Dataset Information

Inositol Monophosphatase: A Bifunctional Enzyme in Mycobacterium smegmatis.

ABSTRACT: Inositol monophosphatase (IMPase) is a crucial enzyme for the biosynthesis of phosphatidylinositol, an essential component in mycobacterial cell walls. IMPase A (ImpA) from Mycobacterium smegmatis is a bifunctional enzyme that also functions as a fructose-1,6-bisphosphatase (FBPase). To better understand the bifunctional nature of this enzyme, point mutagenesis was conducted on several key residues and their enzyme activity was tested. Our results along with active site models support the fact that ImpA is a bifunctional enzyme with residues Gly94, Thr95 hypothesized to be contributing to the FBPase activity and residues Trp220, Asp221 hypothesized to be contributing to the IMPase activity. Double mutants, W220A + D221A reduced both FBPase and IMPase activity drastically while the double mutant G94A + T95A surprisingly partially restored the IMPase activity compared to the single mutants. This study establishes the foundation toward obtaining a better understanding of the bifunctional nature of this enzyme.

SUBMITTER: Goswami R

PROVIDER: S-EPMC6217659 | biostudies-literature | 2018 Oct

REPOSITORIES: biostudies-literature

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Inositol Monophosphatase: A Bifunctional Enzyme in <i>Mycobacterium smegmatis</i>.

Goswami Rajendra R Bondoc Jasper Marc G JMG Wheeler Paul R PR Jafari Alireza A Gonzalez Trinidad T Mehboob Shahila S Movahedzadeh Farahnaz F

ACS omega 20181023 10

Inositol monophosphatase (IMPase) is a crucial enzyme for the biosynthesis of phosphatidylinositol, an essential component in mycobacterial cell walls. IMPase A (ImpA) from <i>Mycobacterium smegmatis</i> is a bifunctional enzyme that also functions as a fructose-1,6-bisphosphatase (FBPase). To better understand the bifunctional nature of this enzyme, point mutagenesis was conducted on several key residues and their enzyme activity was tested. Our results along with active site models support the ...[more]

PMID: 30411052

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Project description:Synthesis of myo-inositol is crucial in multicellular eukaryotes for production of phosphatidylinositol and inositol phosphate signaling molecules. The myo-inositol monophosphatase (IMP) enzyme is required for the synthesis of myo-inositol, breakdown of inositol (1,4,5)-trisphosphate, a second messenger involved in Ca(2+) signaling, and synthesis of L-galactose, a precursor of ascorbic acid. Two myo-inositol monophosphatase -like (IMPL) genes in Arabidopsis encode chloroplast proteins with homology to the prokaryotic IMPs and one of these, IMPL2, can complement a bacterial histidinol 1-phosphate phosphatase mutant defective in histidine synthesis, indicating an important role for IMPL2 in amino acid synthesis. To delineate how this small gene family functions in inositol synthesis and metabolism, we sought to compare recombinant enzyme activities, expression patterns, and impact of genetic loss-of-function mutations for each. Our data show that purified IMPL2 protein is an active histidinol-phosphate phosphatase enzyme in contrast to the IMPL1 enzyme, which has the ability to hydrolyze D-galactose 1-phosphate, and D-myo-inositol 1-phosphate, a breakdown product of D-inositol (1,4,5) trisphosphate. Expression studies indicated that all three genes are expressed in multiple tissues, however, IMPL1 expression is restricted to above-ground tissues only. Identification and characterization of impl1 and impl2 mutants revealed no viable mutants for IMPL1, while two different impl2 mutants were identified and shown to be severely compromised in growth, which can be rescued by histidine. Analyses of metabolite levels in impl2 and complemented mutants reveals impl2 mutant growth is impacted by alterations in the histidine biosynthesis pathway, but does not impact myo-inositol synthesis. Together, these data indicate that IMPL2 functions in the histidine biosynthetic pathway, while IMP and IMPL1 catalyze the hydrolysis of inositol- and galactose-phosphates in the plant cell.

Project description:The pathway for the synthesis of di-myo-inositol-phosphate (DIP) was recently elucidated on the basis of the detection of the relevant activities in cell extracts of Archaeoglobus fulgidus and structural characterization of products by nuclear magnetic resonance (NMR) (N. Borges, L. G. Gonçalves, M. V. Rodrigues, F. Siopa, R. Ventura, C. Maycock, P. Lamosa, and H. Santos, J. Bacteriol. 188:8128-8135, 2006). Here, a genomic approach was used to identify the genes involved in the synthesis of DIP. Cloning and expression in Escherichia coli of the putative genes for CTP:l-myo-inositol-1-phosphate cytidylyltransferase and DIPP (di-myo-inositol-1,3'-phosphate-1'-phosphate, a phosphorylated form of DIP) synthase from several (hyper)thermophiles (A. fulgidus, Pyrococcus furiosus, Thermococcus kodakaraensis, Aquifex aeolicus, and Rubrobacter xylanophilus) confirmed the presence of those activities in the gene products. The DIPP synthase activity was part of a bifunctional enzyme that catalyzed the condensation of CTP and l-myo-inositol-1-phosphate into CDP-l-myo-inositol, as well as the synthesis of DIPP from CDP-l-myo-inositol and l-myo-inositol-1-phosphate. The cytidylyltransferase was absolutely specific for CTP and l-myo-inositol-1-P; the DIPP synthase domain used only l-myo-inositol-1-phosphate as an alcohol acceptor, but CDP-glycerol, as well as CDP-l-myo-inositol and CDP-d-myo-inositol, were recognized as alcohol donors. Genome analysis showed homologous genes in all organisms known to accumulate DIP and for which genome sequences were available. In most cases, the two activities (l-myo-inositol-1-P cytidylyltransferase and DIPP synthase) were fused in a single gene product, but separate genes were predicted in Aeropyrum pernix, Thermotoga maritima, and Hyperthermus butylicus. Additionally, using l-myo-inositol-1-phosphate labeled on C-1 with carbon 13, the stereochemical configuration of all the metabolites involved in DIP synthesis was established by NMR analysis. The two inositol moieties in DIP had different stereochemical configurations, in contradiction of previous reports. The use of the designation di-myo-inositol-1,3'-phosphate is recommended to facilitate tracing individual carbon atoms through metabolic pathways.

Dataset Information

Inositol Monophosphatase: A Bifunctional Enzyme in Mycobacterium smegmatis.

Publications

Inositol Monophosphatase: A Bifunctional Enzyme in <i>Mycobacterium smegmatis</i>.

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