The properties and localization of Saprolegnia monoica chitin synthase differ from those of other fungi

Abstract

a n d Sa I om 6 n B a r t n i c k i -G a rc fa3 The presence of non-fibrillar a-chitin in cellulosic fungi (class Oomycetes) poses intriguing questions as to i t s role, subcellular localization and evolutionary significance. Previous studies reported on the similarity of chitin synthase from Saprolegnia monoica with that of other fungi. The present work describes important dissimilarities. There was no evidence that the chitin synthase of S. monoica was present in small low-density vesicles (chitosomes). Chitin synthase sedimented with membranous components of high specific gravity (sp. gr. 1-177) that could be partially but distinctly separated from membranes harbouring most of the 1,3-&glucan synthase in the cell (sp. gr. 1.158).In contrast to other fungi, the chitin synthase from 5. monoica was greatly stimulated by digitonin: both membrane-bound and dissociated chitin synthase showed little activity in the absence of digitonin. As in other fungi, the chitin synthase from S. monoica was solubilized by digitonin and remained zymogenic after dissociation. However, unlike the enzyme from other fungi, the solubilized chitin synthase of S. monoica had a lower sedimentation coefficient, was not stimulated by phospholipids and was not inhibited by high concentrations of digitonin. Unlike the enzyme from Mucor rouxii, the solubilized chitin synthase from 5. monoica did not bind to a cation exchanger. The enzyme was partially purified by a four-step scheme that included sucrose density-gradient centrifugation, a single passage through a strong anion exchanger and two consecutive passages through a weak anion exchanger. The final preparation contained five to seven polypeptide bands that cochromatographed with the chitin synthase activity, some of which may be part of a presumed chitin synthase macromolecular complex.

Purification of an active, oligomeric chitin synthase complex from the midgut of the tobacco hornworm

“…Previous studies on fungal chitin synthases also reported high molecular mass complexes of about 400-800 kDa when analyzed under non-denaturing conditions (Braun and Calderone, 1979;Hardy and Gooday, 1983;Kang et al, 1984;Leal-Morales et al, 1997;Machida and Saito, 1993;Montgomery et al, 1984;Ruiz-Herrera et al, 1980). However, these complexes were not explicitly assigned as chitin synthase oligomers.…”

The CAZyome of Phytophthora spp.: A comprehensive analysis of the gene complement coding for carbohydrate-active enzymes in species of the genus Phytophthora

“…Even more intriguing is the presence of a fifth member of the GT family 2 that matches very strongly ( E value = 0) the putative chitin synthase gene from Magnaporthe grisea . Although no direct evidence exists favoring the presence of chitin in P. infestans cell wall, the existence of chitin synthase genes has been demonstrated in other oomycetes [54,55], suggesting that chitin is indeed produced in these species. We have cloned this gene from both P. infestans and P. sojae and preliminary experiments indicate that the gene is expressed in cultures grown in vitro .…”

Cell Wall Chitosaccharides Are Essential Components and Exposed Patterns of the Phytopathogenic OomyceteAphanomyces euteiches

“…Both organisms have a glucose-rich cell wall that in addition contains GlcNAc (12), and WGA labeling showed in both organisms that the GlcNAc components are not present only in the inner cell wall layers, in contrast to what is usually observed in true fungi (16,70,84). Leal-Morales et al (50) mentioned that the GlcNAc-containing material was uniformly distributed throughout the mycelial walls of Saprolegnia, a feature that is likely to occur also in Aphanomyces, as the proportion of GlcNAc in KOH-MeOH cell walls, where the amorphous superficial layers have been solubilized by treatment with hot KOH-MeOH, was the same as in crude cell walls. In Saprolegnia, however, the GlcNAc components are true crystalline chitin that accounts for only 0.7% of the cell wall and is not essential for hyphal growth (12), whereas in Aphanomyces these components are noncrystalline chitosaccharides that account for ca.…”

“…However, the cell wall of the closely related hyphochytridiomycetes contains both cellulose and chitin, and chitin was unambiguously detected by biophysical analyses in some oomycete species of the Leptomitale and Saprolegniale orders (2,12). In Saprolegnia monoica, a chitin synthase (CHS) activity was characterized in detail, and two CHS genes were identified, among which one was fully sequenced (50,54). In the Peronosporale and Pythiale orders, partial putative CHS sequences were identified in Plasmopara viticola and Phytophthora capsici (54,87).…”