Functional Characterization and Protein Engineering of Salvia miltiorrhiza Rosmarinic Acid Synthase.

IF 6.2 1区农林科学 Q1 AGRICULTURE, MULTIDISCIPLINARY

Journal of Agricultural and Food Chemistry Pub Date : 2025-09-25 DOI:10.1021/acs.jafc.5c08090

Yue Feng,Zhoulu Wang,Junbo Li,Zhenli Ren,Yanting Wu,Hao Zhan,Xuefei Chen,Yao Wang,Min Shi,Xu Jia,Degang Zhao,Jianbo Zhang,Kunlun Li,Guoyin Kai

{"title":"Functional Characterization and Protein Engineering of Salvia miltiorrhiza Rosmarinic Acid Synthase.","authors":"Yue Feng,Zhoulu Wang,Junbo Li,Zhenli Ren,Yanting Wu,Hao Zhan,Xuefei Chen,Yao Wang,Min Shi,Xu Jia,Degang Zhao,Jianbo Zhang,Kunlun Li,Guoyin Kai","doi":"10.1021/acs.jafc.5c08090","DOIUrl":null,"url":null,"abstract":"Rosmarinic acid (RA) has been incorporated in various nutritious and health-promoting products. Rosmarinic acid synthase (RAS) is a committed enzyme in RA biosynthesis. However, SmRASs from Salvia miltiorrhiza remains functionally uncharacterized. Herein, a group of SmRASs was identified from the S. miltiorrhiza, and recombinant SmRAS1 was demonstrated to convert caffeoyl-CoA and 3,4-dihydroxyphenyllactic acid to form RA. SmRAS1 exhibited maximum activity at 45 °C and pH 8.0 with Km values of 18 μM and 1647 μM for caffeoyl-CoA and 3,4-dihydroxyphenyllactic acid, respectively. Subsequently, three highly catalytic activity SmRAS1 mutations R367W, G293I, and G293Rwere obtained, exhibiting 1.53-2.15 fold higher catalytic efficiency than the wild-type enzyme. Structural analysis and molecular dynamics simulations revealed that enlarged substrate access tunnel and reduced flexibility of residues286-295 located above the substrate pocket enhanced its catalytic activity. These results elucidate the mechanism underlying RA biosynthesis in S. miltiorrhiza and provide a promising RAS for RA biosynthesis in microbes.","PeriodicalId":41,"journal":{"name":"Journal of Agricultural and Food Chemistry","volume":"86 1","pages":""},"PeriodicalIF":6.2000,"publicationDate":"2025-09-25","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Journal of Agricultural and Food Chemistry","FirstCategoryId":"97","ListUrlMain":"https://doi.org/10.1021/acs.jafc.5c08090","RegionNum":1,"RegionCategory":"农林科学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q1","JCRName":"AGRICULTURE, MULTIDISCIPLINARY","Score":null,"Total":0}

引用次数: 0

Abstract

Rosmarinic acid (RA) has been incorporated in various nutritious and health-promoting products. Rosmarinic acid synthase (RAS) is a committed enzyme in RA biosynthesis. However, SmRASs from Salvia miltiorrhiza remains functionally uncharacterized. Herein, a group of SmRASs was identified from the S. miltiorrhiza, and recombinant SmRAS1 was demonstrated to convert caffeoyl-CoA and 3,4-dihydroxyphenyllactic acid to form RA. SmRAS1 exhibited maximum activity at 45 °C and pH 8.0 with Km values of 18 μM and 1647 μM for caffeoyl-CoA and 3,4-dihydroxyphenyllactic acid, respectively. Subsequently, three highly catalytic activity SmRAS1 mutations R367W, G293I, and G293Rwere obtained, exhibiting 1.53-2.15 fold higher catalytic efficiency than the wild-type enzyme. Structural analysis and molecular dynamics simulations revealed that enlarged substrate access tunnel and reduced flexibility of residues286-295 located above the substrate pocket enhanced its catalytic activity. These results elucidate the mechanism underlying RA biosynthesis in S. miltiorrhiza and provide a promising RAS for RA biosynthesis in microbes.

查看原文本刊更多论文

丹参迷迭香酸合成酶的功能表征及蛋白工程研究。

迷迭香酸（RA）已被纳入各种营养和健康促进产品。迷迭香酸合成酶（rorosemary acid synthase， RAS）是类风湿性关节炎生物合成中的一种重要酶。然而，从丹参中提取的smras在功能上仍未被表征。本研究从丹参中鉴定出一组SmRAS1，并证实重组SmRAS1能将咖啡酰辅酶a和3,4-二羟基苯基乳酸转化为RA。SmRAS1对咖啡酰辅酶a和3,4-二羟基苯基乳酸的活性在45°C和pH 8.0时最高，Km值分别为18 μM和1647 μM。随后，获得了三个具有高催化活性的SmRAS1突变体R367W、G293I和g293r，其催化效率比野生型酶高1.53-2.15倍。结构分析和分子动力学模拟表明，位于底物口袋上方的残基286-295增大了底物通道，降低了其柔韧性，增强了其催化活性。这些结果阐明了丹参中RA的生物合成机制，并为微生物中RA的生物合成提供了有前途的RAS。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

求助全文

约1分钟内获得全文求助全文

来源期刊

Journal of Agricultural and Food Chemistry 农林科学-农业综合

CiteScore

9.90

自引率

8.20%

发文量

1375

审稿时长

2.3 months

期刊介绍： The Journal of Agricultural and Food Chemistry publishes high-quality, cutting edge original research representing complete studies and research advances dealing with the chemistry and biochemistry of agriculture and food. The Journal also encourages papers with chemistry and/or biochemistry as a major component combined with biological/sensory/nutritional/toxicological evaluation related to agriculture and/or food.