Crystal Structure of Agaricus bisporus Mushroom Tyrosinase: Identity of the Tetramer Subunits and Interaction with Tropolone

Tyrosinase catalyzes the conversion of phenolic compounds into their quinone derivatives, which are precursors for the formation of melanin, a ubiquitous pigment in living organisms. Because of its importance for browning reactions in the food industry, the tyrosinase from the mushroom Agaricus bisporus has been investigated in depth. In previous studies the tyrosinase enzyme complex was shown to be a H2L2 tetramer, but no clues were obtained of the identities of the subunits, their mode of association, and the 3D structure of the complex. Here we unravel this tetramer at the molecular level. Its 2.3 Å resolution crystal structure is the first structure of the full fungal tyrosinase complex. The complex comprises two H subunits of ~392 residues and two L subunits of ~150 residues. The H subunit originates from the ppo3 gene and has a fold similar to other tyrosinases, but it is ~100 residues larger. The L subunit appeared to be the product of orf239342 and has a lectin-like fold. The H subunit contains a binuclear copper-binding site in the deoxy-state, in which three histidine residues coordinate each copper ion. The side chains of these histidines have their orientation fixed by hydrogen bonds or, in the case of His85, by a thioether bridge with the side chain of Cys83. The specific tyrosinase inhibitor tropolone forms a pre-Michaelis complex with the enzyme. It binds near the binuclear copper site without directly coordinating the copper ions. The function of the ORF239342 subunits is not known. Carbohydrate binding sites identified in other lectins are not conserved in ORF239342, and the subunits are over 25 Å away from the active site, making a role in activity unlikely. The structures explain how calcium ions stabilize the tetrameric state of the enzyme.

Shortening of membrane lipid acyl chains compensates for phosphatidylcholine deficiency in choline-auxotroph yeast

Phosphatidylcholine (PC) is an abundant membrane lipid component in most eukaryotes, including yeast, and has been assigned multiple functions in addition to acting as building block of the lipid bilayer. Here, by isolating S. cerevisiae suppressor mutants that exhibit robust growth in the absence of PC, we show that PC essentiality is subject to cellular evolvability in yeast. The requirement for PC is suppressed by monosomy of chromosome XV or by a point mutation in the ACC1 gene encoding acetyl-CoA carboxylase. Although these two genetic adaptations rewire lipid biosynthesis in different ways, both decrease Acc1 activity, thereby reducing average acyl chain length. Consistently, soraphen A, a specific inhibitor of Acc1, rescues a yeast mutant with deficient PC synthesis. In the aneuploid suppressor, feedback inhibition of Acc1 through acyl-CoA produced by fatty acid synthase (FAS) results from upregulation of lipid synthesis. The results show that budding yeast regulates acyl chain length by fine-tuning the activities of Acc1 and FAS and indicate that PC evolved by benefitting the maintenance of membrane fluidity

Quantitative trait locus mapping for bruising sensitivity and cap color of Agaricus bisporus (button mushrooms)

White button mushrooms discolor after mechanical damage of the cap skin. This hampers the development of a mechanical harvest system for the fresh market. To unravel the genetic basis for bruising sensitivity, two haploid populations (single spore cultures) were generated derived from crosses between parental lines differing in discoloration after mechanical damage (bruising sensitivity). The haploids were crossed with different homokaryotic tester lines to generate mushrooms and allow assessment of the bruising sensitivity in different genetic backgrounds. Bruising sensitivity appears to be a polygenic highly heritable trait (H2: 0.88-0.96) and a significant interaction between genotypes and tester lines and genotypes and flushes was found. Using SNP markers evenly spread over all chromosomes, a very low recombination was found between markers allowing only assignment of QTL for bruising sensitivity to chromosomes and not to sub-regions of chromosomes. The cap color of the two parental lines of population 1 is white and brown respectively. A major QTL for bruising sensitivity was assigned to chromosome 8 in population 1 that also harbors the main determinant for cap color (brown versus white). Splitting offspring in white and non-white mushrooms made minor QTL for bruising sensitivity on other chromosomes (e.g. 3 and 10) more prominent. The one on chromosome 10 explained 31% phenotypic variation of bruising sensitivity in flush 2 in the subpopulations of population 1. The two parental lines of population 2 are both white. Major QTL of bruising sensitivity were detected on chromosome 1 and 2, contributing totally more than 44% variation of the bruising sensitivity in flush 1 and 54% variation of that in flush 2. A considerable consistency was found in QTL for bruising sensitivity in the different populations studied across tester lines and flushes indicating that this study will provide a base for breeding cultivars that are less sensitive for bruising allowing the use of mechanical harvest and automatic postharvest handling for produce for the fresh market. The low recombination between homologous chromosomes, however, underlines the need to introduce a normal recombination pattern found in a subspecies of the button mushroom

