Abstract
To recycle reduced sulfur to methionine in the methionine salvage pathway (MSP), 5-methylthioribulose-1-phosphate is converted to 2-keto-4-methylthiobutyrate, the methionine precursor, by four steps; dehydratase, enolase, phosphatase, and dioxygenase reactions (catalyzed by MtnB, MtnW, MtnX and MtnD, respectively, in Bacillus subtilis). It has been proposed that the MtnBD fusion enzyme in Tetrahymena thermophila catalyzes four sequential reactions from the dehydratase to dioxygenase steps, based on the results of molecular biological analyses of mutant yeast strains with knocked-out MSP genes, suggesting that new catalytic function can be acquired by fusion of enzymes. This result raises the question of how the MtnBD fusion enzyme can catalyze four very different reactions, especially since there are no homologous domains for enolase and phosphatase (MtnW and MtnX, respectively, in B. subtilis) in the peptide. Here, we tried to identify the domains responsible for catalyzing the four reactions using recombinant proteins of full-length MtnBD and each domain alone. UV-visible and 1H-NMR spectral analyses of reaction products revealed that the MtnB domain catalyzes dehydration and enolization and the MtnD domain catalyzes dioxygenation. Contrary to a previous report, conversion of 5-methylthioribulose-1-phosphate to 2-keto-4-methylthiobutyrate was dependent on addition of an exogenous phosphatase from B. subtilis. This was observed for both the MtnB domain and full-length MtnBD, suggesting that MtnBD does not catalyze the phosphatase reaction. Our results suggest that the MtnB domain of T. thermophila MtnBD acquired the new function to catalyze both the dehydratase and enolase reactions through evolutionary gene mutations, rather than fusion of MSP genes.
Highlights
Duplication, fusion, mutation, and homologous recombination of genes encoding enzymes have contributed to the emergence, diversification, and development of metabolic pathways during evolution [1,2,3]
These results suggested that the reaction of T. thermophila MtnBD proceeds through the MTRu-1-P dehydration in a similar manner to that of the B. subtilis MtnB. 1H-nuclear Magnetic Resonance (NMR) analysis showed that MTRu-1-P was completely converted into HK-MTPenyl-1-P after 6 h (Fig. 2H)
Our results highlighted that the MtnB domain of T. thermophila MtnBD catalyzes MTRu-1-P dehydratase/enolase reactions and the MtnD domain catalyzes the DHK-MTPene dioxygenase reaction
Summary
Duplication, fusion, mutation, and homologous recombination of genes encoding enzymes have contributed to the emergence, diversification, and development of metabolic pathways during evolution [1,2,3]. Recent progress in genome analysis has shown that genes encoding proteins of known function may be found in other species as fusion genes encoding a single multifunctional protein [4,5,6,7]. This event, known as gene fusion/protein fusion, is one of several molecular evolutionary processes [8], [9]. Higher eukaryotes often utilize multifunctional fusion enzymes that catalyze several biosynthetic steps. This trend has been observed for metabolic enzymes catalyzing fatty acid and purine biosynthesis [10], [11]. The advantages of fusion enzymes include decreased regulatory load on metabolic pathways and enhanced catalytic efficiency of sequential reactions without the loss of metabolic intermediates
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