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Green Chemical Engineering 2021年第2期2卷 217-223页

作者： Xian-Tai Zhou Hong-Bing Ji Fine Chemical Industry Research Institute Sun Yat-sen UniversityGuangzhou510275China School of Chemical Engineering Guangdong University of Petrochemical TechnologyMaoming525000China

substrate specificity is a hallmark of enzymatic *** this work,the biomimetic catalytic oxidation of styrene and cyclohexanone by iron(III)porphyrins and molecular oxygen was carried out,and remarkable differences in efficiency were *** specificity of the substrates for biomimetic catalytic oxidation was investigated by kinetics and mechanistic *** studies revealed that the oxidation of styrene followed Michaelis-Menten kinetics with KM=8.99 mol L^(-1),but the oxidation of cyclohexanone followed first-order kinetics with kobs=1.46×10^(-4) s^(-1),indicating that the styrene epoxidation by metalloporphyrins exhibited characteristics of enzyme-like catalysis,while the oxidation of cyclohexanone was in agreement with the general rules of chemical *** catalytic mechanisms for the two substrates were discussed by operando electron paramagnetic resonance spectroscopy,operando UV-vis spectroscopy,and KI/starch *** specificity was concluded to be attributed to the stability of high-valence species and oxygen transfer rate.

关键词： substrate specificity Kinetics Oxidation Metalloporphyrins Mechanism

Helicase activity and substrate specificity of RecQ5β

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Chinese Physics B 2017年第6期26卷 499-507页

作者：尤菁徐雅楠李辉吕袭明李伟王鹏业窦硕星奚绪光 Beijing National Laboratory for Condensed Matter Physics and CAS Key Laboratory of Soft Matter Physics Institute of Physics Chinese Academy of Sciences (CAS) Beijing 100190 China School of Physical Sciences University of Chinese Academy of Sciences Beijing 100049 China College of Life Science Inner Mongolia University for Nationalities Tongliao 028000 China College of Life Sciences Northwest Agriculture and Forestry University Yangling 712100 China LBPA IDA ENS Cachan CNRS Universite Paris-Saclay Cachan F-94235 France

RecQ5β is an essential DNA helicase in humans, playing important roles in DNA replication, repair, recombination and transcription. The unwinding activity and substrate specificity of RecQ5β is still elusive. Here, we used stopped-flow kinetic method to measure the unwinding and dissociation kinetics of RecQ5β with several kinds of DNA substrates, and found that RecQ5β could well unwind ss/ds DNA, forked DNA and Holiday junction, but was compromised in unwinding blunt DNA and G-quadruplex. Rec5β has the preferred unwinding specificity for certain DNA substrates containing the junction point, which may improve the binding affinity and unwinding activity of RecQ5β. Moreover, from a comparison with the truncated RecQ5β～（1-467）, we discovered that the C-terminal domain might strongly influence the unwinding activity and binding affinity of RecQ5β. These results may shed light on the physiological functions and working mechanisms of RecQ5β helicase.

关键词： helicase RecQ5β DNA substrate specificity unwinding kinetics

Switch of substrate specificity of hyperthermophilic acylaminoacyl peptidase by combination of protein and solvent engineering

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Protein & Cell 2011年第6期2卷 497-506页

作者： Chang Liu Guangyu Yang Lie Wu Guohe Tian Zuoming Zhang Yan Feng State Key Laboratory of Microbial Metabolism School of Life Sciences and BiotechnologyShanghai Jiao Tong UniversityShanghai 200240China Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education Jilin UniversityChangchun 130023China Changchun Institute of Biological Products China National Biotec GroupChangchun 130061China

