Allosamidin is a known inhibitor of class 18 chitinases. We show that allosamidin is a competitive inhibitor of the fungal chitinase CiX1 from Coccidioides immitis, with a K i of 60 nM. We report the X-ray structure of the complex and show that upon inhibitor binding the side-chain of Asp169 rotates to form an ion pair with the oxazolinium cation. The mechanism of action is thought to involve protonation of the leaving group by Glu171 and substrate assistance by the sugar acetamido moiety to form an oxazoline-like intermediate. We converted both amino acid residues to the corresponding amide and found that each mutation effectively abolishes enzyme activity. X-ray structures show the mutant enzymes retain the basic wild-type structure and that the loss of mutant activity is due to their altered chemical properties. The high affinity of allosamidin, and its similarity to the putative reaction intermediate, suggests it is a transition state analog. This helps validate our contention that the role of Asp169 is to electrostatically stabilize the reaction transition state.
The endochitinase from Coccidioides immitis (CiX1) is a member of the class 18 chitinase family. Here we show the enzyme functions by a retaining catalytic mechanism; that is, the -conformation of the chitin substrate linkages is preserved after hydrolysis. The pattern of cleavage of N-acetyglucosamine (GlcNAc) oligosaccharide substrates has been determined. (GlcNAc) 6 is predominantly cleaved into (GlcNAc) 2 and (GlcNAc) 4 , where the (GlcNAc) 2 group arises from the nonreducing end of the substrate and is formed as the -anomer. With time, transglycosylation occurs, generating (GlcNAc) 8 from the product dimer and fresh hexamer. Similar patterns are seen for the cleavage of (GlcNAc) 5 and (GlcNAc) 4 where dimers cleaved from the nonreducing end reflect the most common binding and hydrolysis pattern. Intrinsic fluorescence measurements suggest the dissociation constant for (GlcNAc) 4 is 50 µM. Synthetic substrates with fluorescent leaving groups exhibit complicated profiles in the relationship between initial velocity and substrate concentration, making it difficult to obtain the values of kinetic constants. An improved theoretical analysis of the time-course of (GlcNAc) 6 degradation allows the unitary free energy of binding of the individual subsites of the enzyme to be estimated. The free energy values obtained are consistent with the dissociation constant obtained by fluorescence measurements, and generate a model of substrate interaction that can be tested against the crystal structure of the enzyme.
The X-ray structure of chitinase from the fungal pathogen Coccidioides immitis has been solved to 2.2 Å resolution. Like other members of the class 18 hydrolase family, this 427 residue protein is an eight-stranded b0a-barrel. Although lacking an N-terminal chitin anchoring domain, the enzyme closely resembles the chitinase from Serratia marcescens. Among the conserved features are three cis peptide bonds, all involving conserved active site residues. The active site is formed from conserved residues such as tryptophans 47, 131, 315, 378, tyrosines 239 and 293, and arginines 52 and 295. Glu171 is the catalytic acid in the hydrolytic mechanism; it was mutated to a Gln, and activity was abolished. Allosamidin is a substrate analog that strongly inhibits the class 18 enzymes. Its binding to the chitinase hevamine has been observed, and we used conserved structural features of the two enzymes to predict the inhibitors binding to the fungal enzyme.
Chitinase is necessary for fungal growth and cell division and, therefore, is an ideal target for the design of inhibitors which may act as antifungal agents. A chitinase from the fungal pathogen Coccidioides immitis has been expressed as a fusion protein with gluathione-S-transferase (GST), which aids in puri®cation. After cleavage from GST, chitinase was crystallized from 30% PEG 4000 in 0.1 M sodium acetate pH 4.6. The crystals have a tetragonal crystal lattice and belong to space group P4 1 2 1 2 or P4 3 2 1 2 and diffract to 2.2 A Ê resolution. The unit-cell parameters are a = b = 91.2, c = 95.4 A Ê ; there is only one chitinase molecule in the asymmetric unit.
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