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, ...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.展开更多
In the blood fluke Schistosoma mansoni a functionally active, monomeric, phospholipid hydroperoxide glutathione peroxidase (PHGPx) has been purified and characterized. This enzyme contains a catalytically active selen...In the blood fluke Schistosoma mansoni a functionally active, monomeric, phospholipid hydroperoxide glutathione peroxidase (PHGPx) has been purified and characterized. This enzyme contains a catalytically active selenocysteine. The protein has been shown to be the product of a cloned gene, previously referred to as a glutathione peroxidase gene. S. mansoni PHGPx has been found 5 times more abundant in female than in male worm extract. As in vertebrate PHGPx, homology alignment indicates that the residues involved in the glutathione binding by the tetrameric cellular glutathione peroxidase are mutated in the S. mansoni enzyme. Thus, this aspect appears a landmark of the PHGPx-type of glutathione peroxidases,which might be of functional relevance展开更多
Deubiquitinating enzymes(DUBs) or deubiquitinases facilitate the escape of multiple proteins from ubiquitin-proteasome degradation and are critical for regulating protein expression levels in vivo.Therefore,dissecting...Deubiquitinating enzymes(DUBs) or deubiquitinases facilitate the escape of multiple proteins from ubiquitin-proteasome degradation and are critical for regulating protein expression levels in vivo.Therefore,dissecting the underlying mechanism of DUB recognition is needed to advance the development of drugs related to DUB signaling pathways.To data,extensive studies on the ubiquitin chain specificity of DUBs have been reported,but substrate protein recognition is still not clearly understood.As a breakthrough,the scaffolding role may be significant to substrate protein selectivity.From this perspective,we systematically characterized the scaffolding proteins and complexes contributing to DUB substrate selectivity.Furthermore,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.展开更多
Substrate specificity is a hallmark of enzymatic catalysis.In this work,the biomimetic catalytic oxidation of styrene and cyclohexanone by iron(III)porphyrins and molecular oxygen was carried out,and remarkable differ...Substrate specificity is a hallmark of enzymatic catalysis.In 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 observed.The specificity of the substrates for biomimetic catalytic oxidation was investigated by kinetics and mechanistic studies.Kinetics 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 catalysis.Different catalytic mechanisms for the two substrates were discussed by operando electron paramagnetic resonance spectroscopy,operando UV-vis spectroscopy,and KI/starch experiments.Substrate specificity was concluded to be attributed to the stability of high-valence species and oxygen transfer rate.展开更多
The inherent evolvability of promiscuous enzymes endows them with great potential to be artificially evolved for novel functions.Previously,we succeeded in transforming a promiscuous acylaminoacyl peptidase(apAAP)from...The inherent evolvability of promiscuous enzymes endows them with great potential to be artificially evolved for novel functions.Previously,we succeeded in transforming a promiscuous acylaminoacyl peptidase(apAAP)from the hyperthermophilic archaeon Aeropyrum pernix K1 into a specific carboxylesterase by making a single mutation.In 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 engineering.After 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 identified.Additionally,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 type.Its 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%DMSO.The structural basis for the improved activity was studied by substrate docking and molecular dynamics simulation.It 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 substrates.In 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.展开更多
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...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.展开更多