The inherent evolvability of promiscuous enzymes endows them with great potential to be artificially evolved for novel ***,we succeeded in transforming a promiscuous acylaminoacyl peptidase(apAAP)from the hyperthermophilic archaeon Aeropyrum pernix K1 into a specific carboxylesterase by making a single *** order to fulfill the urgent requirement of thermostable lipolytic enzymes,in this paper we describe how the substrate preference of apAAP can be further changed from p-nitrophenyl caprylate(pNP-C8)to p-nitrophenyl laurate(pNP-C12)by protein and solvent *** one round of directed evolution and subsequent saturation mutagenesis at selected residues in the active site,three variants with enhanced activity towards pNP-C12 were ***,a combined mutant W474V/F488G/R526V/T560W was generated,which had the highest catalytic efficiency(kcat/Km)for pNP-C12,about 71-fold higher than the wild *** activity was further increased by solvent engineering,resulting in an activity enhancement of 280-fold compared with the wild type in the presence of 30%*** structural basis for the improved activity was studied by substrate docking and molecular dynamics *** was revealed that W474V and F488G mutations caused a significant change in the geometry of the active center,which may facilitate binding and subsequent hydrolysis of bulky *** conclusion,the combination of protein and solvent engineering may be an effective approach to improve the activities of promiscuous enzymes and could be used to create naturally rare hyperthermophilic enzymes.

关键词： acylaminoacyl peptidase esterase substrate specificity protein engineering solvent engineering

Deubiquitination complex platform:A plausible mechanism for regulating the substrate specificity of deubiquitinating enzymes

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Acta Pharmaceutica Sinica B 2023年第7期13卷 2955-2962页

作者： Yi-Zheng Fang Li Jiang Qiaojun He Ji Cao Bo Yang Institute of Pharmacology and Toxicology Zhejiang Province Key Laboratory of Anti-Cancer Drug ResearchCollege of Pharmaceutical SciencesZhejiang UniversityHangzhou 310058China The Innovation Institute for Artificial Intelligence in Medicine Zhejiang UniversityHangzhou 310018China Cancer Center of Zhejiang University Hangzhou 310058China Center for Drug Safety Evaluation and Research of Zhejiang University Hangzhou 310058China

Deubiquitinating enzymes(DUBs) or deubiquitinases facilitate the escape of multiple proteins from ubiquitin-proteasome degradation and are critical for regulating protein expression levels in ***,dissecting the underlying mechanism of DUB recognition is needed to advance the development of drugs related to DUB signaling *** data,extensive studies on the ubiquitin chain specificity of DUBs have been reported,but substrate protein recognition is still not clearly *** a breakthrough,the scaffolding role may be significant to substrate protein *** this perspective,we systematically characterized the scaffolding proteins and complexes contributing to DUB substrate ***,we proposed a deubiquitination complex platform(DCP) as a potentially generic mechanism for DUB substrate recognition based on known examples,which might fill the gaps in the understanding of DUB substrate specificity.

关键词： Deubiquitinating enzyme(DUB) Deubiquitination substrate specificity Complex Scaffold Protein partner

Study on substrate specificity at Subsites for Severe Acute Respiratory Syndrome Coronavirus 3CL Protease

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Acta Biochimica et Biophysica Sinica 2005年第12期37卷 807-813页

作者： Yu-Fei SHAN~1 Gen-Jun XU~(1,2)1 Institute of Bichemistry and Cell Biology.Shanghai Institutes for Biological Sciences,Chinese Academy of Sciences,Shanghai 200031,China 2 College of Life Science Zhejiang Sci-Tech University,Hangzhou 310018,China Institute of Biochemistry and Cell Biology Shanghai Institutes for Biological SciencesChinese Academy of SciencesShanghai 200031China College of Life Science Zhejiang Sci-Tech UniversityHangzhou 310018China

Autocleavage assay and peptide-based cleavage assay were used to study the substratespecificity of 3CL protease from the severe acute respiratory syndrome *** was found that therecognition between the enzyme and its substrates involved many positions in the substrate,at least includingresidues from P4 to P2′.The deletion of either P4 or P2′residue in the substrate would decrease its cleavageefficiency *** contrast to the previous suggestion that only small residues in substrate could beaccommodated to the S1′subsite,we have found that bulky residues such as Tyr and Trp were also *** addition,based on both peptide-based assay and autocleavage assay,Ile at the P1′position could not behydrolyzed,but the mutant L27A could hydrolyze the Ile peptide *** suggested that there was astereo hindrance between the S1′subsite and the side chain of Ile in the *** 20 amino acids exceptPro could be the residue at the P2′position in the substrate,but the cleavage efficiencies were *** specificity information of the enzyme is helpful for potent anti-virus inhibitor design and usefulfor other coronavirus studies.

