Samantha R Summers, Sarah Alamdari, Casey J Kraft, Roman Brunecky, Jim Pfaendtner, Joel L Kaar
{"title":"远端和活性位点残基的取代降低了酸热菌降解纤维素的产物E1的抑制作用。","authors":"Samantha R Summers, Sarah Alamdari, Casey J Kraft, Roman Brunecky, Jim Pfaendtner, Joel L Kaar","doi":"10.1093/protein/gzab031","DOIUrl":null,"url":null,"abstract":"Cellulases are largely afflicted by inhibition from their reaction products, especially at high-substrate loading, which represents a major challenge for biomass processing. This challenge was overcome for endoglucanase 1 (E1) from Acidothermus cellulolyticus by identifying a large conformational change involving distal residues upon binding cellobiose. Having introduced alanine substitutions at each of these residues, we identified several mutations that reduced cellobiose inhibition of E1, including W212A, W213A, Q247A, W249A and F250A. One of the mutations (W212A) resulted in a 47-fold decrease in binding affinity of cellobiose as well as a 5-fold increase in the kcat. The mutation further increased E1 activity on Avicel and dilute-acid treated corn stover and enhanced its productivity at high-substrate loadings. These findings were corroborated by funnel metadynamics, which showed that the W212A substitution led to reduced affinity for cellobiose in the +1 and +2 binding sites due to rearrangement of key cellobiose-binding residues.","PeriodicalId":54543,"journal":{"name":"Protein Engineering Design & Selection","volume":null,"pages":null},"PeriodicalIF":2.6000,"publicationDate":"2021-02-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"1","resultStr":"{\"title\":\"Substitution of distal and active site residues reduces product inhibition of E1 from Acidothermus Cellulolyticus.\",\"authors\":\"Samantha R Summers, Sarah Alamdari, Casey J Kraft, Roman Brunecky, Jim Pfaendtner, Joel L Kaar\",\"doi\":\"10.1093/protein/gzab031\",\"DOIUrl\":null,\"url\":null,\"abstract\":\"Cellulases are largely afflicted by inhibition from their reaction products, especially at high-substrate loading, which represents a major challenge for biomass processing. This challenge was overcome for endoglucanase 1 (E1) from Acidothermus cellulolyticus by identifying a large conformational change involving distal residues upon binding cellobiose. Having introduced alanine substitutions at each of these residues, we identified several mutations that reduced cellobiose inhibition of E1, including W212A, W213A, Q247A, W249A and F250A. One of the mutations (W212A) resulted in a 47-fold decrease in binding affinity of cellobiose as well as a 5-fold increase in the kcat. The mutation further increased E1 activity on Avicel and dilute-acid treated corn stover and enhanced its productivity at high-substrate loadings. These findings were corroborated by funnel metadynamics, which showed that the W212A substitution led to reduced affinity for cellobiose in the +1 and +2 binding sites due to rearrangement of key cellobiose-binding residues.\",\"PeriodicalId\":54543,\"journal\":{\"name\":\"Protein Engineering Design & Selection\",\"volume\":null,\"pages\":null},\"PeriodicalIF\":2.6000,\"publicationDate\":\"2021-02-15\",\"publicationTypes\":\"Journal Article\",\"fieldsOfStudy\":null,\"isOpenAccess\":false,\"openAccessPdf\":\"\",\"citationCount\":\"1\",\"resultStr\":null,\"platform\":\"Semanticscholar\",\"paperid\":null,\"PeriodicalName\":\"Protein Engineering Design & Selection\",\"FirstCategoryId\":\"99\",\"ListUrlMain\":\"https://doi.org/10.1093/protein/gzab031\",\"RegionNum\":4,\"RegionCategory\":\"生物学\",\"ArticlePicture\":[],\"TitleCN\":null,\"AbstractTextCN\":null,\"PMCID\":null,\"EPubDate\":\"\",\"PubModel\":\"\",\"JCR\":\"Q3\",\"JCRName\":\"BIOCHEMISTRY & MOLECULAR BIOLOGY\",\"Score\":null,\"Total\":0}","platform":"Semanticscholar","paperid":null,"PeriodicalName":"Protein Engineering Design & Selection","FirstCategoryId":"99","ListUrlMain":"https://doi.org/10.1093/protein/gzab031","RegionNum":4,"RegionCategory":"生物学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"Q3","JCRName":"BIOCHEMISTRY & MOLECULAR BIOLOGY","Score":null,"Total":0}
Substitution of distal and active site residues reduces product inhibition of E1 from Acidothermus Cellulolyticus.
Cellulases are largely afflicted by inhibition from their reaction products, especially at high-substrate loading, which represents a major challenge for biomass processing. This challenge was overcome for endoglucanase 1 (E1) from Acidothermus cellulolyticus by identifying a large conformational change involving distal residues upon binding cellobiose. Having introduced alanine substitutions at each of these residues, we identified several mutations that reduced cellobiose inhibition of E1, including W212A, W213A, Q247A, W249A and F250A. One of the mutations (W212A) resulted in a 47-fold decrease in binding affinity of cellobiose as well as a 5-fold increase in the kcat. The mutation further increased E1 activity on Avicel and dilute-acid treated corn stover and enhanced its productivity at high-substrate loadings. These findings were corroborated by funnel metadynamics, which showed that the W212A substitution led to reduced affinity for cellobiose in the +1 and +2 binding sites due to rearrangement of key cellobiose-binding residues.
期刊介绍:
Protein Engineering, Design and Selection (PEDS) publishes high-quality research papers and review articles relevant to the engineering, design and selection of proteins for use in biotechnology and therapy, and for understanding the fundamental link between protein sequence, structure, dynamics, function, and evolution.