Tyrosine aminotransferase(TAT)catalyzes the transamination of tyrosine and other aromatic amino acids.The enzyme is thought to play a role in tyrosinemia type II,hepatitis and hepatic carcinoma recovery.The objective ...Tyrosine aminotransferase(TAT)catalyzes the transamination of tyrosine and other aromatic amino acids.The enzyme is thought to play a role in tyrosinemia type II,hepatitis and hepatic carcinoma recovery.The objective of this study is to investigate its biochemical and structural characteristics and substrate specificity in order to provide insight regarding its involvement in these diseases.Mouse TAT(mTAT)was cloned from a mouse cDNA library,and its recombinant protein was produced using Escherichia coli cells and purified using various chromatographic techniques.The recombinant mTAT is able to catalyze the transamination of tyrosine usingα-ketoglutaric acid as an amino group acceptor at neutral pH.The enzyme also can use glutamate and phenylalanine as amino group donors and p-hydroxyphenylpyruvate,phenylpyruvate and alpha-ketocaproic acid as amino group acceptors.Through macromolecular crystallography we have determined the mTAT crystal structure at 2.9Åresolution.The crystal structure revealed the interaction between the pyridoxal-5′-phosphate cofactor and the enzyme,as well as the formation of a disulphide bond.The detection of disulphide bond provides some rational explanation regarding previously observed TAT inactivation under oxidative conditions and reactivation of the inactive TAT in the presence of a reducing agent.Molecular dynamics simulations using the crystal structures of Trypanosoma cruzi TAT and human TAT provided further insight regarding the substrate-enzyme interactions and substrate specificity.The biochemical and structural properties of TAT and the binding of its cofactor and the substrate may help in elucidation of the mechanism of TAT inhibition and activation.展开更多
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 solutio...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.展开更多
Diterpene glycosyltransferase UGT76G1 from Stevia rebaudiana (SrUGT76G1) is key to the generation ofeconomically important steviol glycosides (SGs), a group of natural sweeteners with high-intensity sweetness. SrUGT76...Diterpene glycosyltransferase UGT76G1 from Stevia rebaudiana (SrUGT76G1) is key to the generation ofeconomically important steviol glycosides (SGs), a group of natural sweeteners with high-intensity sweetness. SrUGT76G1 accommodates a wide range of steviol-derived substrates and many other small molecules. We report here the crystal structures of SrUGT76G1 in complex with multiple ligands to answer howthis enzyme recognizes diterpenoid aglycones and catalyzes the 1,3-sugar chain branching. A spaciouspocket for sugar-acceptor binding was observed from the determined SrUGT76G1 structures, which canexplain its broad substrate spectrum. Residues Gly87 and Leu204 lining the pocket play key roles in switching between diterpenoid and flavonoid glucosylation. An engineered mutant of SrUGT76G1, T284S, couldcatalyze a selectively increased production of next-generation sweetener rebaudioside M, with diminishedside product of rebaudioside I. Taken together, these resutls provide significant insights into molecularbasis of the substrate specificity of scarcely documented diterpenoid glycosyltransferases and wouldfacilitate the structure-guided glycoengineering to produce diversified diterpenoids with new activities.展开更多
Disaccharide phosphorylases(DSPs)are carbohydrate-active enzymes with outstanding potential for the biocatalytic conversion of common table sugar into products with attractive properties.They are modular enzymes that ...Disaccharide phosphorylases(DSPs)are carbohydrate-active enzymes with outstanding potential for the biocatalytic conversion of common table sugar into products with attractive properties.They are modular enzymes that form active homo-oligomers.From a mechanistic as well as a structural point of view,they are similar to glycoside hydrolases or glycosyltransferases.As the majority of DSPs show strict stereo-and regiospecificities,these enzymes were used to synthesize specific disaccharides.Currently,protein engineering of DSPs is pursued in different laboratories to broaden the donor and acceptor substrate specificities or improve the industrial particularity of naturally existing enzymes,to eventually generate a toolbox of new catalysts for glycoside synthesis.Herein we review the characteristics and classifications of reported DSPs and the glycoside products that they have been used to synthesize.展开更多