关键词： SARS coronavirus 3CL protease substrate specificity autocleavage binding site inhibitor design

COMPARISONS OF ESTERASE PROFILES AND substrate specificity BETWEEN ORGANO PHOSP HATE - SUSCEPTIBLE AND RESISTANT CLONES OF THE GREENBUG （SCHIZAPHIS GRAMINUM）

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COMPARISONS OF ESTERASE PROFILES AND SUBSTRATE SPECIFICITY B...

Molecular mechanism of substrate specificity for heparan sulfate2-O-sulfotransferase

中国昆虫学会2000年学术年会

作者： HE Feng - qin (Institute of Zoology, The Chinese Academy of Sciences, Beijing 100080)ZHU Kun - yan (Department of Zoology, Kansas State University, Manhattan, KS 66506)

＜正＞ The profiles and substrate specificity of esterases were compared between an organophosphosphate （OF） - susceptible （OSS） and a resistant （OR - 2） clones of the greenbug （Schizaphis graminum） using five selected esterase substrates. Five substrates generated similar esterase profiles, and showed a distinct peak of the esterase activity in the same position in the OR - 2 clone, wherease the OSS clone did not show such a distinct peak. ( - naphythyl acetate was the best substrate followed by ( - naphthyl butyrate, p -nitrophenyl acetate, and phenyl acetate for discriminating these two greenbug clones. The major esterase peak contained three esterases with no significant difference in their affinity to ( - naphthyl acetate and sensitivity to inhibition by paraoxon and PHMB. These esterases apparently were B - type esterases with some A - type esterase properties based on their substrate and inhibitor specificity.

关键词： greenbug (Schizaphis graminum) organophosphate - resistance esterase substrate specificity

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Molecular mechanism of substrate specificity for heparan sul...

FROM STRUCTURE TO FUNCTION:INSIGHTS INTO THE CATALYTIC substrate specificity AND THERMOSTABILITY DISPLAYED BY BACILLUS SUBTILIS MANNANASE BCMAN

2014年中国药学大会暨中国药师周

作者：刘纯慧 Juzheng Sheng Juno M.Krahn Lalith Perera Yongmei Xu Po-Hung Hsieh Wenfang Dou Jian Liu Lars C.Pedersen Institute of Biochemical and Biotechnological Drug School of Pharmaceutical SciencesShandong University Division of Chemical Biology and Medicinal Chemistry Eshelman School of PharmacyUniversity of North Carolina Laboratory of Structural Biology National Institute of Environmental Health SciencesNational Institutes of HealthResearch Triangle ParkNC Lab of Pharmaceutical Engineering School of Pharmaceutical SciencesJiangnan University

Heparan sulfate（HS）is an abundant polysaccharide in the animal kingdom with essential physiological *** is composed of sulfated saccharides that are biosynthesized through a complex pathway involving multiple *** vivo regulation of this process remains *** 2-Osulfotransferase（2OST）is a key enzyme in this ***,we report the crystal structure of the ternary complex of2OST,3’-phosphoadenosine 5’-phosphate,and a heptasaccharide *** site-directed mutagenesis and specific oligosaccharide substrate sequences,we probed the molecular basis of specificity and 2OST’s position in the ordered HS biosynthesis *** studies revealed that Arg80,Lys350,and Arg190 of 2OST interact with the N-sulfo groups near the modification site,consistent with the dependence of 2OST on *** contrast,6-O-sulfo groups on HS are likely excluded by steric and electrostatic repulsion within the active site supporting the hypothesis that2-O-sulfation occurs prior to *** results provide the structural evidence for understanding the sequence of enzymatic events in this pathway.

关键词： Heparan sulfate 2-O-sulfatransferase substrate specificity crystal structure site-directed mutagenesis oligosaccharide

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FROM STRUCTURE TO FUNCTION:INSIGHTS INTO THE CATALYTIC SUBST...

