Methylcobalamin-Dependent Radical SAM <i>C</i>-Methyltransferase Fom3 Recognizes Cytidylyl-2-hydroxyethylphosphonate and Catalyzes the Nonstereoselective C-Methylation in Fosfomycin Biosynthesis

Sato, Shusuke,Kudo, Fumitaka,Kim, Seung-Young,Kuzuyama, Tomohisa,Eguchi, Tadashi American Chemical Society 2017 Biochemistry Vol.56 No.28

<P>A methylcobalamin (MeCbl)-dependent radical <I>S</I>-adenosyl-<SMALL>l</SMALL>-methionine (SAM) methyltransferase Fom3 was found to catalyze the C-methylation of cytidylyl-2-hydroxyethylphosphonate (HEP-CMP) to give cytidylyl-2-hydroxypropylphosphonate (HPP-CMP), although it was originally proposed to catalyze the C-methylation of 2-hydroxyethylphosphonate to give 2-hydroxypropylphosphonate in the biosynthesis of a unique C–P bond containing antibiotic fosfomycin in <I>Streptomyces</I>. Unexpectedly, the Fom3 reaction product from HEP-CMP was almost a 1:1 diastereomeric mixture of HPP-CMP, indicating that the C-methylation is not stereoselective. Presumably, only the CMP moiety of HEP-CMP is critical for substrate recognition; on the other hand, the enzyme does not fix the 2-hydroxy group of the substrate and either of the prochiral hydrogen atoms at the C2 position can be abstracted by the 5′-deoxyadenosyl radical generated from SAM to form the substrate radical intermediates, which react with MeCbl to afford the corresponding products. This strict substrate recognition mechanism with no stereoselectivity of a MeCbl-dependent radical SAM methyltransferase is remarkable in natural product biosynthetic chemistry, because such a hidden clue for selective substrate recognition is likely to be found in the other biosynthetic pathways.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/bichaw/2017/bichaw.2017.56.issue-28/acs.biochem.7b00472/production/images/medium/bi-2017-00472r_0005.gif'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/bi7b00472'>ACS Electronic Supporting Info</A></P>

Microintaglio Printing Enables to Reproduce the Central Dogma of Molecular Biology on Microarray Chips

Takanori Ichiki,Shusuke Sato,Manish Biyani,Shingo Ueno 한국표면공학회 2013 한국표면공학회 학술발표회 초록집 Vol.2013 No.11

Role of oxytocin in improving the welfare of farm animals — A review

Siyu Chen,Shusuke Sato 아세아·태평양축산학회 2017 Animal Bioscience Vol.30 No.4

Recently, increasing attention has been paid to the welfare of farm animals, which have been evaluated using behavioral and physiological measures. However, so far, the measures have almost always been used to estimate poor welfare. In this review, firstly we focus on how oxytocin (OT) relates to positive social behavior, pleasure, and stress tolerance, and second on which management factors stimulate OT release. OT induces maternal and affiliative behaviors and has an anti-stress effect. Further, OT is produced during enjoyable events, and has positive feedback on its own release as well. Therefore, to some extent, the relationship of OT to positive normal behavior was mutually beneficial—heightened OT concentration owing to comfortable rearing conditions induces positive social behavior, which in turn may increase OT concentration. Hence, studies on animal welfare should pay more attention to increasing comfort and the stress tolerance, rather than only focusing on when stress occurs in farm animals.

The Effects of Components of Grazing System on Welfare of Fattening Pigs

Tozawa, Akitsu,Tanaka, Shigefumi,Sato, Shusuke Asian Australasian Association of Animal Productio 2016 Animal Bioscience Vol.29 No.3

The objective of this study was to clarify the most effective component of grazing for improving welfare of fattening pigs. This study compared welfare indicators of 20 fattening pigs aged 100 to 124 days (the prior period) and 138 to 164 days (the latter period) in an indoor housing system (IS), an outdoor pasturing system (OP), a concrete floor paddock system (CF), a concrete floor paddock system with fresh grass (FG), or a soil floor paddock system (SF). The last three treatments include important components of a grazing system: extra space, grass feed, and soil floor. Behavior, wounds on the body, and performances, measured as average daily gain (ADG) and feed conversion ratio, were observed. CF pigs behaved similarly to IS pigs. FG pigs showed higher levels of foraging, chewing and activity. SF pigs engaged in higher levels of foraging, exploring, activity, and rooting, and showed a similar amount of playing behavior as OP pigs. ADG was the same in all treatments at the prior period, and increased in the order FG, IS, CF, SF, and OP at the latter. The behaviors and performance of SF pigs resembled those of OP which seemed to indicate a consistently higher standard of welfare than the other treatments. In conclusion, the existence of a soil floor is the most important component of a pasture for improving the welfare of pigs.