Successful import of hundreds of nucleus-encoded proteins is essential for chloroplast biogenesis. The import of cytosolic precursor proteins relies on the Toc- （translocon at the outer chloroplast membrane） and Ti...Successful import of hundreds of nucleus-encoded proteins is essential for chloroplast biogenesis. The import of cytosolic precursor proteins relies on the Toc- （translocon at the outer chloroplast membrane） and Tic- （translocon at the inner chloroplast membrane） complexes. In Arabidopsis thaliana, precursor recognition is mainly mediated by outer membrane receptors belonging to two gene families： Toc34/33 and Toc159/132/120/90. The role in import and precursor selectivity of these receptors has been intensively studied, but the function of Toc90 still remains unclear. Here, we report the ability of Toc90 to support the import of Toc159 client proteins. We show that the overexpression of Toc90 partially complements the albino knockout of Toc159 and restores photoautotrophic growth. Several lines of evidence including proteome profiling demonstrate the import and accumulation of proteins essential for chloroplast biogenesis and functionality.展开更多
Human NUDT16(hNUDT16)is a decapping enzyme initially identified as the human homolog to the Xenopus laevis X29.As a metalloenzyme,hNUDT16 relies on divalent cations for its cap-hydrolysis activity to remove m7 GDP and...Human NUDT16(hNUDT16)is a decapping enzyme initially identified as the human homolog to the Xenopus laevis X29.As a metalloenzyme,hNUDT16 relies on divalent cations for its cap-hydrolysis activity to remove m7 GDP and m227GDP from RNAs.Metal also determines substrate specificity of the enzyme.So far,only U8 small nucleolar RNA(snoRNA)has been identified as the substrate of hNUDT16 in the presence of Mg2+.Here we demonstrate that besides U8,hNUDT16 can also actively cleave the m7 GDP cap from mRNAs in the presence of Mg2+or Mn2+.We further show that hNUDT16 does not preferentially recognize U8 or mRNA substrates by our cross-inhibition and quantitative decapping assays.In addition,our mutagenesis analysis identifies several key residues involved in hydrolysis and confirms the key role of the REXXEE motif in catalysis.Finally an investigation into the subcellular localization of hNUDT16 revealed its abundance in both cytoplasm and nucleus.These findings extend the substrate spectrum of hNUDT16 beyond snoRNAs to also include mRNA,demonstrating the pleiotropic decapping activity of hNUDT16.展开更多
Protein succinylation is a biochemical reaction in which a succinyl group(-CO-CH2-CH2-CO-)is attached to the lysine residue of a protein molecule.Lysine succinylation plays important regulatory roles in living cells.H...Protein succinylation is a biochemical reaction in which a succinyl group(-CO-CH2-CH2-CO-)is attached to the lysine residue of a protein molecule.Lysine succinylation plays important regulatory roles in living cells.However,studies in this field are limited by the difficulty in experimentally identifying the substrate site specificity of lysine succinylation.To facilitate this process,several tools have been proposed for the computational identification of succinylated lysine sites.In this study,we developed an approach to investigate the substrate specificity of lysine succinylated sites based on amino acid composition.Using experimentally verified lysine succinylated sites collected from public resources,the significant differences in position-specific amino acid composition between succinylated and non-succinylated sites were represented using the Two Sample Logo program.These findings enabled the adoption of an effective machine learning method,support vector machine,to train a predictive model with not only the amino acid composition,but also the composition of k-spaced amino acid pairs.After the selection of the best model using a ten-fold crossvalidation approach,the selected model significantly outperformed existing tools based on an independent dataset manually extracted from published research articles.Finally,the selected model was used to develop a web-based tool,SuccSite,to aid the study of protein succinylation.Two proteins were used as case studies on the website to demonstrate the effective prediction of succinylation sites.We will regularly update SuccSite by integrating more experimental datasets.SuccSite is freely accessible at http://csb.cse.yzu.edu.tw/SuccSite/.展开更多
