N,N′ Bisformyl Dityrosine is an in vivo Precursor of the Yeast Ascospore Wall

Abstract

The amino acid, dityrosine, is a major component of the spore wall surface of the yeast Saccharomyces cerevisiae, where it is part of a highly cross-linked macromolecular network of yet unknown chemical structure, consisting mostly of glucosamine, dityrosine and few other amino acids. Biosynthesis of the dityrosine moiety of this network consists of several steps, including the chemical modification of free L-tyrosine and the subsequent oxidative cross-linking of the modified tyrosine residues (catalyzed by a cytochrome P-450), leading to soluble dityrosine-containing spore wall precursors. We isolated, purified and characterized the dityrosine-containing precursor that appears late in spore wall synthesis and that is thought to be directly incorporated into the maturing spore wall. Chemical and spectroscopic analyses showed that this precursor is N,N'-bisformyl dityrosine. In addition, we identified a tyrosine-containing spore wall precursor as N-formyl tyrosine. The elucidation of the chemical structure of soluble spore wall precursors is crucial for the characterization of the function of the enzymes involved in maturation of the spore surface, e.g. by in vitro systems. A dityrosine-containing fragment, which was solubilized from mature spore walls by partial hydrolysis, was identified as N-formyl dityrosine. Mature spore walls contain significant amounts of N-formyl dityrosine and N,N'-bisformyl dityrosine. This supports the assumption that the dityrosine-containing macromolecular network on the spore surface has an unusual, non-peptidic structure.

Yeast ascospore wall assembly requires two chitin deacetylase isozymes

“…It was previously shown that the outermost layer of the yeast spore wall consists of a dityrosine-rich polymer closely associated with the underlying chitosan layer. It was speculated that chitosan is a prerequisite for the addition of the dityrosine layer [2]. In accordance with this assumption we detected only very low levels of dityrosine when most of the chitin was acetylated.…”

“…CDA from the fungus M. rouxii [14], which is inhibited by acetic acid [3], is able to deacetylate chitin oligosaccharides with a degree of polymerization higher than 3 and it fully deacetylates only (GlnNAc) 4 and (GlnNAc) 5 . C. lindemuthianum CDA, on the other hand, which is not inhibited by acetic acid [4], is able to deacetylate (GlnNAc) 2 and fully deacetylates (GlnNAc) 3 to (GlnNAc) 5 [13]. Such di¡erences in speci¢city might contribute to the formation of a set of partially deacetylated chitin oligomers important in a number of cases [15^17].…”

“…spore walls In previous studies [2,6] it was speculated that the deacetylation of the chitin layer by the action of the two chitin deacetylases plays an important role in the subsequent addition of the external dityrosine-rich layer of the walls. In order to investigate this role we compared the levels of dityrosine, total glucosamine and glucosamine corresponding to chitosan in wild type and CDA deletion strains.…”

“…The two outer layers confer on the spores the characteristic resistance against environmental stress. [1,2] Chitin deacetylase (CDA) is the enzyme that catalyzes the conversion of chitin to chitosan by deacetylation of N-acetyl-D-glucosamine residues. So far, CDA enzymes have been isolated from the fungi Mucor rouxii and Colletotrichum lindemuthianum [3,4] and genes encoding CDA have been studied in M. rouxii and Saccharomyces cerevisiae [5,6].…”

Sporulation‐specific expression of the yeast DIT1/DIT2 promoter is controlled by a newly identified repressor element and the short form of Rim101p

“…The CEN plasmids carrying the constructs were introduced into the yeast strain W303-euro-a. After growth on selective medium, cells were analysed for the presence of dityrosine, which is the product of the reactions mediated by Dit1p and Dit2p and hence an indicator of the ectopic expression of DIT1 and DIT2 [22]. Cells harbouring the DIT1 and DIT2 genes with their wild-type promoter region did not contain detectable amounts of dityrosine (Fig.…”