High-resolution (1.5 Å) crystal structure of phospholipase C from Bacillus cereus

Abstract

Both the phosphatidylinositol-hydrolysing and the phosphatidylcholine-hydrolysing phospholipases C have been implicated in the generation of second messengers in mammalian cells. The phosphatidylcholine-hydrolysing phospholipase C (PLC) from Bacillus cereus, a monomeric protein containing 245 amino-acid residues, is similar to some of the corresponding mammalian proteins. This, together with the fact that the bacterial enzyme can mimic the action of mammalian PLC in causing, for example, enhanced prostaglandin biosynthesis, suggests that B. cereus PLC can be used as a model for the hitherto poorly characterized mammalian PLCs. We report here the three-dimensional structure of B. cereus PLC at 1.5 A resolution. The enzyme is an all-helix protein belonging to a novel structural class and contains, at least in the crystalline state, three Zn2+ in the active site. We also present preliminary results from a study at 1.9 A resolution of the complex between PLC and inorganic phosphate (Pi) which indicate that the substrate binds directly to the metal ions.

UDP-glucose Deficiency Causes Hypersensitivity to the Cytotoxic Effect of Clostridium perfringens Phospholipase C

“…Ca 2ϩ is required for the interaction of the toxin with membranes, whereas Zn 2ϩ plays a structural role. Crystallographic studies have revealed that the B. cereus PC-PLC has two tightly bound and one loosely bound Zn 2ϩ ions, which are required for the enzymatic activity (48). These Zn 2ϩ ions are coordinated to several residues conserved in the C. perfringens PLC, which suggests that these two enzymes have the same catalytic mechanism.…”

Spectroscopic and mechanistic studies of dinuclear metallohydrolases and their biomimetic complexes

“…However, this closed shell configuration causes problems for the (bio)chemist studying Zn(II) systems as this configuration is spectroscopically silent. The problem is often solved by replacing, in vitro, Zn(II) with Co(II) or Cu(II) and utilising the excellent spectroscopic properties of these ions, 109 or with Cd(II) and using 113 Cd-NMR spectroscopy as a probe. 110 In addition to dinuclear Zn(II) hydrolases, mono-and trinuclear enzymes are also common in nature.…”

“…80 The natural abundance of 113 Cd is 12.22% 153 and it is very sensitive to environment changes due to the large surrounding electron cloud. It is also a spin 1/2 nucleus which generally yields sharp NMR signals, making 113 Cd NMR spectroscopy a useful method for structure elucidation in biological, inorganic and organometallic cadmiumcontaining samples. 110,154 Cd(II) is known to be extremely toxic to mammals, hence it is generally viewed as an element that is not used by nature.…”

Phosphate Diester Cleavage, DNA Interaction and Cytotoxic Activity of a Bimetallic Bis(1,4,7‐triazacyclononane) Zinc Complex

“…and examples of hydrolytically active zinc enzymes include P1 nuclease, [10] alkaline phosphatase, [11] phospholipase C, [12] and aminopeptidase. [13] The catalytic role of zinc in these enzymes includes Lewis acid activation of the substrate and the formation of Zn-OH which can act as an efficient nucleophile or as a general base catalyst at physiological pH.…”