Dralll is a type liP restriction endonucleases （REases） that recognizes and creates a double strand break within the gapped palindromic sequence CACTNNN^GTG of double-stranded DNA indicates nicking on the bottom st...Dralll is a type liP restriction endonucleases （REases） that recognizes and creates a double strand break within the gapped palindromic sequence CACTNNN^GTG of double-stranded DNA indicates nicking on the bottom strand; indicates nicking on the top strand）. However, wild type Dralll shows significant star activity. In this study, it was found that the prominent star site is CATSGTT;GTG, consisting of a star 5＇ half （CAT） and a canonical 3＇ half （GTG）. Dralll nicks the 3＇ canonical half site at a faster rate than the 5＇ star half site, in contrast to the similar rate with the canonical full site. The crystal structure of the Dralll protein was solved. It indicated, as supported by mutagenesis, that Dralll possesses a ~13a- metal HNH active site. The structure revealed extensive intra-molecular interactions between the N-terminal domain and the C-terminal domain containing the HNH active site. Disruptions of these interactions through site- directed mutagenesis drastically increased cleavage fidelity. The understanding of fidelity mechanisms will enable generation of high fidelity REases.展开更多
l-Lactate dehydrogenases can reduce alpha-keto carboxylic acids asymmetrically and generally have a broad substrate spectrum.l-Lactate dehydrogenase gene(LF-l-LDH0845)with reducing activity towards 3,4-dihydroxyphenyl...l-Lactate dehydrogenases can reduce alpha-keto carboxylic acids asymmetrically and generally have a broad substrate spectrum.l-Lactate dehydrogenase gene(LF-l-LDH0845)with reducing activity towards 3,4-dihydroxyphenylpyruvate and phenylpyruvate was obtained from Lactobacillus fermentum JN248.To change the substrate specificity of LDH0845 and improve its catalytic activity towards large substrates,site-directed mutation of Tyr221 was performed by analyzing the amino acids in the active center.Kinetic parameters show that the kcat values of Y221F mutant on 3,4-dihydroxyphenylpyruvate,4-methyl-2-oxopentanoate,and glyoxylate are 1.21 s^(−1),1.35 s^(−1),and 0.72 s^(−1),respectively,which are 420%,150%and 130%of the wild-type LDH0845.This study shows that the mutations of Y221 can significantly change the substrate specificity of LDH0845,making it become a potential tool enzyme for the reduction of alpha-keto carboxylic acids with large functional groups.展开更多
基金supported by the National Natural Science Foundation of China(Grant Nos.11674383,11474346,and 11274374)the National Basic Research Program of China(Grant No.2013CB837200)the National Key Research and Development Program of China(Grant No.2016YFA0301500)
文摘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.
文摘In the blood fluke Schistosoma mansoni a functionally active, monomeric, phospholipid hydroperoxide glutathione peroxidase (PHGPx) has been purified and characterized. This enzyme contains a catalytically active selenocysteine. The protein has been shown to be the product of a cloned gene, previously referred to as a glutathione peroxidase gene. S. mansoni PHGPx has been found 5 times more abundant in female than in male worm extract. As in vertebrate PHGPx, homology alignment indicates that the residues involved in the glutathione binding by the tetrameric cellular glutathione peroxidase are mutated in the S. mansoni enzyme. Thus, this aspect appears a landmark of the PHGPx-type of glutathione peroxidases,which might be of functional relevance
基金supported by National Natural Science Foundation of China(No.U21A20420 to Bo Yang)Zhejiang Provincial Natural Science Foundation(No.LR22H310002 to Ji Cao,China)。
文摘Deubiquitinating enzymes(DUBs) or deubiquitinases facilitate the escape of multiple proteins from ubiquitin-proteasome degradation and are critical for regulating protein expression levels in vivo.Therefore,dissecting the underlying mechanism of DUB recognition is needed to advance the development of drugs related to DUB signaling pathways.To data,extensive studies on the ubiquitin chain specificity of DUBs have been reported,but substrate protein recognition is still not clearly understood.As a breakthrough,the scaffolding role may be significant to substrate protein selectivity.From this perspective,we systematically characterized the scaffolding proteins and complexes contributing to DUB substrate selectivity.Furthermore,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.
基金support from the National Natural Science Foundation of China(No.21938001 and 21878344)the National Key Research and Development Program of China(2016YFA0602900)+3 种基金the National Natural Science Foundation of ChinaSINOPEC Joint Fund(No.U1663220)Guangdong Provincial Key R&D Programme(2019B110206002)the Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program(2017BT01C102)Research and Innovation Team Construction Project of Guangdong University of Petrochemical Technology.
