Structural dynamics of the Ca2+-regulated cutinase towards structure-based improvement of PET degradation activity.

We previously revealed the structural basis of Ca2+ dependent regulation of a polyethylene terephthalate (PET)-degrading enzyme, Cut190, and proposed a unique reaction cycle in which the enzyme repeatedly binds and releases Ca2+. Here, we report crystal structures of Cut190 mutants with high thermal stability complexed with PET-like ligands that contain aromatic rings. The structural information has allowed us to perform further computational analyses using a PET-trimer bound model. Our multicanonical molecular dynamics simulations and subsequent analyses of the free energy landscapes revealed a novel intermediate form that occurs during the enzymatic reaction cycle. Furthermore, the computational analyses were used to investigate the effect of the point mutations F77L and F81L in the Ca2+-binding site, which showed that the former stabilizes the engaged and open forms to improve transition between the open and active forms, while the latter extremely increases the open form. Subsequent experiments showed that the F77L mutation increased the activity, while the F81L mutation decreased the activity. Our computational analysis has enabled us to explore the dynamics of Cut190 on a completely new level, providing key insights into how the balance between the various conformations influences the reaction cycle and ultimately how to improve the reaction cycle.

Genetic analysis of pungency deficiency in Japanese chili pepper 'Shishito' (Capsicum annuum) revealed its unique heredity and brought the discovery of two genetic loci involved with the reduction of pungency.

The sensation of pungency generated by capsaicinoids is a characteristic trait of chili peppers (Capsicum spp.), and the presence or absence of pungency is central in determining its usage as a spice or a vegetable. In the present study, we aimed to clarify the heredity and genetic factors involved in the deficiency of pungency (quite low pungency) that is uniquely observed in the Japanese chili pepper 'Shishito' (Capsicum annuum). First, the F2 population ('Shishito' × pungent variety 'Takanotsume') was used for segregation analysis, and pungency level was investigated using capsaicinoid quantification with high-performance liquid chromatography. Also, restriction site associated DNA sequencing of the F2 population was performed, and genetic map construction and quantitative trait locus (QTL) mapping were implemented. The results indicated that the F2 population showed varying capsaicinoid content and two major QTLs were detected, Shql3 and Shql7, which explained 39.8 and 19.7% of the genetic variance, respectively. According to these results, the quite low pungency of 'Shishito' was a quantitative trait that involved at least the two loci. Further, this trait was completely separate from general non-pungent traits controlled by individual recessive genes, as described in previous studies. The present study is the first report to investigate the genetic mechanism of pungency deficiency in Japanese chili peppers, and our results provide new insights into the genetic regulation of pungency in chili pepper.

The pungent-variable sweet chili pepper 'Shishito' (Capsicum annuum) provides insights regarding the relationship between pungency, the number of seeds, and gene expression involving capsaicinoid biosynthesis.

Pungent traits caused by capsaicinoids are characteristic of chili peppers (Capsicum spp.), and the pungent-variable sweet chili pepper 'Shishito' (Capsicum annuum) is unique in being known for the pungency in fruits with few seeds. In the present study, we tried to clarify the relationship between the number of seeds and pungency in 'Shishito'. First, we investigated the pungency of 'Shishito' by simple sensory evaluations and quantifications of capsaicinoids by high-performance liquid chromatography. As a result, few-seeded fruits had a larger fluctuation of capsaicinoid content than many-seeded ones. This indicates that the number of seeds, in particular a decrease of the seeds, has some sort of connection with the pungency of 'Shishito'. Then, we analyzed the relationship between pungency and gene expression involving capsaicinoid biosynthesis at the individual fruit level. We vertically separated the placental septum in which capsaicinoids are synthesized and performed quantitative reverse transcription-polymerase chain reaction (qRT-PCR) for 18 genes involved in capsaicinoid biosynthesis. We also used the placental septum for capsaicinoid quantification so that the gene expression levels and capsaicinoid contents in the same fruits were obtained, and their correlations were analyzed using 20 biological replicates. Among the 18 genes, expression levels of 11 genes (WRKY9, CaMYB31, AT3, BCAT, BCKDH, KAS I, KAS III, ACL, CaKR1, FAT, and pAMT) had a significant positive correlation with the capsaicinoid concentration, and they were considered to upregulate capsaicinoid biosynthesis. These results provide new insights regarding the environmental variation of the pungency traits in chili peppers and the relationship between pungency, the number of seeds, and gene expression involved in capsaicinoid biosynthesis.