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Enzymes have evolved to rapidly and selectively hydrolyze diverse natural and anthropogenic polymers, but only a limited group of related enzymes have been shown to hydrolyze synthetic polyamides. In this work, we synthesized and characterized a panel of 95 diverse enzymes from the N-terminal nucleophile hydrolase superfamily with 30-50% pairwise amino acid identity. We found that nearly 40% of the enzymes had substantial nylon hydrolase activity, in many cases comparable to that of the best-characterized nylon hydrolase, NylC. There was no relationship between phylogeny and activity, nor any evidence of prior selection for nylon hydrolase activity. Several newly-identified hydrolases showed significant substrate selectivity, generating up to 20-fold higher product titers with Nylon 6,6 versus Nylon 6. Finally, we determined the crystal structure and oligomerization state of a Nylon 6,6-selective hydrolase to elucidate structural factors that could affect activity and selectivity. These new enzymes provide insights into the widespread potential for nylon hydrolase evolution and opportunities for analysis and engineering of improved hydrolases., Competing Interests: Conflict of Interest E.E.D, J.F.C, P.M.B.S-V, S.H.C, and J.K.M. are inventors on a patent application involving enzymes described in this work.

Nylon hydrolase (NylC), a member of the N-terminal nucleophile (Ntn) hydrolase superfamily, is responsible for the degradation of various aliphatic nylons, including nylon-6 and nylon-66. NylC is initially expressed as an inactive precursor (36 kDa), but the precursor is autocatalytically cleaved at Asn266/Thr267 to generate an active enzyme composed of 27 and 9 kDa subunits. We isolated various mutants with amino acid changes at the catalytic centre. X-ray crystallographic analysis revealed that the NylC precursor forms a doughnut-shaped quaternary structure composed of four monomers (molecules A-D) with D2 symmetry. Catalytic residues in the precursor are covered by loop regions at the A/B interface (equivalent to the C/D interface). However, the catalytic residues are exposed to the solvent environment through autocleavage followed by movements of the loop regions. T267A, D306A and D308A mutations resulted in a complete loss of autocleavage. By contrast, in the T267S mutant, autocleavage proceeded slowly at a constant reaction rate (k = 2.8 × 10 -5  s -1 ) until complete conversion, but the reaction was inhibited by K189A and N219A mutations. Based on the crystallographic and molecular dynamic simulation analyses, we concluded that the Asp308-Asp306-Thr267 triad, resembling the Glu-Ser-Ser triad conserved in Ntn-hydrolase family enzymes, is responsible for autocleavage and that hydrogen-bonding networks connecting Thr267 with Lys189 and Asn219 are required for increasing the nucleophilicity of Thr267-OH in both the water accessible and water inaccessible systems. Furthermore, we determined that NylC employs the Asp308-Asp306-Thr267 triad as catalytic residues for substrate hydrolysis, but the reaction requires Lys189 and Tyr146 as additional catalytic/substrate-binding residues specific for nylon hydrolysis., (© 2023 Federation of European Biochemical Societies.)

Successful sexual reproduction of plants requires temperature-sensitive processes, and temperature stress sometimes causes developmental asynchrony between male and female reproductive tissues. In Arabidopsis thaliana, self-pollination occurs when the stamen and pistil lengths are aligned in a single flower so that pollens at the stamen tip are delivered to the stigma at the pistil tip. Although intercellular signalling acts in several reproduction steps, how signalling molecules, including secreted peptides, contribute to the synchronous growth of reproductive tissues remains limited. Here, we show that the mutant of the secreted peptide EPIDERMAL PATTERNING FACTOR LIKE 6 (EPFL6), which shows no phenotypes at a moderate temperature, fails in fruit production at a cool temperature due to insufficient elongation of stamens. EPFL6 is expressed in stamen filaments and promotes filament elongation to achieve the alignment of stamen and pistil lengths at a cool temperature. We also found that, at a moderate temperature, all EPFL6-subfamily genes are required for stamen elongation. Furthermore, we showed that ERECTA (ER), known as a common receptor for EPFL-family peptides, mediates the stamen-pistil growth coordination. Lastly, we provided evidence that modulation of ER activity rescues the reproduction failure caused by insufficient stamen elongation by realigning the stamen and pistil lengths., (© 2022 John Wiley & Sons Ltd.)

Aims/introduction: The aim of this study was to develop a scale to evaluate disease stigma in patients with lifestyle-related chronic non-communicable diseases (LCNCDs), which we named the Kanden Institute Stigma Scale (KISS), and to consider its possible clinical application for patients with diabetes., Materials and Methods: An initial 90 questions were drafted and categorized into six subscales according to the manifestations of stigma. The final version of the KISS was developed as a 24-item questionnaire comprising four items for each subscale., Results: A total of 539 outpatients including 452 patients with diabetes and 87 patients without diabetes were recruited. Construct validity was confirmed by assessing the correlation with previously established measures. Confirmatory factor analysis showed the KISS to have good model fitness (adjusted goodness-of-fit index = 0.856). Test-retest reproducibility analysis showed that the intraclass coefficient of the first and a second KISS was 0.843 (P < 0.001), indicating excellent reproducibility. The KISS showed higher scores for patients with diabetes than for patients without diabetes (12.23 ± 0.49 vs 5.76 ± 0.73, P < 0.05). The KISS score was significantly higher in type 1 and type 2 diabetes patients taking insulin therapy than in type 2 diabetes patients not taking insulin (P < 0.05)., Conclusion: The KISS is a validated and reliable questionnaire for assessment of stigma among patients with diabetes as well as other lifestyle-related chronic non-communicable diseases, and might contribute to identifying and rectifying diabetes stigma, as well promoting awareness among health care professionals of this very consequential health problem., (© 2022 The Authors. Journal of Diabetes Investigation published by Asian Association for the Study of Diabetes (AASD) and John Wiley & Sons Australia, Ltd.)

Background: The appearance quality of the eggplant (Solanum melongena L.) fruit is an important trait that influences its commercial value. It is known that quality traits such as anthocyanin composition and fruit surface pattern are categorical and are inherited simply. However, research examples of gene mapping for the composition (anthocyanin accumulation profile) and the surface pattern in eggplant fruit are limited., Methods and Results: To map loci for these traits including the accumulation profiles of two anthocyanins, a widely spreading anthocyanin, delphinidin 3-(p-coumaroyl) rutinoside-5-glucoside (nasunin), and the relatively rare delphinidin 3-glucoside (D3G), we used two F 2 intracrossed populations (LWF2 and N28F2). For the LWF2 population, mapping was achieved by reconstructing the linkage map created by Fukuoka et al. [1]. In the case of the N28F2 population, we constructed a linkage map consisting of 13 linkage groups using 238 simple sequence repeats, 75 single-nucleotide polymorphisms. Using the two F 2 populations, the nasunin accumulating profile, the striped pattern on the fruit surface, the colors of flowers, fruit, and calyxes, and the D3G accumulating profile were genetically mapped. Furthermore, by utilizing the eggplant reference genome information, mutations in the causative candidate genes for those loci were identified., Conclusion: Overall, the results of this study suggest that inactivation of key enzymes of anthocyanin metabolism and the gene orthologous to the tomato u gene are potential causes of observed variety in eggplant appearance traits., (© 2022. The Author(s), under exclusive licence to Springer Nature B.V.)