Screening for a new Streptomyces strain capable of efficient keratin degradation

第二届全国“跨学科蛋白质研究”学术讨论会

作者：闫小雪安晓敏桂璐璐梁栋材生物大分子国家重点实验室中国科学院生物物理研究所

BCman,αβ-mannanase from the plant root beneficial bacterium *** Z-2,has a potential to be used in the production of Mannooligosaccharide (MOS),which shows defense induction activity on both melon and tobacco,and pla... 详细信息

关键词： endo-β-mannanase substrate specificity thermostability shallow-dish-shaped active center X-ray crystallography

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Journal of Environmental Sciences 2007年第9期19卷 1125-1128页

作者： CHAO Ya-peng XIE Fu-hong YANG Jing LU Jing-hua QIAN Shi-jun State Key Laboratories of Transducer Technology Institute of Microbiology Chinese Academy of Sciences Beijing 100080 China.

Keratinous wastes could be degraded by some microorganisms in nature. Native human foot skin （NHFS） was used as sole nitrogen source to screen microorganisms with keratin-degrading capability. From approximately 200 strains, a strain of Streptomyces sp. strain No. 16 was found to possess the strongest keratinolytic activity, and the total activity in the culture was 110 KU/ml with specific activity of 2870 KU/mg protein （KU： keratinase unit）. substrate specificity test indicated that the crude keratinase could degrade keratin azure, human hair, cock feathers and collagen. The optimal pH of the crude keratinase ranged from 7.5 to 10 and the temperature ranged from 40℃ to 55℃. Metal chelating agent ethylenediamine tetraacetic acid obviously stimulated the keratinolytic activity but suppressed the proteolytic activity. To our knowledge, this is the first report on specific induction of keratinases by NHFS from an actinomycete. Moreover, excellent characteristics of its crude keratinase may lead to the potential application in waste treatment and recovery, poultry and leather industry, medicine, and cosmetic development.

关键词： keratinolytic capability Streptomyces sp. native human foot skin (NHFS) keratinase substrate specificity

Characterization of extracellular phosphatase activities in periphytic biofilm from paddy field

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Pedosphere 2021年第1期31卷 116-124页

作者： Shujie CAI Kaiying DENG Jun TANG Rui SUN Hailong LU Jiuyu LI Yonghong WU Renkou XU Institute of Soil Science Chinese Academy of SciencesNanjing 210008China College of Resources and Environment University of Chinese Academy of SciencesBeijing 100049China

Periphytic biofilms exist widely in paddy fields, but their influences on the hydrolysis of organic phosphorus(P) have rarely been investigated. In this study,a periphytic biofilm was incubated in a paddy soil solution, and hydrolysis kinetic parameters(half-saturation constant(Km) and maximum catalytic reaction rate(Vmax)), optimal environmental conditions, substrate specificity, and response to different P regimes of the phosphatase activities in the periphytic biofilm were determined, in order to characterize extracellular phosphatase activities in periphytic biofilms from paddy fields. The results indicated that the periphytic biofilm could produce an acid phosphomonoesterase(PMEase), an alkaline PMEases, and a phosphodiesterase(PDEase). These three phosphatases displayed high substrate affinity, with Km values ranging from 141.03 to 212.96 μmol L^(-1). The Vmax/Km ratios for the phosphatases followed the order of alkaline PMEase > acid PMEase > PDEase, which suggested that the PMEases, especially the alkaline PMEase, had higher catalytic efficiency. The optimal pH was 6.0 for the acid PMEase activity and 8.0 for the PDEase activity, and the alkaline PMEase activity increased with a pH increase from 7.0 to 12.0. The optimal temperature was 50℃ for the PMEases and 60℃ for the PDEase. The phosphatases showed high catalytic efficiency for condensed P over a wide pH range and for orthophosphate monoesters at pH 11.0, except for inositol hexakisphosphate at pH 6.0. The inorganic P supply was the main factor in the regulation of phosphatase activities. These findings demonstrated that the periphytic biofilm tested had high hydrolysis capacity for organic and condensed P,especially under P-limited conditions.

关键词： hydrolysis capacity kinetic parameters optimal environmental conditions phosphodiesterase phosphomonoesterase phosphorus hydrolysis phosphorus supply substrate specificity