Oligopeptides production by a method involving an enzymatic reaction and a subsequent chemical reaction

We previously reported that an amide bond is unexpectedly formed1) by an acyl-CoA synthetase, AcsA, which plays an essential role in acid utilization in the nitrile-degrative pathway2). Although AcsA essentially catalyzes the formation of a carbon-sulfur bond (the ligation of an acid with CoA), it surprisingly synthesized N-acyl-l-cysteine when a suitable acid and l-cysteine are used as substrates. Furthermore, this unexpected enzyme activity was also observed for acetyl-CoA synthetase and firefly luciferase, both of which belong to the same superfamily of adenylate-forming enzymes. However, the mechanism underlying the carbon-nitrogen bond synthesis remained unknown. Next, we succeeded in producing N-(D-alanyl)-l-cysteine (a dipeptide) from D-alanine and l-cysteine by using DltA, which is homologous to the adenylation domain of nonribosomal peptide synthetase (NRPS) and belongs to the superfamily of adenylate-forming enzymes. To elucidate the mechanism of these surprising reaction, DltA was used. When cysteine derivatives with a protected amino group N-Boc-l-Cys was used instead of l-cysteine, we confirmed the formation of an thioester intermediate. Thereby, we proposed the following unprecedented reaction mechanism underlying these carbon-nitrogen bond synthetic reactions by the thioester-bond-synthesizing enzymes: (i) the formation of S-acyl-l-cysteine as an intermediate via its “enzymatic activity” and (ii) subsequent “chemical“ S→N acyl transfer in the intermediate, resulting in peptide formation3). Step (ii) of this reaction mechanism is identical to the corresponding reaction in native chemical ligation, a method of chemical peptide synthesis, whereas step (i) is not. We predicted that enzymes belonging to the superfamily of adenylate-forming enzymes can synthesize peptide/amide compounds by the same mechanism. Accordingly, we tried to express and purify DhbE, a stand-alone adebylation domain of NRPS, for production of valuable peptide/amide compounds. The purified DhbE synthesized N-aromatic acyl-l-cysteine4). Here, we reported the first demonstration of the N-acylation by “internal” adenylation domains in the multidomain enzyme DhbF. The adenylation domain of NRPS originally is responsible for its selective substrate recognition and activation of the substrate. DhbF is an NRPS involved in bacillibactin synthesis and consists of multiple domains (adenylation domain, condensation domain, peptidyl carrier protein domain, and thioesterase domain). DhbFA1 and DhbFA2 (here named) are “internal” adenylation domains in DhbF. Here, we firstly succeeded in expressing and purifying “internal” adenylation domain DhbFA1 or DhbFA2 separately. When glycine and l-cysteine were used as substrates of DhbFA1, the formation of N-glycyl-l-cysteine (Gly-Cys) was observed. When l-threonine and l-cysteine were used as substrates of DhbFA2, N-l-threonyl-l-cysteine (Thr-Cys) was formed. Furthermore, DhbFA1 or DhbFA2 synthesizes not only dipeptides but also various oligopeptides. Because many adenylation domains that could activate the respective substrates are present in the natural world, we can synthesize various peptides or amides by using adenylation domains or enzymes belonging to the superfamily of adenylate-forming enzymes. References: 1. Abe, T. et al., J. Biol. Chem. 283, 11312-11321 (2008). 2. Hashimoto, Y. et al., J. Biol. Chem. 280, 8660-8667 (2005). 3. Abe, T. et al., J. Biol. Chem. 291, 1735-1750 (2016). 4. Abe, T. et al., J. Antibiot. 70, 435-442 (2017)

From Sound to Voice: Understanding Sayaka Murata's Novel Convenience Store Woman

In Sayaka Murata's novel Convenience Store Woman, the heroine, Keiko Furukura, is a woman chained by the three shackles of being a virgin, unmarried and a part-time worker. To Furukura, the convenience store is a holy place that enables her function as a "normal part of society". There are various kinds of sounds in the store such as music. However, her encounter with Shiraha causes discord and her belief in the convenience store is shaken. Led by a "heavenly voice" in the convenience store, her conscience is awakened just as she quits the job and is about to surrender to society. She turns down a proposal from Shiraha and becomes a convenience store woman in a true sense. This novel expresses the author's reflection on rebellion against modern society and the concern of humanity with a sense of humor

Perceptual Continuity and Naturalness of Expressive Strength in Singing Voices Based on Speech Morphing

This paper experimentally shows the importance of perceptual continuity of the expressive strength in vocal timbre for natural change in vocal expression. In order to synthesize various and continuous expressive strengths with vocal timbre, we investigated gradually changing expressions by applying the STRAIGHT speech morphing algorithm to singing voices. Here, a singing voice without expression is used as the base of morphing, and singing voices with three different expressions are used as the target. Through statistical analyses of perceptual evaluations, we confirmed that the proposed morphing algorithm provides perceptual continuity of vocal timbre. Our results showed the following: (i) gradual strengths in absolute evaluations, and (ii) a perceptually linear strength provided by the calculation of corrected intervals of the morph ratio by the inverse (reciprocal) function of an equation that approximates the perceptual strength. Finally, we concluded that applying continuity was highly effective for achieving perceptual naturalness, judging from the results showing that (iii) our gradual transformation method can perform well for perceived naturalness

SYNTHESIS OF ZANTHOXYLINE AND ITS RELATED COMPOUNDS: REVISION OF THE REPORTED STRUCTURE

Benzo[c]phenanthridine alkaloid, zanthoxyline, was synthesized through an intramolecular biaryl coupling reaction of N-naphthylbenzamide derivative using a palladium reagent. Comparison of the NMR data for the synthetic product with that reported previously revealed that the proposed structure was incorrect. Synthesis of the related compounds suggested that the correct structure of zanthoxyline is the previously known decarine

Selective disappearance of medial back muscles in a case of myotonic dystrophy type 1

Here, we report a unique case of late-onset myotonic dystrophy type 1 in a 64-year-old woman, with selective disappearance of the medial lower back muscles. We compared the clinical features of this patient with those of a cohort of 29 patients with myotonic dystrophy type 1 to clarify the correlation between clinical features and lower back muscle atrophy. After classification into three subgroups according to muscle atrophy pattern, medial muscle atrophy was present in 17.2% of the patients. Affected patients were older at onset than non-affected patients, and limb muscle power and respiratory function decreased with atrophy progression