Faculty of Arts & Science
Biochemistry

Email: steven.mosimann@uleth.ca

Webpage: http://people.uleth.ca/~steven.mosimann/

Research

One research focus within my group is the development of methods for the large-scale production of a series of bioactive, myo-inositol polyphosphates (IPP, Fig. 1) that are not commercially available or are prohibitively expensive. Of the more than 20 different IPPs detected in eucaryotes, at least eight function as cofactors and signal transduction molecules. The most (and only) abundant IPP is myo-inositol hexakisphosphate (IP6 or phytate) which occurs at exceptionally high-levels in grain seeds (up to 2% of the dry weight)¹.

In order to produce IPPs of interest in a cost effective and environmentally friendly manner, we are bioengineering the substrate specificity of a recently discovered family of myo-inositol polyphosphatases (IPPases) that utilize InsP₆ as a substrate. These enzymes remove phosphoryl groups one-by-one following a preferred, ordered pathway (Figure 2)².  To date, this collaborative research program has identified related IPPases in more than 30 different procaryotic organisms.  The naturally-occurring IPPases have been utilized to prepare otherwise unavailable IPPs, including Ins(1,2,4,5,6)P₅, Ins(2,4,5,6)P₄ and several InsP₃s (2,4,5 and 2,4,6).

In an effort to understand the substrate specificity and kinetic mechanism of these IPPases, we have determined the X-ray crystallographic structure of several IPPases, both alone and in complex with naturally occurring substrates (Figure 3)^3-5.  These studies have allowed us to identify specific interactions between the enzyme and substrate that determine the substrate specificity.  Using this information, we are able to rationally predict site-directed mutations that alter the substrate specificity of these enzymes.  Currently, we are  bioengineering IPPases, that will allow us to prepare additional IPPs that are otherwise unavailable.  In parallel with this work, we are also extending our structural studies to include less phosphorylated forms of IPP.  These studies will complement our earlier work and allow us to complete our identification of specificity determinants within the IPPase active site.

To further our understanding of the substrate specificity of these enzymes, we have developed IPPase constructs that can be labeled with a fluorophore (5-iodoacetamidofluorescein)⁵. These constructs are currently being used in both substrate binding assay and pre-steady state kinetic studies in collaboration with Dr. HJ Wieden.

Selected Publications

R.J. Gruninger, S. Dobing, A.D. Smith, L.M. Bruder, H.J. Wieden, L.B. Selinger, and S.C. Mosimann. Substrate binding in protein tyrosine phosphatase-like inositol polyphosphatases. J. Biol. Chem., 287(13) :9722-9730 (2012)

R.J. Gruninger, L.B. Selinger and S.C. Mosimann. Structural analysis of a multifunctional, tandemly repeated inositol polyphosphatase. J Mol Biol., 392(1):75-86 (2009).*

* Special Edition: 50 years of structure determination

R.J. Gruninger, L.B. Selinger and S.C. Mosimann. Effect of ionic strength and oxidation of the P-loop conformation of the protein tyrosine phosphatase-like phytase, PhyAsr. FEBS Journal., 275:3783-3792 (2008)

A. Puhl, R.J. Gruninger, T. Janzen, R. Griener, S.C. Mosimann and L.B. Selinger. Kinetic and structural analysis of a protein tyrosine phosphatase-like myo-inositol polyphosphatase.  Prot .Sci., 16: 1368-1378 (2007)

Protein Data Bank Entries

2011 – 3O3L

2010 – 3MOZ,3LOL

2008 – 3D1Q, 3D1O, 3D1H,2PSZ,2PTO

Current Group Members

1. Lisza Bruder (NSERC CGM)
2. Ben Farnell
3. Peter Van Herk
4. Justin Yamashita
5. Ben Van Driesten
6. Derek Waldner