crystallization communications Acta Cryst. (2011). F67, 471–474 doi:10.1107/S1744309111003496 471 Acta Crystallographica Section F Structural Biology and Crystallization Communications ISSN 1744-3091 Cloning, overexpression, purification, crystallization and preliminary X-ray diffraction analysis of an inositol monophosphatase family protein (SAS2203) from Staphylococcus aureus MSSA476 Sudipta Bhattacharyya, Debajyoti Dutta, Ananta Kumar Ghosh and Amit Kumar Das* Department of Biotechnology, Indian Institute of Technology Kharagpur, Kharagpur, West Bengal 721 302, India Correspondence e-mail: amitk@hijli.iitkgp.ernet.in Received 7 December 2010 Accepted 26 January 2011 The gene product of the sas2203 ORF of Staphylococcus aureus MSSA476 encodes a 30 kDa molecular-weight protein with a high sequence resemblance (29% identity) to tetrameric inositol monophosphatase from Thermotoga maritima. The protein was cloned, expressed, purified to homogeneity and crystallized. Crystals appeared in several conditions and good diffraction-quality crystals were obtained from 0.2 M Li 2 SO 4 , 20% PEG 3350, 0.1 M HEPES pH 7.0 using the sitting-drop vapour-diffusion method. A complete diffraction data set was collected to 2.6 A ˚ resolution using a Rigaku MicroMax-007 HF Cu K X-ray generator and a Rigaku R-AXIS IV ++ detector. The diffraction data were consistent with the orthorhombic space group P2 1 2 1 2 1 , with unit-cell parameters a = 49.98, b = 68.35, c = 143.79 A ˚ ,  =  =  = 90  , and the crystal contained two molecules in the asymmetric unit. 1. Introduction Inositol monophosphatases are ubiquitous proteins with molecular mass ’ 30 kDa which are abundant in bacteria, unicellular eukary- otes and plant and animal cells. This group of enzymes belongs to the FIG superfamily of proteins, which also includes fructose-1,6- bisphosphatases (FBPases; both the major and the glpX-encoded variants), inositol monophosphatases (IMPases) and inositol poly- phosphatases (IPPases). The IMPase group of proteins have two well conserved motifs: motifs A, W-x 0,1 (IV)-D-P-(IL)-D-x-T-x 2 -(FYI)-x- (HK), and B, W-D-x 2 -(AG)-(AG)-x-(AIL)-(ILV)-(ALV)-x 3 -G-(AG) (Neuwald et al., 1991). Most of the members of the IMPase family of proteins exhibit Mg 2+ -activated Li + -inhibited phosphatase activity towards many phosphorylated compounds ranging from inositol monophosphate to sugar and nucleotide phosphates. The degree of Li + inhibition as well as the substrate specificity of this group of enzymes varies greatly depending on the source organism and the molecular basis of this variation is still a matter of controversy. However, in accordance with recent structural studies, the Li + - sensitivity of IMPases can be related to the length and the flexibility of the active-site mobile loop containing the residues binding the third Mg 2+ ion (Johnson et al., 2001; Stieglitz et al., 2002; Li et al., 2010). In IMPases, the participation of the third Mg 2+ ion in phos- phoester hydrolysis is crucial as it can activate the water nucleophile in order to initiate the reaction (Li et al., 2010). Li + , which has almost the same ionic radius as Mg 2+ , can effectively replace the latter, but owing to the difference in their preferred coordination number (four versus six) and the geometries of their coordination complexes (tetrahedral versus octahedral) Li + cannot place the water nucleo- phile in the proper position and hence the phosphatase activity is severely impaired. IMPases have long been identified as one of the key enzymes involved in the phosphatidyl inositol signalling cascade in eukaryotic systems. The role of this enzyme in human beings is well documented as it plays a pivotal role in manic depressive disorder and has been found to be the primary target of Li + -based therapies (Berridge et al. , 1989). However, the role of IMPase in prokaryotes is not precisely known as inositol and its derivatives are not as abundant in # 2011 International Union of Crystallography All rights reserved