文摘Substrate specificity is a hallmark of enzymatic catalysis.In 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 observed.The specificity of the substrates for biomimetic catalytic oxidation was investigated by kinetics and mechanistic studies.Kinetics 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 catalysis.Different catalytic mechanisms for the two substrates were discussed by operando electron paramagnetic resonance spectroscopy,operando UV-vis spectroscopy,and KI/starch experiments.Substrate specificity was concluded to be attributed to the stability of high-valence species and oxygen transfer rate.
基金supported by the National Basic Research Program of China(973 Program)the National Natural Science Foundation of China.
文摘The inherent evolvability of promiscuous enzymes endows them with great potential to be artificially evolved for novel functions.Previously,we succeeded in transforming a promiscuous acylaminoacyl peptidase(apAAP)from the hyperthermophilic archaeon Aeropyrum pernix K1 into a specific carboxylesterase by making a single mutation.In 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 engineering.After 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 identified.Additionally,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 type.Its 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%DMSO.The structural basis for the improved activity was studied by substrate docking and molecular dynamics simulation.It 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 substrates.In 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.
文摘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.
基金supported in part by a research grant from NINDS(NS062836)。
文摘Tyrosine aminotransferase(TAT)catalyzes the transamination of tyrosine and other aromatic amino acids.The enzyme is thought to play a role in tyrosinemia type II,hepatitis and hepatic carcinoma recovery.The objective of this study is to investigate its biochemical and structural characteristics and substrate specificity in order to provide insight regarding its involvement in these diseases.Mouse TAT(mTAT)was cloned from a mouse cDNA library,and its recombinant protein was produced using Escherichia coli cells and purified using various chromatographic techniques.The recombinant mTAT is able to catalyze the transamination of tyrosine usingα-ketoglutaric acid as an amino group acceptor at neutral pH.The enzyme also can use glutamate and phenylalanine as amino group donors and p-hydroxyphenylpyruvate,phenylpyruvate and alpha-ketocaproic acid as amino group acceptors.Through macromolecular crystallography we have determined the mTAT crystal structure at 2.9Åresolution.The crystal structure revealed the interaction between the pyridoxal-5′-phosphate cofactor and the enzyme,as well as the formation of a disulphide bond.The detection of disulphide bond provides some rational explanation regarding previously observed TAT inactivation under oxidative conditions and reactivation of the inactive TAT in the presence of a reducing agent.Molecular dynamics simulations using the crystal structures of Trypanosoma cruzi TAT and human TAT provided further insight regarding the substrate-enzyme interactions and substrate specificity.The biochemical and structural properties of TAT and the binding of its cofactor and the substrate may help in elucidation of the mechanism of TAT inhibition and activation.
基金supported by the State Key Basic Research Program of China (No. 2015CB158200)the National Natural Science Foundation of China (No. 41877102)+1 种基金the Interdisciplinary Innovation Team of Chinese Academy of Sciences (CAS)the Youth Innovation Promotion Association of CAS (No. 2014272)。
文摘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.
基金This work was financially supported by the National Key R&D Program of China(2018YFA0900600)the Strategic Priority Research Program "Molecular Mechanism of Plant Growth and Development" of CAS(XDB27020202,XDB27020103)+5 种基金the National Natural Science Foundation of China(31700263,31670099,31700261)grants from the Shanghai Science and Technology Commission(19XD1424500)J.L.is supported by the Foundation of Youth Innovation Promotion Association of the Chinese Academy of SciencesThis work was also financially supported by the Construction of the Registry and Database of Bioparts for Synthetic Biology of the Chinese Academy of Science(no.ZSYS-016)the International Partnership Program of Chinese Academy of Science(no.153D31KYSB20170121)the National Key Laboratory of Plant Molecular Genetics,SIPPI,CAS.
