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O -Methyltransferases (OMTs) play important roles in antitumor lignan biosynthesis. To date, six OMTs catalyzing the methylation of dibenzylbutyrolactone lignans as biosynthetic precursors of antitumor lignans have been identified. However, there is still no systematic understanding of the diversity and regularity of the biosynthetic mechanisms among various plant lineages. Herein, we report the characterization of two OMTs from Anthriscus sylvestris and Thujopsis dolabrata var. hondae [designated as AsSecoNorYatein (SNY) OMT and TdSNYOMT] together with the six known OMTs to evaluate their diversity and regularity. Although A. sylvestris 5- O -methylthujaplicatin (SecoNorYatein) and 4- O -demethylyatein (NorYatein) OMT (AsSNYOMT) and TdSNYOMT accept 5- O -methylthujaplicatin and 4- O -demethylyatein as substrates, phylogenetic analysis indicated that these two OMTs shared low amino acid sequence identity, 33.8%, indicating a signature of parallel evolution. The OMTs and the six previously identified OMTs were found to be diverse in terms of their substrate specificity, regioselectivity and amino acid sequence identity, indicating independent evolution in each plant species. Meanwhile, two-entropy analysis detected four amino acid residues as being specifically acquired by dibenzylbutyrolactone lignan OMTs. Site-directed mutation of AsSNYOMT indicated that two of them contributed specifically to 5- O -methylthujaplicatin methylation. The results provide a new example of parallel evolution and the diversity and regularity of OMTs in plant secondary (specialized) metabolism. [ABSTRACT FROM AUTHOR]

Tetrahydrofuran ring formation from dibenzylbutyrolactone lignans is a key step in the biosynthesis of aryltetralin lignans including deoxypodophyllotoxin and podophyllotoxin. Previously, Fe(II)- and 2-oxoglutarate-dependent dioxygenase (2-ODD) from Podophyllum hexandrum (Himalayan mayapple, Berberidaceae) was found to catalyze the cyclization of a dibenzylbutyrolactone lignan, yatein, to give deoxypodophyllotoxin and designated as deoxypodophyllotoxin synthase (DPS). Recently, we reported that the biosynthesis of deoxypodophyllotoxin and podophyllotoxin evolved in a lineage-specific manner in phylogenetically unrelated plant species such as P. hexandrum and Anthriscus sylvestris (cow parsley, Apiaceae). Therefore, a comprehensive understanding of the characteristics of DPSs that catalyze the cyclization of yatein to deoxypodophyllotoxin in various plant species is important. However, for plant species other than P. hexandrum , the isolation of the DPS enzyme gene and the type of the enzyme, e.g. whether it is 2-ODD or another type of enzyme such as cytochrome P-450, have not been reported. In this study, we report the identification and characterization of A. sylvestris DPS (AsDPS). Phylogenetic analysis showed that AsDPS belonged to the 2-ODD superfamily and shared moderate amino acid sequence identity (40.8%) with P. hexandrum deoxypodophyllotoxin synthase (PhDPS). Recombinant protein assay indicated that AsDPS and PhDPS differ in terms of the selectivity of substrate enantiomers. Protein modeling using AlphaFold2 and site-directed mutagenesis indicated that the Tyr305 residue of AsDPS probably contributes to substrate recognition. This study advances our understanding of the podophyllotoxin biosynthetic pathway in A. sylvestris and provides new insight into 2-ODD involved in plant secondary (specialized) metabolism. [ABSTRACT FROM AUTHOR]

Mucosa‐associated lymphoid tissue lymphoma‐translocation gene 1 (MALT1)‐deficiency is a rare combined immunodeficiency characterized by recurrent infections, dermatitis and enteropathy. We herein investigate the immunological profiles of our patient and previously reported children with MALT1‐deficiency. A mutation analysis was performed by targeted panel sequencing for primary immunodeficiency. Lymphocyte subset, activation and B cell differentiation were analyzed by flow cytometry and t‐distributed stochastic neighbor embedding. Pneumocystis pneumonia developed in a 6‐month‐old Japanese infant with atopic dermatitis, enteritis and growth restriction. This infant showed agammaglobulinemia without lymphopenia. At 8 years of age, the genetic diagnosis of MALT1‐deficiency was confirmed on a novel homozygous mutation of c.1102G>T, p.E368X. T cell stimulation tests showed impairments in the production of interleukin‐2, phosphorylation of nuclear factor kappa B (NF‐κB) p65 and differentiation of B cells. In combination with the literature data, we found that the number of circulatory B cells, but not T cells, were inversely correlated with the age of patients. The hematopoietic cell transplantation (HCT) successfully reconstituted the differentiation of mature B cells and T cells. These data conceptualize that patients with complete MALT1‐deficiency show aberrant differentiation and depletion of B cells. The early diagnosis and HCT lead to a cure of the disease phenotype associated with the loss‐of‐function mutations in human CARD11. [ABSTRACT FROM AUTHOR]