tailieunhanh - Báo cáo khoa học: A novel isoform of pantothenate synthetase in the Archaea

The linear biosynthetic pathway leading froma-ketoisovalerate to panto-thenate (vitamin B5) and on to CoA comprises eight steps in the Bacteria and Eukaryota. Genes for up to six steps of this pathway can be identified by sequence homology in individual archaeal genomes. | ễFEBS Journal A novel isoform of pantothenate synthetase in the Archaea Silvia Ronconi Rafal Jonczyk and Ulrich Genschel Lehrstuhl fur Genetik Technische Universitat Munchen Freising Germany Keywords archaealmetabolism CoA biosynthesis evolution of metabolism Methanosarcina mazei pantothenate synthetase Correspondence U. Genschel Lehrstuhlfur Genetik Technische Universitat Munchen Am Hochanger 8 85350 Freising Germany Fax 49 8161 715636 Tel 49 8161 715644 E-mail genschel@ Received 7 February 2008 revised 17 March 2008 accepted 19 March 2008 doi The linear biosynthetic pathway leading from a-ketoisovalerate to pantothenate vitamin B5 and on to CoA comprises eight steps in the Bacteria and Eukaryota. Genes for up to six steps of this pathway can be identified by sequence homology in individual archaeal genomes. However there are no archaeal homologs to known isoforms of pantothenate synthetase PS or pantothenate kinase. Using comparative genomics we previously identified two conserved archaeal protein families as the best candidates for the missing steps. Here we report the characterization of the predicted PS gene from Methanosarcina mazei which encodes a hypothetical protein MM2281 with no obvious homologs outside its own family. When expressed in Escherichia coli MM2281 partially complemented an auxotrophic mutant without PS activity. Purified recombinant MM2281 showed no PS activity on its own but the enzyme enabled substantial synthesis of 14C 4 -phosphopantothenate from 14C b-alanine pantoate and ATP when coupled with E. coli pantothenate kinase. ADP but not AMP was detected as a coproduct of the coupled reaction. MM2281 also transferred the 14C-label from 14C b-alanine to pantothenate in the presence of panto-ate and ADP presumably through isotope exchange. No exchange took place when pantoate was removed or ADP replaced with AMP. Our results indicate that MM2281 represents a novel type of PS that forms ADP and is .