Mutational and computational analysis of the role of conserved residues in the active site of a family 18 chitinase

Contains fulltext : 58658.pdf (publisher's version ) (Closed access)Glycoside hydrolysis by retaining family 18 chitinases involves a catalytic acid (Glu) which is part of a conserved DXDXE sequence motif that spans strand four of a (betaalpha)(8) barrel (TIM barrel) structure. These glycoside hydrolases are unusual in that the positive charge emerging on the anomeric carbon after departure of the leaving group is stabilized by the substrate itself (the N-acetyl group of the distorted -1 sugar), rather than by a carboxylate group on the enzyme. We have studied seven conserved residues in the catalytic center of chitinase B from Serratia marcescens. Putative roles for these residues are proposed on the basis of the observed mutational effects, the pH-dependency of these effects, pK(a) calculations and available structural information. The results indicate that the pK(a) of the catalytic acid (Glu144) is 'cycled' during catalysis as a consequence of substrate-binding and release and, possibly, by a back and forth movement of Asp142 between Asp140 and Glu144. Rotation of Asp142 towards Glu144 also contributes to an essential distortion of the N-acetyl group of the -1 sugar. Two other conserved residues (Tyr10 and Ser93) are important because they stabilize the charge on Asp140 while Asp142 points towards Glu144. Asp215, lying opposite Glu144 on the other side of the scissile glycosidic bond, contributes to catalysis by promoting distortion of the -1 sugar and by increasing the pK(a) of the catalytic acid. The hydroxyl group of Tyr214 makes a major contribution to the positioning of the N-acetyl group of the -1 sugar. Taken together, the results show that catalysis in family 18 chitinases depends on a relatively large number of (partly mobile) residues that interact with each other and the substrate

Kinetic, inhibition and structural studies on 3-oxoacyl-ACP reductase from <i>Plasmodium</i> <i>falciparum</i>, a key enzyme in fatty acid biosynthesis

Type II fatty acid biosynthesis represents an attractive target for the discovery of new antimalarial drugs. Previous studies have identified malarial ENR (enoyl acyl-carrier-protein reductase, or FabI) as the target for the antiseptic triclosan. In the present paper, we report the biochemical properties and 1.5 Å (1 Å=0.1 nm) crystal structure of OAR (3-oxoacyl acyl-carrier-protein reductase, or FabG), the second reductive step in fatty acid biosynthesis and its inhibition by hexachlorophene. Under optimal conditions of pH and ionic strength, Plasmodium falciparum OAR displays kinetic properties similar to those of OAR from bacteria or plants. Activity with NADH is &lt;3% of that with NADPH. Fluorescence enhancement studies indicate that NADPH can bind to the free enzyme, consistent with kinetic and product inhibition studies suggesting a steady-state ordered mechanism. The crystal structure reveals a tetramer with a sulphate ion bound in the cofactor-binding site such that the side chains of the catalytic triad of serine, tyrosine and lysine are aligned in an active conformation, as previously observed in the Escherichia coli OAR–NADP+ complex. A cluster of positively charged residues is positioned at the entrance to the active site, consistent with the proposed recognition site for the physiological substrate (3-oxoacyl-acyl-carrier protein) in E coli OAR. The antibacterial and anthelminthic agent hexachlorophene is a potent inhibitor of OAR (IC50 2.05 μM) displaying non-linear competitive inhibition with respect to NADPH. Hexachlorophene (EC50 6.2 μM) and analogues such as bithionol also have antimalarial activity in vitro, suggesting they might be useful leads for further development

The copper catalysed reaction of sodium methoxide with aryl bromides. A mechanistic study leading to a facile synthesis of anisole derivatives

The copper catalysed reaction of unactivated aryl bromides with sodium methoxide has been investigated by studying a number of parameters (copper catalyst, cosolvent, concentration and relative ratio of the reactants, additives and aryl bromide substituents) which influence this reaction. The ipso-substitution reaction was found to proceed via an intimate electron transfer mechanism involving a cuprate-like intermediate, Na[Cu(OMe){2}]. A convenient synthesis of methyl aryl ethers from aryl bromides and concentrated sodium methoxide solutions in dimethyl formamide and methanol is presented. Also an attempt to extend this reaction to the use of chlorine derivatives was made