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.
Saccharomyces cerevisiae possesses three isozymes of 5,10-methylenetetrahydrofolate dehydrogenase (MTD). The NAD-dependent enzyme is the first monofunctional form found in eukaryotes. Here we report its crystallization in a form suitable for high-resolution structure. The space group is P4(2)2(1)2 with cell constants a = b = 75.9, c = 160.0 A, and there is one 36 kDa molecule in the asymmetric unit. Crystals diffract to 2.9 A resolution.
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.
Saccharomyces cerevisiae possesses three isozymes of 5,10-methylenetetrahydrofolate dehydrogenase (MTD). The NAD-dependent enzyme is the first monofunctional form found in eukaryotes. Here we report its crystallization in a form suitable for high-resolution structure. The space group is P4(2)2(1)2 with cell constants a = b = 75.9, c = 160.0 A, and there is one 36 kDa molecule in the asymmetric unit. Crystals diffract to 2.9 A resolution.
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