Productivity and Quality of Mulberry Leaves under Cattle Grazing in a Wasted Mulberry Plantation

Shin-ichiro Ogura,Shusuke Sato,Shigefumi Tanaka,Shin Matsumoto,Kunihiro Abe,Toshio Shimizu,Yoshihiro Takahashi,Hiroshi Yoshida 한국초지조사료학회 2009 한국초지조사료학회 학술대회논문집 Vol.2009 No.08

Mulberry (Morus sp.) plantations have been deteriorating in Japan due to decline of silk industry. In mountainous areas, large part of the mulberry plantations has been wasted. Cattle grazing in the wasted mulberry plantations is a practical system to reuse the wasted areas for food production, because mulberry leaves are highly nutritive (Ezenwa and Kitahara, 2001), productive (Kitahara et al., 2002) and palatable to cattle (Ogura et al., 2008). In this study, the productivity and quality of mulberry leaves were investigated for 3 years in a wasted mulberry plantation grazed with beef cows, in Minamisanriku town, Miyagi, Japan. Cattle grazing started in September 2005 (paddock A, 44 a) and June 2006 (paddock B, 96 a). For each paddock, two pregnant cows were grazed from June to August-October in 2006-2008. In the paddocks A and B, four mulberry trees were chosen and the leaf mass and chemical composition were measured in mid-June and late August (pre-grazing leaf mass) during 2006-2008. A non-grazed paddock was also measured in 2007-2008. Leaf productivity of mulberry trees significantly decreased with cattle grazing; pre-grazing leaf mass changed from 246.8 g to 10.4 g DM/tree and 286.2 g to 17.6 g DM/tree in the paddock A and B, respectively (Fig 1). In contrast, 149.2-365.1 g DM/tree of leaves was produced in the non-grazed paddock. Chemical composition was stable during the three years in all paddocks. It is concluded that cattle grazing gives severe damage to leaf production of mulberry trees in three years.

Effect of Suckling Systems on Serum Oxytocin and Cortisol Concentrations and Behavior to a Novel Object in Beef Calves

Siyu Chen,Shigefumi Tanaka,Shin-ichiro Ogura,노상건,Shusuke Sato 아세아·태평양축산학회 2015 Animal Bioscience Vol.28 No.11

We investigated differences between effects of natural- and bucket-suckling methods on basal serum oxytocin (OT) and cortisol concentrations, and the effect of OT concentration on affiliative and investigative behavior of calves to a novel object. Ten Japanese Black calves, balanced with birth order, were allocated evenly to natural-suckling (NS) and bucket suckling (BS) groups. Blood samples were collected at the ages of 1 and 2 months (1 week after weaning) calves, and serum OT and cortisol concentrations were measured using enzyme-linked immunosorbent assay and enzymeimmunoassay tests, respectively. Each calf at the age of 2 months (2 weeks after weaning) was released into an open-field with a calf decoy, and its investigative and affiliative behaviors were recorded for 20 minutes. In 1-month-old calves, the basal serum OT concentration (25.5±4.9 [mean±standard deviation, pg/mL]) of NS was significantly higher than that of BS (16.9±6.7) (p<0.05), whereas the basal cortisol concentration (5.8±2.5 [mean±standard deviation, ng/mL]) of NS was significantly lower than that in BS (10.0±2.8) (p<0.05). Additionally, a negative correlation was noted between serum OT and cortisol concentrations in 1-month-old calves (p = 0.06). Further, the higher serum OT concentration the calves had at 1 month old, the more investigative the calves were at 2 months old but not affiliative in the open-field with a calf decoy. Thus, we concluded that the natural suckling method from a dam elevates the basal serum OT concentration in calves, and high serum OT concentrations induce investigative behavior and attenuate cortisol concentrations.