文摘Diterpene glycosyltransferase UGT76G1 from Stevia rebaudiana (SrUGT76G1) is key to the generation ofeconomically important steviol glycosides (SGs), a group of natural sweeteners with high-intensity sweetness. SrUGT76G1 accommodates a wide range of steviol-derived substrates and many other small molecules. We report here the crystal structures of SrUGT76G1 in complex with multiple ligands to answer howthis enzyme recognizes diterpenoid aglycones and catalyzes the 1,3-sugar chain branching. A spaciouspocket for sugar-acceptor binding was observed from the determined SrUGT76G1 structures, which canexplain its broad substrate spectrum. Residues Gly87 and Leu204 lining the pocket play key roles in switching between diterpenoid and flavonoid glucosylation. An engineered mutant of SrUGT76G1, T284S, couldcatalyze a selectively increased production of next-generation sweetener rebaudioside M, with diminishedside product of rebaudioside I. Taken together, these resutls provide significant insights into molecularbasis of the substrate specificity of scarcely documented diterpenoid glycosyltransferases and wouldfacilitate the structure-guided glycoengineering to produce diversified diterpenoids with new activities.
基金This work was supported by the Key Research Program of the Chinese Academy of Sciences(Grant No.ZDRW-ZS-2016-3)the National Natural Science Foundation of China(Grant No.21778073)Tianjin Synthetic Biotechnology Innovation Capacity Improvement Project(Grant No.TSBICIP-KJGG-003).
文摘Disaccharide phosphorylases(DSPs)are carbohydrate-active enzymes with outstanding potential for the biocatalytic conversion of common table sugar into products with attractive properties.They are modular enzymes that form active homo-oligomers.From a mechanistic as well as a structural point of view,they are similar to glycoside hydrolases or glycosyltransferases.As the majority of DSPs show strict stereo-and regiospecificities,these enzymes were used to synthesize specific disaccharides.Currently,protein engineering of DSPs is pursued in different laboratories to broaden the donor and acceptor substrate specificities or improve the industrial particularity of naturally existing enzymes,to eventually generate a toolbox of new catalysts for glycoside synthesis.Herein we review the characteristics and classifications of reported DSPs and the glycoside products that they have been used to synthesize.
文摘Successful import of hundreds of nucleus-encoded proteins is essential for chloroplast biogenesis. The import of cytosolic precursor proteins relies on the Toc- （translocon at the outer chloroplast membrane） and Tic- （translocon at the inner chloroplast membrane） complexes. In Arabidopsis thaliana, precursor recognition is mainly mediated by outer membrane receptors belonging to two gene families： Toc34/33 and Toc159/132/120/90. The role in import and precursor selectivity of these receptors has been intensively studied, but the function of Toc90 still remains unclear. Here, we report the ability of Toc90 to support the import of Toc159 client proteins. We show that the overexpression of Toc90 partially complements the albino knockout of Toc159 and restores photoautotrophic growth. Several lines of evidence including proteome profiling demonstrate the import and accumulation of proteins essential for chloroplast biogenesis and functionality.
基金the Natural Science Foundation of China(No.30870118)。
文摘Human NUDT16(hNUDT16)is a decapping enzyme initially identified as the human homolog to the Xenopus laevis X29.As a metalloenzyme,hNUDT16 relies on divalent cations for its cap-hydrolysis activity to remove m7 GDP and m227GDP from RNAs.Metal also determines substrate specificity of the enzyme.So far,only U8 small nucleolar RNA(snoRNA)has been identified as the substrate of hNUDT16 in the presence of Mg2+.Here we demonstrate that besides U8,hNUDT16 can also actively cleave the m7 GDP cap from mRNAs in the presence of Mg2+or Mn2+.We further show that hNUDT16 does not preferentially recognize U8 or mRNA substrates by our cross-inhibition and quantitative decapping assays.In addition,our mutagenesis analysis identifies several key residues involved in hydrolysis and confirms the key role of the REXXEE motif in catalysis.Finally an investigation into the subcellular localization of hNUDT16 revealed its abundance in both cytoplasm and nucleus.These findings extend the substrate spectrum of hNUDT16 beyond snoRNAs to also include mRNA,demonstrating the pleiotropic decapping activity of hNUDT16.
