Chitosan synthesis by the tandem action of chitin synthetase and chitin deacetylase from Mucor rouxii

Davis, Laurel L.; Bartnicki-García, S.

doi:10.1021/bi00301a005

Cited by 157 publications

(90 citation statements)

References 24 publications

(29 reference statements)

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“…The kinetics of chitin synthetase would affect not only chitin but also chitosan synthesis, since the latter is formed by deacetylation of nascent chitin (Davis & Bartnicki-, Garcia, 1984 (Linz & Orlowski, 1982) ; in Mucor racernosus, protein synthesis commences immediately upon addition of nutrient medium (Linz & Orlowski, 1982). Apparently, sporangiospores of these species of Mucor are also poised to begin chitin synthesis immediately at the onset of germination.…”

Section: Levels Of Chitin Synthetase During Developmentmentioning

confidence: 99%

Chitosomes and chitin synthetase in the asexual life cycle of Mucor rouxii: spores, mycelium and yeast cells

Kamada

Bracker²,

Bartnicki-García³

1991

Journal of General Microbiology

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To help understand the subcellular machinery responsible for cell wall formation in a fungus, we determined the abundance and subcellular distribution of chitin synthetase (chitin synthase, EC 2.4.1.16) and chitosomes in the asexual life cycle of Mucor rouxii. Cell-free extracts of ungerminated sporangiospores, hyphaelmycelium in exponential and stationary phase, and yeast cells were fractionated by isopycnic centrifugation in sucrose density gradients. The total amount of chitin synthetase per cell increased exponentially during aerobic germination of spores. In all developmental stages, the profile of chitin synthetase activity encompassed a broad range of sucrose density (d = 1.12-1.22) with two distinct zones: a low-density chitosome zone (d = approx. 1.12-1.16) and a highdensity, mixed-membrane zone (d = approx. 1.16-1.22). Chitosomes were a major reservoir of chitin synthetase in all stages of the life cycle, including ungerminated spores. Two kinds of chitin synthetase profiles were recognized and correlated with the growth state. In nongrowing cells (ungerminated sporangiospores and stationary-phase mycelium), the profile was skewed toward lower densities with a sharp chitosome peak at d = 1.12-1.13. In actively growing cultures (aerobic mycelium or anaerobic yeast cells), the entire profile of chitin synthetase was displaced toward higher densities; the average buoyant density of chitosomes was higher (d= 1-14-1.16), and more chitin synthetase was associated with denser (d= 1.16-1.23) membrane fractions. In all life cycle stages, chitosomal chitin synthetase was almost completely zymogenic. In contrast to the enzyme from spores or from growing cells, samples of chitosomal chitin synthetase from stationary-phase mycelium were unstable and contained a high proportion of larger vesicles in addition to the typical microvesicles. The presence of chitosomes in ungerminated spores indicates that these cells are poised to begin synthesizing somatic (= vegetative) cell walls at the onset of germination. The increased buoyant density of chitosomes in actively growing cultures suggests that the composition of these microvesicles changes significantly as they mobilize chitin synthetase to the cell surface.

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Section: Levels Of Chitin Synthetase During Developmentmentioning

confidence: 99%

Chitosomes and chitin synthetase in the asexual life cycle of Mucor rouxii: spores, mycelium and yeast cells

Kamada

Bracker²,

Bartnicki-García³

1991

Journal of General Microbiology

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“…Thus, they were classified into family 46 of glycosyl hydrolases. 1 So far, no information concerning essential catalytic residues is available for this enzyme family. However, in the case of glycosyl hydrolases, most, if not all, catalytic amino acids are aspartate or glutamate residues conserved in regions sharing amino acid sequence similarities (14,17).…”

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confidence: 99%

Site-directed Mutagenesis of Evolutionary Conserved Carboxylic Amino Acids in the Chitosanase from Streptomyces sp. N174 Reveals Two Residues Essential for Catalysis

Boucher

Fukamizo

Honda

et al. 1995

Journal of Biological Chemistry

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The comparison of four sequences of prokaryotic chitosanases, belonging to the family 46 of glycosyl hydrolases, revealed a conserved N-terminal module of 50 residues, including five invariant carboxylic residues. To verify if some of these residues are important for catalytic activity in the chitosanase from Streptomyces sp. N174, these 5 residues were replaced by site-directed mutagenesis. Substitutions of Glu-22 or Asp-40 with sterically conservative (E22Q, D40N) or functionally conservative (E22D, D40E) residues reduced drastically specific activity and k cat , while K m was only slightly changed. The other residues examined, Asp-6, Glu-36, and Asp-37, retained significant activity after mutation. Circular dichroism studies of the mutant chitosanases confirmed that the observed effects are not due to changes in secondary structure. These results suggested that Glu-22 and Asp-40 are directly involved in the catalytic center of the chitosanase and the other residues are not essential for catalytic activity.

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“…Studies performed to understand the way chitosan biosynthesis proceeds in fungi suggest that chitin synthase is operating in tandem with chitin deacetylase, the first enzyme synthesizing chitin by the polymerization of N-acetylglucosaminyl residues from uridine 5'-diphospho-N-acetylglucosamine (UDP-GlcNAc), and the second enzyme hydrolyzing the N-acetamido groups in the chitin chains, acting more efficiently on nascent than on microfibrillar chitin (12,13).…”

mentioning

confidence: 99%

Bioconversion of chitin to chitosan: purification and characterization of chitin deacetylase from Mucor rouxii.

Kafetzopoulos

Martinou

Bouriotis

1993

Proc. Natl. Acad. Sci. U.S.A.

218

145

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Chitin deacetylase, the enzyme that catalyzes the hydrolysis of acetamido groups of N-acetylglucosamine in chitin, has been purified to homogeneity from mycelial extracts of the fungus Mucor rouxii and further characterized. The enzyme exhibits a low pI (-3). Its apparent molecular mass was determined to be =75 kDa by sodium dodecyl sulfate/polyacrylamide gel electrophoresis and -80 kDa by size-exclusion chromatography, suggesting that the enzyme exists as a monomer. Carbohydrate analysis of purified chitin deacetylase revealed that the enzyme is a high-mannose glycoprotein and that its carbohydrate content is -30% by weight. Chitin deacetylase is active on several chitinous substrates and chitin derivatives. The enzyme requires at least four N-acetylglucosamine residues (chitotetraose) for catalysis, and it is inhibited by carboxylic acids, particularly acetic acid. When glycol chitin (a water-soluble chitin derivative) was used as substrate, the optimum temperature for enzyme activity was determined to be -.50°C and the optimum pH was -4.5.

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Chitosan synthesis by the tandem action of chitin synthetase and chitin deacetylase from Mucor rouxii

Cited by 157 publications

References 24 publications

Chitosomes and chitin synthetase in the asexual life cycle of Mucor rouxii: spores, mycelium and yeast cells

Chitosomes and chitin synthetase in the asexual life cycle of Mucor rouxii: spores, mycelium and yeast cells

Site-directed Mutagenesis of Evolutionary Conserved Carboxylic Amino Acids in the Chitosanase from Streptomyces sp. N174 Reveals Two Residues Essential for Catalysis

Bioconversion of chitin to chitosan: purification and characterization of chitin deacetylase from Mucor rouxii.

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