Properties of chitin synthetase in isolated chitosomes from yeast cells of Mucor rouxii.

Chitin synthetase was isolated and purified 120-fold from the supernatant fraction (54,500 X g) of broken yeast cells of Mucor rouxii. The purified preparations consisted mainly of chitin synthetase particles (chitosomes) with an average size larger than 7 X 10(6) daltons (by gel filtration) and an average sedimentation coefficient of 105 S. The samples also contained other enzyme complexes (fatty acid synthetase, pyruvate dehydrogenase, and, depending on method, ribosomes). Nearly all of the chitosomal chitin synthetase occurred in a zymogenic form that required proteolytic activation. In most properties, the chitosomal enzyme was similar to crude enzyme (54,000 X g sediment): kinetics, activation by proteases, response to metals, stimulation by N-acetylglucosamine, and inhibition by polyoxin or UDP. One mamor difference was the much greater stability of the chitosomal chitin synthetase zymogen against spontaneous activation and destruction. Product (chitin microfibril) and enzyme (chitin synthetase) remained associated in a complex that was readily separated by centrifugation.     

An analysis of core deformations in protein superfamilies

An analysis is presented on how structural cores modify their shape across homologous proteins, and whether or not a relationship exists between these structural changes and the vibrational normal modes that proteins experience as a result of the topological constraints imposed by the fold. A set of 35 representative, well-populated protein families is studied. The evolutionary directions of deformation are obtained by using multiple structural alignments to superimpose the structures and extract a conserved core, together with principal components analysis to extract the main deformation modes from the three-dimensional superimposition. In parallel, a low-resolution normal mode analysis technique is employed to study the properties of the mechanical core plasticity of these same families. We show that the evolutionary deformations span a low dimensional space of 4-5 dimensions on average. A statistically significant correspondence exists between these principal deformations and the approximately 20 slowest vibrational modes accessible to a particular topology. We conclude that, to a significant extent, the structural response of a protein topology to sequence changes takes place by means of collective deformations along combinations of a small number of low-frequency modes. The findings have implications in structure prediction by homology modeling.     

Stabilization of chitin synthetase and purification of chitosomes from several mycelial Mucorales

Stability of chitin synthetase in cell-free extracts from mycelial fungi was markedly improved by the presence of sucrose in the homogenization media. Breakage of mycelium in sucrose-containing buffer yielded enzyme preparations from which chitosomal chitin synthetase could be purified by a procedure involving ammonium sulfate precipitation, gel filtration and centrifugation in sucrose density gradients. Purified chitosomes catalyzed the synthesis of chitin microfibrils in vitro upon incubation with substrate and activators. Chitosomal chitin synthetase from the filamentous form of M. rouxii was similar to the enzyme from yeast cells, except for the poorer stability and diminished sensitivity to GlcNAc activation of the former.     

Nikkomycin-resistant mutants ofMucor rouxii: physiological and biochemical properties

We isolated three nikkomycin-resistant mutants of the dimorphic fungusM. rouxii which were physiologically characterized regarding their response to yeast-phase inducing conditions and their sensitivity to bacilysin. Mutant strains G21 and G23, showed a qualitatively normal, though delayed, dimorphic transition and partial cross-resistance to bacilysin. Mutant strain G27 showed an altered dimorphism, producing a high proportion (50%) of hyphal cells, and a wild-type sensitivity to bacilysin. Cell-free extracts from this mutant exhibited an activity of both basal and protease-activated chitin synthetase which was overexpressed as compared with the parental strain and mutants G21 and G23. Results are discussed in terms of the different genetic background of the mutants.Abbreviations NTG N-methyl-N-nitro-N-nitrosoguanidine - UDP-GlcNAc uridine 5-diphospho-N-acetylglucosamine - GlcNAc N-acetylglucosamine     

Chitin synthetase activity in a developmental mutant of Phycomyces blakesleeanus

Chitin synthetase activity was analyzed in vitro and in vivo in two morphogenetic stages, namely, dormant spore cells and germlings of the wild type strain and the developmental mutant S356 of Phycomyces blakesleeanus. In vitro experiments showed a much higher specific activity in dormant spores of the mutant strain than in those of the wild-type. This difference was restricted to the dormant spore phase since germlings exhibited comparable levels of activity to those detected in the wild-type strain. Although no correlation was observed between chitin synthesis in vitro and in vivo in mutant spores, germination of these cells was accompanied by an earlier expression of chitin synthetase in vivo. Germination of mutant spores in liquid medium produced morphologically aberrant germlings. Contrary to the extended mycelial growth of the wild-type strain in solid medium, the mutant grew with a typical colonial morphology. Results are discussed in relation to the possible basis of the mutant phenotype.     