Main Phenolic Compounds of the Melanin Biosynthesis Pathway in Bruising-Tolerant and Bruising-Sensitive Button Mushroom (Agaricus bisporus) Strains

Browning is one of the most common postharvest changes in button mushrooms, which often results in economic losses. Phenolic compounds, which are associated with browning, were extracted from the nonbruised and bruised skin tissue of various button mushrooms with a sulfite-containing solution and analyzed with UHPLC-PDA-MS. In total, 34 phenolic compounds were detected. Only small differences in the total phenolic content between bruising-tolerant and -sensitive strains were observed. The contents of γ-l-glutaminyl-4-hydroxybenzene (GHB) and γ-l-glutaminyl-3,4-dihydroxybenzene (GDHB) correlated with bruising sensitivity; for example, <i>R</i>² values of 0.85 and 0.98 were found for nonbruised brown strains, respectively. In nonbruised skin tissue of the strains with brown caps, the GHB and GDHB contents in sensitive strains were on average 20 and 15 times higher, respectively, than in tolerant strains. GHB and GDHB likely participate in the formation of brown GHB–melanin, which seemed to be the predominant pathway in bruising-related discoloration of button mushrooms

Main Phenolic Compounds of the Melanin Biosynthesis Pathway in Bruising-Tolerant and Bruising-Sensitive Button Mushroom (Agaricus bisporus) Strains

Browning is one of the most common postharvest changes in button mushrooms, which often results in economic losses. Phenolic compounds, which are associated with browning, were extracted from the nonbruised and bruised skin tissue of various button mushrooms with a sulfite-containing solution and analyzed with UHPLC-PDA-MS. In total, 34 phenolic compounds were detected. Only small differences in the total phenolic content between bruising-tolerant and -sensitive strains were observed. The contents of γ-l-glutaminyl-4-hydroxybenzene (GHB) and γ-l-glutaminyl-3,4-dihydroxybenzene (GDHB) correlated with bruising sensitivity; for example, <i>R</i>² values of 0.85 and 0.98 were found for nonbruised brown strains, respectively. In nonbruised skin tissue of the strains with brown caps, the GHB and GDHB contents in sensitive strains were on average 20 and 15 times higher, respectively, than in tolerant strains. GHB and GDHB likely participate in the formation of brown GHB–melanin, which seemed to be the predominant pathway in bruising-related discoloration of button mushrooms

Shortening of membrane lipid acyl chains compensates for phosphatidylcholine deficiency in choline-auxotroph yeast

Phosphatidylcholine (PC) is an abundant membrane lipid component in most eukaryotes, including yeast, and has been assigned multiple functions in addition to acting as building block of the lipid bilayer. Here, by isolating S. cerevisiae suppressor mutants that exhibit robust growth in the absence of PC, we show that PC essentiality is subject to cellular evolvability in yeast. The requirement for PC is suppressed by monosomy of chromosome XV or by a point mutation in the ACC1 gene encoding acetyl-CoA carboxylase. Although these two genetic adaptations rewire lipid biosynthesis in different ways, both decrease Acc1 activity, thereby reducing average acyl chain length. Consistently, soraphen A, a specific inhibitor of Acc1, rescues a yeast mutant with deficient PC synthesis. In the aneuploid suppressor, feedback inhibition of Acc1 through acyl-CoA produced by fatty acid synthase (FAS) results from upregulation of lipid synthesis. The results show that budding yeast regulates acyl chain length by fine-tuning the activities of Acc1 and FAS and indicate that PC evolved by benefitting the maintenance of membrane fluidity