title: Recombinant expression of a functional myo-inositol-1-phosphate synthase (MIPS) in Mycobacterium smegmatis.
abstract: Myo-inositol-1-phosphate synthase (MIPS, E.C. 5.5.1.4) catalyzes the first step in inositol production-the conversion of glucose-6-phosphate (Glc-6P) to myo-inositol-1-phosphate. While the three dimensional structure of MIPS from Mycobacterium tuberculosis has been solved, biochemical studies examining the in vitro activity have not been reported to date. Herein we report the in vitro activity of mycobacterial MIPS expressed in E. coli and Mycobacterium smegmatis. Recombinant expression in E. coli yields a soluble protein capable of binding the NAD(+) cofactor; however, it has no significant activity with the Glc-6P substrate. In contrast, recombinant expression in M. smegmatis mc(2)4517 yields a functionally active protein. Examination of structural data suggests that MtMIPS expressed in E. coli adopts a fold that is missing a key helix containing two critical (conserved) Lys side chains, which likely explains the inability of the E. coli expressed protein to bind and turnover the Glc-6P substrate. Recombinant expression in M. smegmatis may yield a protein that adopts a fold in which this key helix is formed enabling proper positioning of important side chains, thereby allowing for Glc-6P substrate binding and turnover. Detailed mechanistic studies may be feasible following optimization of the recombinant MIPS expression protocol in M. smegmatis.
authors.affiliation: Department of Biochemistry, Virginia Tech, Blacksburg, VA, 24061, USA.
authors.forename: Xinyi
authors.initials: X
authors.lastname: Huang
authors.affiliation: Department of Biochemistry, Virginia Tech, Blacksburg, VA, 24061, USA. mhernick@acp.edu.|Department of Pharmaceutical Sciences, Appalachian College of Pharmacy, Oakwood, VA, 24631, USA. mhernick@acp.edu.
authors.forename: Marcy
authors.identifier: http://orcid.org/0000-0002-2839-5767
authors.initials: M
authors.lastname: Hernick
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism; D015870:Gene Expression; D007700:Kinetics; D020102:Mycobacterium smegmatis / Q000201:enzymology* / Q000235:genetics; D007296:Myo-Inositol-1-Phosphate Synthase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D009243:NAD / Q000378:metabolism; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
chemical_list: D001426:Bacterial Proteins; D009243:NAD; D007296:Myo-Inositol-1-Phosphate Synthase
keywords: Mycobacteria; Mycobacterium smegmatis; Mycobacterium tuberculosis; Myo-inositol-1-phosphate synthase (MIPS); Recombinant expression