基金the Warshel Institute for Computational Biology,School of Life and Health Sciences,The Chinese University of Hong Kong,Shenzhen,China for financially supporting this research
文摘Protein succinylation is a biochemical reaction in which a succinyl group(-CO-CH2-CH2-CO-)is attached to the lysine residue of a protein molecule.Lysine succinylation plays important regulatory roles in living cells.However,studies in this field are limited by the difficulty in experimentally identifying the substrate site specificity of lysine succinylation.To facilitate this process,several tools have been proposed for the computational identification of succinylated lysine sites.In this study,we developed an approach to investigate the substrate specificity of lysine succinylated sites based on amino acid composition.Using experimentally verified lysine succinylated sites collected from public resources,the significant differences in position-specific amino acid composition between succinylated and non-succinylated sites were represented using the Two Sample Logo program.These findings enabled the adoption of an effective machine learning method,support vector machine,to train a predictive model with not only the amino acid composition,but also the composition of k-spaced amino acid pairs.After the selection of the best model using a ten-fold crossvalidation approach,the selected model significantly outperformed existing tools based on an independent dataset manually extracted from published research articles.Finally,the selected model was used to develop a web-based tool,SuccSite,to aid the study of protein succinylation.Two proteins were used as case studies on the website to demonstrate the effective prediction of succinylation sites.We will regularly update SuccSite by integrating more experimental datasets.SuccSite is freely accessible at http://csb.cse.yzu.edu.tw/SuccSite/.
文摘Dralll is a type liP restriction endonucleases （REases） that recognizes and creates a double strand break within the gapped palindromic sequence CACTNNN^GTG of double-stranded DNA indicates nicking on the bottom strand; indicates nicking on the top strand）. However, wild type Dralll shows significant star activity. In this study, it was found that the prominent star site is CATSGTT;GTG, consisting of a star 5＇ half （CAT） and a canonical 3＇ half （GTG）. Dralll nicks the 3＇ canonical half site at a faster rate than the 5＇ star half site, in contrast to the similar rate with the canonical full site. The crystal structure of the Dralll protein was solved. It indicated, as supported by mutagenesis, that Dralll possesses a ~13a- metal HNH active site. The structure revealed extensive intra-molecular interactions between the N-terminal domain and the C-terminal domain containing the HNH active site. Disruptions of these interactions through site- directed mutagenesis drastically increased cleavage fidelity. The understanding of fidelity mechanisms will enable generation of high fidelity REases.
基金the National Key Scientific Instrument and Equipment Development Project of China(2013YQ17052504)the Program for Changjiang Scholars and Innovative Research Team in the University of Ministry of Education of China(IRT_15R55)+1 种基金the Natural Science Foundation of Shaanxi province(2019JQ-725)the Postgraduate Research and Practice Innovation Program of Jiangsu Province(KYCX19_1841,KYCX19_1842)for financial support.
文摘l-Lactate dehydrogenases can reduce alpha-keto carboxylic acids asymmetrically and generally have a broad substrate spectrum.l-Lactate dehydrogenase gene(LF-l-LDH0845)with reducing activity towards 3,4-dihydroxyphenylpyruvate and phenylpyruvate was obtained from Lactobacillus fermentum JN248.To change the substrate specificity of LDH0845 and improve its catalytic activity towards large substrates,site-directed mutation of Tyr221 was performed by analyzing the amino acids in the active center.Kinetic parameters show that the kcat values of Y221F mutant on 3,4-dihydroxyphenylpyruvate,4-methyl-2-oxopentanoate,and glyoxylate are 1.21 s^(−1),1.35 s^(−1),and 0.72 s^(−1),respectively,which are 420%,150%and 130%of the wild-type LDH0845.This study shows that the mutations of Y221 can significantly change the substrate specificity of LDH0845,making it become a potential tool enzyme for the reduction of alpha-keto carboxylic acids with large functional groups.
