TETRAHEDRON LETTERS Tetrahedron Letters 39 (1998) 6069-6072 Pergamon Recombinant Bacillus subtilis Whole Cell System as a Catalyst for Enzymatic Synthesis of Cyclic Inositol Phosphate Anil K. Tyagi and Ram A. Vishwakarma* Bio-organic Chemistry Laboratory National Institute of Immunology Aruna Asaf All Marg, JNU Complex New Delhi 110067, India Received 1 May 1998; accepted 15 June 1998 Abstract: Whole cell culture of recombinant bacteria Bacillus subtilis, over expressing the phosphoinositide specific phospholipase C (PI-PLC) gene of Bacillus thuringiensis has been used as an efficient catalyst for practical muhigram scale synthesis of D-myo-inositol 1,2-cyclic monophosphate directly from phospholipid mixtures containing phosphatidyi-inositol. This synthesis does not require protein purification and growing bacterial culture can be directly used. The reaction can be monitored by in-situ 31p NMR. © 1998 Published by Elsevier Science Ltd. All rights reserved. Phosphoinositide specific phospholipase C (PI-PLC, EC 3.1.4.10) enzyme catalyses the cleavage of the sn-3-phosphodiester bond of phosphatidylinositol (PI, 1 in Scheme-l) into lipid soluble diacyl glycerol (DAG, 3) and water soluble inositol-l-phosphate [I(1)P, 4]. The enzyme plays a central role in cellular signal transduction cascades by generating second messenger molecules; DAG which activates protein kinase C (PKC) isozymes and inositol 1,4,5-triphosphate which promotes Ca 2+ ion release from the internal o stores of cells and further signal transduction J[ J .~---~ .... ~ ~ .~ / OH O O - / v \ ~ ~ events. 1,2 PI-PLC class of enzymes are .fi..~ " [ HO O--P--O / O ,~ ~ ~\ ~ / ~ ~\ produced by a number of gram positive no'~ 'on o bacteria e.g. Bacillus species, on 1 Staphylococcus aureus and Listeria ] P,-PLe w t monocytogens, and also by' higher eukaryotes. The physiological role of °o o bacterial PI-PLCs, which are much smaller Ho..~ ~ ..~b o ~u\~ .... . /~, ~ ...... . . in size (~ 300 amino acid residues) as ,,~.~ J .o ~ o H O { / 'OH \/ \ / "]l/~" \ /~\~ " ...... "\~ " • " /" / compared to their mammalian counterparts, is on o 3 not well understood. However it is 2 H~O --..[ interesting that the bacterial, and not p,-pLc mammalian PI-PLC, can cleave the glycosyl OH O phosphatidyl inositol (GPI) anchored Ho .~ 02' oH membrane proteins and carbohydrates 3. "~ ( ~--- HO" ~"[" 'OH - There has been significant interest on these on enzymes for their biological mechanism, 4 and 4 Schme-1 a number of them have been cloned and sequenced. 5 The X-ray structural, 6 site-directed mutagenesis 7 and isotope labelling8 studies have shown that bacterial PI-PLC combines two activities; a phosphotransferase activity producing D-myo-inositol 1,2-cyclic monophosphate [l(l:2cyc)P] (2) with overall retention of configuration at the phosphorus atom, and a 0040-4039/98/$ - see front matter © 1998 Published by Elsevier Science Ltd. All rights reserved. Pll: S0040-4039(98)01250-7