Crystal structure of a novel homodimeric <scp>l</scp>‐ribulose 3‐epimerase from <i>Methylomonus</i> sp.

Yoshida, Hiromi; Yoshihara, Akihide; Kato, Shiro; Mochizuki, Susumu; Akimitsu, Kazuya; Izumori, Ken; Kamitori, Shigehiro

doi:10.1002/2211-5463.13159

Cited by 10 publications

(4 citation statements)

References 52 publications

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“…This is different from the in vivo condition, and the highest activity of the recombinant enzyme could be observed at a relatively higher temperature. Such a stabilization of the recombinant enzyme has been reported elsewhere (Yoshida et al 2021 ). In the analysis of thermal stability (Fig.…”

Section: Discussionsupporting

confidence: 75%

X-ray structure and characterization of a probiotic Lactobacillus rhamnosus Probio-M9 L-rhamnose isomerase

Yoshida,

Yamamoto,

Kurahara

et al. 2024

Appl Microbiol Biotechnol

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A recombinant L-rhamnose isomerase (L-RhI) from probiotic Lactobacillus rhamnosus Probio-M9 (L. rhamnosus Probio-M9) was expressed. L. rhamnosus Probio-M9 was isolated from human colostrum and identified as a probiotic lactic acid bacterium, which can grow using L-rhamnose. L-RhI is one of the enzymes involved in L-rhamnose metabolism and catalyzes the reversible isomerization between L-rhamnose and L-rhamnulose. Some L-RhIs were reported to catalyze isomerization not only between L-rhamnose and L-rhamnulose but also between D-allulose and D-allose, which are known as rare sugars. Those L-RhIs are attractive enzymes for rare sugar production and have the potential to be further improved by enzyme engineering; however, the known crystal structures of L-RhIs recognizing rare sugars are limited. In addition, the optimum pH levels of most reported L-RhIs are basic rather than neutral, and such a basic condition causes non-enzymatic aldose-ketose isomerization, resulting in unexpected by-products. Herein, we report the crystal structures of L. rhamnosus Probio-M9 L-RhI (LrL-RhI) in complexes with L-rhamnose, D-allulose, and D-allose, which show enzyme activity toward L-rhamnose, D-allulose, and D-allose in acidic conditions, though the activity toward D-allose was low. In the complex with L-rhamnose, L-rhamnopyranose was found in the catalytic site, showing favorable recognition for catalysis. In the complex with D-allulose, D-allulofuranose and ring-opened D-allulose were observed in the catalytic site. However, bound D-allose in the pyranose form was found in the catalytic site of the complex with D-allose, which was unfavorable for recognition, like an inhibition mode. The structure of the complex may explain the low activity toward D-allose. Key points • Crystal structures of LrL-RhI in complexes with substrates were determined. • LrL-RhI exhibits enzyme activity toward L-rhamnose, D-allulose, and D-allose. • The LrL-RhI is active in acidic conditions.

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Section: Discussionsupporting

confidence: 75%

X-ray structure and characterization of a probiotic Lactobacillus rhamnosus Probio-M9 L-rhamnose isomerase

Yoshida,

Yamamoto,

Kurahara

et al. 2024

Appl Microbiol Biotechnol

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“…( Itoh et al, 1994 ), but only 25.4% and 24.2% sequence similarity with the LREases from Methylomonas sp. (MdLRE) ( Yoshida et al, 2021 ) and T. maritima (TmLRE) ( Shin et al, 2017 ), respectively. Notably, the residues E150, D183, H209, and E244, which are involved in substrate recognition and metal coordination, were absolutely conserved with those of other reported DAEases.…”

Section: Resultsmentioning

confidence: 99%

Biochemical characterization, structure-guided mutagenesis, and application of a recombinant D-allulose 3-epimerase from Christensenellaceae bacterium for the biocatalytic production of D-allulose

Guan,

Zhu,

Wang

et al. 2024

Front. Bioeng. Biotechnol.

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D-Allulose has become a promising alternative sweetener due to its unique properties of low caloric content, moderate sweetness, and physiological effects. D-Allulose 3-epimerase (DAEase) is a promising enzyme for D-Allulose production. However, the low catalytic efficiency limited its large-scale industrial applications. To obtain a more effective biocatalyst, a putative DAEase from Christensenellaceae bacterium (CbDAE) was identified and characterized. The recombinant CbDAE exhibited optimum activity at pH 7.5°C and 55°C, retaining more than 60% relative activity from 40°C to 70°C, and the catalytic activity could be significantly increased by Co2+ supplementation. These enzymatic properties of purified CbDAE were compared with other DAEases. CbDAE was also found to possess desirable thermal stability at 55°C with a half-life of 12.4 h. CbDAE performed the highest relative activity towards D-allulose and strong affinity for D-fructose but relatively low catalytic efficiency towards D-fructose. Based on the structure-guided design, the best double-mutation variant G36N/W112E was obtained which reached up to 4.21-fold enhancement of catalytic activity compared with wild-type (WT) CbDAE. The catalytic production of G36N/W112E with 500 g/L D-fructose was at a medium to a higher level among the DAEases in 3.5 h, reducing 40% catalytic reaction time compared to the WT CbDAE. In addition, the G36N/W112E variant was also applied in honey and apple juice for D-allulose conversion. Our research offers an extra biocatalyst for D-allulose production, and the comprehensive report of this enzyme makes it potentially interesting for industrial applications and will aid the development of industrial biocatalysts for D-allulose.

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“…The crystal structure of LRI from Methylomonus sp. has been analyzed and can subsequently be used to obtain mutants [ 112 ].…”

Section: Enzymatic Biotransformation Of D -Allulosementioning

confidence: 99%

Research Advances of d-allulose: An Overview of Physiological Functions, Enzymatic Biotransformation Technologies, and Production Processes

Xia

Cheng

et al. 2021

Foods

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d-allulose has a significant application value as a sugar substitute, not only as a food ingredient and dietary supplement, but also with various physiological functions, such as improving insulin resistance, anti-obesity, and regulating glucolipid metabolism. Over the decades, the physiological functions of d-allulose and the corresponding mechanisms have been studied deeply, and this product has been applied to various foods to enhance food quality and prolong shelf life. In recent years, biotransformation technologies for the production of d-allulose using enzymatic approaches have gained more attention. However, there are few comprehensive reviews on this topic. This review focuses on the recent research advances of d-allulose, including (1) the physiological functions of d-allulose; (2) the major enzyme families used for the biotransformation of d-allulose and their microbial origins; (3) phylogenetic and structural characterization of d-allulose 3-epimerases, and the directed evolution methods for the enzymes; (4) heterologous expression of d-allulose ketose 3-epimerases and biotransformation techniques for d-allulose; and (5) production processes for biotransformation of d-allulose based on the characterized enzymes. Furthermore, the future trends on biosynthesis and applications of d-allulose in food and health industries are discussed and evaluated in this review.

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Crystal structure of a novel homodimeric l‐ribulose 3‐epimerase from Methylomonus sp.

Cited by 10 publications

References 52 publications

X-ray structure and characterization of a probiotic Lactobacillus rhamnosus Probio-M9 L-rhamnose isomerase

X-ray structure and characterization of a probiotic Lactobacillus rhamnosus Probio-M9 L-rhamnose isomerase

Biochemical characterization, structure-guided mutagenesis, and application of a recombinant D-allulose 3-epimerase from Christensenellaceae bacterium for the biocatalytic production of D-allulose

Research Advances of d-allulose: An Overview of Physiological Functions, Enzymatic Biotransformation Technologies, and Production Processes

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