtnW, MtnX, and MtnD, based on the results of complementation experiments with yeast knockout mutants of MSP genes [14]. Salim and co-workers proposed that the fusion of proteins is an evolutionary mechanism to gain a new function and has reduced the number of enzymes in the MSP of T. thermophila .…”