Effect of Suckling Systems on Serum Oxytocin and Cortisol Concentrations and Behavior to a Novel Object in Beef Calves

Chen, Siyu,Tanaka, Shigefumi,Ogura, Shin-ichiro,Roh, Sanggun,Sato, Shusuke Asian Australasian Association of Animal Productio 2015 Animal Bioscience Vol.28 No.11

We investigated differences between effects of natural- and bucket-suckling methods on basal serum oxytocin (OT) and cortisol concentrations, and the effect of OT concentration on affiliative and investigative behavior of calves to a novel object. Ten Japanese Black calves, balanced with birth order, were allocated evenly to natural-suckling (NS) and bucket suckling (BS) groups. Blood samples were collected at the ages of 1 and 2 months (1 week after weaning) calves, and serum OT and cortisol concentrations were measured using enzyme-linked immunosorbent assay and enzymeimmunoassay tests, respectively. Each calf at the age of 2 months (2 weeks after weaning) was released into an open-field with a calf decoy, and its investigative and affiliative behaviors were recorded for 20 minutes. In 1-month-old calves, the basal serum OT concentration ($25.5{\pm}4.9$ [mean${\pm}$standard deviation, pg/mL]) of NS was significantly higher than that of BS ($16.9{\pm}6.7$) (p<0.05), whereas the basal cortisol concentration ($5.8{\pm}2.5$ [mean${\pm}$standard deviation, ng/mL]) of NS was significantly lower than that in BS ($10.0{\pm}2.8$) (p<0.05). Additionally, a negative correlation was noted between serum OT and cortisol concentrations in 1-month-old calves (p = 0.06). Further, the higher serum OT concentration the calves had at 1 month old, the more investigative the calves were at 2 months old but not affiliative in the open-field with a calf decoy. Thus, we concluded that the natural suckling method from a dam elevates the basal serum OT concentration in calves, and high serum OT concentrations induce investigative behavior and attenuate cortisol concentrations.

Fosfomycin Biosynthesis <i>via</i> Transient Cytidylylation of 2-Hydroxyethylphosphonate by the Bifunctional Fom1 Enzyme

Cho, Su-Hee,Kim, Seung-Young,Tomita, Takeo,Shiraishi, Taro,Park, Jin-Soo,Sato, Shusuke,Kudo, Fumitaka,Eguchi, Tadashi,Funa, Nobutaka,Nishiyama, Makoto,Kuzuyama, Tomohisa AMERICAN CHEMICAL SOCIETY 2017 ACS CHEMICAL BIOLOGY Vol.12 No.8

<P>Fosfomycin is a wide-spectrum phosphonate antibiotic that is used clinically to treat cystitis, tympanitis, <I>etc</I>. Its biosynthesis starts with the formation of a carbon–phosphorus bond catalyzed by the phosphoenolpyruvate phosphomutase Fom1. We identified an additional cytidylyltransferase (CyTase) domain at the Fom1 <I>N</I>-terminus in addition to the phosphoenolpyruvate phosphomutase domain at the Fom1 <I>C</I>-terminus. Here, we demonstrate that Fom1 is bifunctional and that the Fom1 CyTase domain catalyzes the cytidylylation of the 2-hydroxyethylphosphonate (HEP) intermediate to produce cytidylyl-HEP. On the basis of this new function of Fom1, we propose a revised fosfomycin biosynthetic pathway that involves the transient CMP-conjugated intermediate. The identification of a biosynthetic mechanism <I>via</I> such transient cytidylylation of a biosynthetic intermediate fundamentally advances the understanding of phosphonate biosynthesis in nature. The crystal structure of the cytidylyl-HEP-bound CyTase domain provides a basis for the substrate specificity and reveals unique catalytic elements not found in other members of the CyTase family.</P><P><B>Graphic Abstract</B> <IMG SRC='http://pubs.acs.org/appl/literatum/publisher/achs/journals/content/acbcct/2017/acbcct.2017.12.issue-8/acschembio.7b00419/production/images/medium/cb-2017-004199_0006.eps'></P><P><A href='http://pubs.acs.org/doi/suppl/10.1021/cb7b00419'>ACS Electronic Supporting Info</A></P>