Core deformations in protein families: a physical perspective

An analysis is presented on how structural cores change shape within protein families, and whether or not there is a relationship between these structural changes and the vibrational modes that proteins experiment due to topological constraints. A set of 13 representative and well-populated protein families are studied. The evolutionary directions of deformation are obtained by applying a new multiple structural alignment technique to superimpose the structures and extract a conserved core, together with Principal Components Analysis (PCA) to extract the main deformation modes. A low-resolution Normal Mode Analysis (NMA) technique is used in parallel to study the properties of the mechanical core plasticity of the same proteins. We find that the evolutionary deformations span a low dimensional space. A statistically significant correspondence exists between these principal deformations and the vibrational modes accessible to a particular topology. We conclude that, to a significant extent, the structures of evolving proteins seem to respond to sequence changes by collective deformations along combinations of low-frequency modes. The findings have implications in structure prediction by homology modeling.     

Altered expression of chitin synthetase activity and biochemical changes in the cell wall in a developmental mutant ofPhycomyces

The Phycomyces developmental mutant S356 elaborates spores which show a much poorer viability and a higher affinity for Calcofluor White than the wild-type spores. Protease-activated extracts of the mutant spores showed higher levels of chitin synthetase activity than the parental strain-derived spores. High levels of enzyme activity in the mutant extracts, but not in the corresponding wild-type extracts, could be detected in the absence of an exogenous protease. The high basal active chitin synthetase is not the result of activation by endogeneous proteases during cell breakage since protease inhibitors did not reduce, but rather increased, the activity levels. The analysis of cell wall composition in the mutant spores revealed significant changes in the proportion of uronic acids and protein but not in chitin. The mutant phenotype is discussed in relation to the developmental stage at which the alterations connected with cell wall metabolism occurred.     

Chitinase activity in germinating cells ofMucor rouxii

Chitinase activity in germinating cells (4 h cultures) ofMucor rouxii was studied. The enzyme activity was recovered in a high speed supernatant of cell homogenates. No activity was detected in the mixed membrane fraction or in the cell walls. Maximum activity was observed at pH 7.6 and at 30–35°C using the chromogenic assay with chitin azure. The latter was digested by GS-chitinase in a manner dependent on substrate concentration and time of incubation. As with other chitinases, GS-chitinase was much more effective against nascent than against preformed chitin. The main product of nascent chitin digestion was diacetylchitobiose, although significant amounts of the trimer were also detected in the hydrolyzates. Allosamidin, an insect and fungal chitinase inhibitor, strongly inhibited hydrolysis of nascent chitin but not of chitin azure by GS-chitinase. The drug failed to inhibit the germination and the ensuing growth of the fungus. Results are discussed in terms of the possible role of GS-chitinase in germination.Abbreviations UDP-GlcNAc uridine 5-diphospho-N-acetylglucosamine - GlcNAc N-acetylglucosamine - GlcNAcP N-acetylglucosamine phosphate - PMB phosphate-magnesium buffer - C₂ N, N-diacetylchitobiose - C₃ N, N, N-triacetylchitotriose - RVB remazol brilliant blue - DPC descending paper chromatography - MU(Ch)₃ 4-methylumbelliferyl-N - N N-triacetylchitotriose     

Isolation and biochemical analysis of Mucor bacilliformis monomorphic mutants.

Fourteen stable mutants of Mucor bacilliformis which grew yeastlike under both aerobic and anaerobic conditions were isolated after treatment of growing mycelium with N-methyl-N'-nitro-N-nitrosoguanidine. Biochemical characterization of the mutants included determination of growth in different carbon and nitrogen sources, determination of sensitivity of respiration to cyanide and salicylhydroxamate, analysis of cytochrome spectra, determination of glutamate dehydrogenases, glutamine synthase, and ornithine decarboxylase activities, and measurement of cyclic AMP levels. Data showed that all mutants were defective in some aspect of oxidative metabolism and had low levels of ornithine decarboxylase, whereas other characters were variable. It was concluded that morphological transition in M. bacilliformis is probably associated with mitochondrial functions and expression of ornithine decarboxylase, but may be independent of cyclic AMP and glutamate dehydrogenase levels. The importance of genetic studies in the analysis of dimorphism is stressed.     

Sterol composition of chitosomes from yeast cells of Mucor rouxii: Comparison with whole cells

Abstract The free sterol composition of chitosomes and whole yeast cells of Mucor rouxii was determined by gas-liquid chromatography (GLC). The results obtained showed that, in contrast to whole cells, chitosomes are particularly rich in ergosterol: 87% vs. 29% of total free sterols. Three other sterols were present in varying amounts in both chitosomes and whole cells. These were identified by GLC as cholesterol, stigmasterol and sitosterol. These results indicate that the yeast cells preferentially accumulate ergosterol in chitosomes providing these microvesicles with a unique composition that may be important for the structure of the organelle.