Objective In Corynebacterium crenatum,the adjacent D311 and D312 of N-acetyl-L-glutamate kinase(NAGK),as a key rate-limiting enzyme of L-arginine biosynthesis under substrate regulatory control by arginine,were initia...Objective In Corynebacterium crenatum,the adjacent D311 and D312 of N-acetyl-L-glutamate kinase(NAGK),as a key rate-limiting enzyme of L-arginine biosynthesis under substrate regulatory control by arginine,were initially replaced with two arginine residues to investigate the L-arginine feedback inhibition for NAGK.Methods NAGK enzyme expression was evaluated using a plasmid-based method.Homologous recombination was employed to eliminate the pro B.Results The IC50 and enzyme activity of NAGK M4,in which the D311 R and D312 R amino acid substitutions were combined with the previously reported E19 R and H26 E substitutions,were 3.7-fold and 14.6% higher,respectively,than those of the wild-type NAGK.NAGK M4 was successfully introduced into the C.crenatum MT genome without any genetic markers;the L-arginine yield of C.crenatum MT-M4 was 26.2% higher than that of C.crenatum MT.To further improve upon the L-arginine yield,we constructed the mutant C.crenatum MT-M4 ?pro B.The optimum concentration of L-proline was also investigated in order to determine its contribution to L-arginine yield.After L-proline was added to the medium at 10 mmol/L,the L-arginine yield reached 16.5 g/L after 108 h of shake-flask fermentation,approximately 70.1% higher than the yield attained using C.crenatum MT.Conclusion Feedback inhibition of L-arginine on NAGK in C.crenatum is clearly alleviated by the M4 mutation of NAGK,and deletion of the pro B in C.crenatum from MT to M4 results in a significant increase in arginine production.展开更多
Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31(WSV340/WSSV396), an envelope protein of white spot syndrome virus(WSSV), contains...Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31(WSV340/WSSV396), an envelope protein of white spot syndrome virus(WSSV), contains an Arg-Gly-Asp(RGD) peptide domain known as a cellular attachment site. At present, the process of VP31 interacting with shrimp host cells has not been explored. Therefore, the VP31 gene was cloned into p ET30a(+), expressed in Escherichia coli strain BL21 and purifi ed with immobilized metal ion affi nity chromatography. Four gill cellular proteins of shrimp( Fenneropenaeus c hinensis) were pulled down by an affi nity column coupled with recombinant VP31(r VP31), and the amino acid sequences were identifi ed with MALDI-TOF/TOF mass spectrometry. Hemocyanin, beta-actin, arginine kinase(AK), and an unknown protein were suggested as the putative VP31 receptor proteins. SDS-PAGE showed that AK is the predominant binding protein of VP31. An i n vitro binding activity experiment indicated that recombinant AK's(r AK) binding activity with r VP31 is comparable to that with the same amount of WSSV. These results suggested that AK, as a member of the phosphagen kinase family, plays a role in WSSV infection. This is the fi rst evidence showing that AK is a binding protein of VP31. Further studies on this topic will elucidate WSSV infection mechanism in the future.展开更多
Objective: To determine exon/intron organization of the Toxocara canis(T. canis) AK(TCAK) and to test green and black tea and several other chemicals against the activity of recombinant TCAK in the guanidino-specific ...Objective: To determine exon/intron organization of the Toxocara canis(T. canis) AK(TCAK) and to test green and black tea and several other chemicals against the activity of recombinant TCAK in the guanidino-specific region by site-directed mutants. Methods: Amplification of genomic DNA fragments containing introns was carried out by PCRs. The open-reading frame(1 200 bp) of TCAK(wild type) was cloned into the BamH 1/SalI site of pM AL-c2X. The maltose-binding protein-TCAK fusion protein was expressed in Escherichia coli TB1 cells. The purity of the expressed enzyme was verified by SDS-PAGE. Mutations were introduced into the guanidino-specific region and other areas of pM AL/TCAK by PCR. Enzyme activity was measured with an NADH-linked assay at 25℃ for the forward reaction(phosphagen synthesis). Results: Arginine kinase in T. canis has a seven-exon/six-intron gene structure. The lengths of the introns ranged from 542 bp to 2 500 bp. All introns begin with gt and end with ag. Furthermore, we measured the enzyme activity of site-directed mutants of the recombinant TCAK. The K_m value of the mutant(Alanine to Serine) decreased indicating a higher affinity for substrate arginine than the wild-type. The K_m value of the mutant(Serine to Glycine) increased to 0.19 mM. The Km value(0.19 mM) of the double mutant(Alanine-Serine to Serine-Glycine) was slightly greater than in the wild-type(0.12 mM). In addition, several other chemicals were tested; including plant extract Azadiracta indica(A. indica), an aminoglycoside antibiotic(aminosidine), a citrus flavonoid glycoside(rutin) and a commercially available catechin mixture against TCAK. Green and black tea(1:10 dilution) produced 15% and 25% inhibition of TCAK, respectively. The extract of A. indica produced 5% inhibition of TCAK. Moreover, green and black tea produced a non-competitive type of inhibition and A. indica produced a mixed-type of inhibition on TCAK. Conclusions: Arginine kinase in T. canis has a seven-exon/six-intron gene structure. However, further studies are needed to identify a specific compound within the extract causing the inhibitory effect and also to determine the molecular mechanisms behind inhibition of arginine kinase in T. canis.展开更多
The complexities of energy transfer mechanisms in the flagella of mammalian sperm flagella have been intensively investigated and demonstrate significant diversity across species.Enzymatic shuttles,particularly adenyl...The complexities of energy transfer mechanisms in the flagella of mammalian sperm flagella have been intensively investigated and demonstrate significant diversity across species.Enzymatic shuttles,particularly adenylate kinase(AK)and creatine kinase(CK),are pivotal in the efficient transfer of intracellular ATP,showing distinct tissue-and species-specificity.Here,the expression profiles of AK and CK were investigated in mice and found to fall into four subgroups,of which Subgroup III AKs were observed to be unique to the male reproductive system and conserved across chordates.Both AK8 and AK9 were found to be indispensable to male reproduction after analysis of an infertile male cohort.Knockout mouse models showed that AK8 and AK9 were central to promoting sperm motility.Immunoprecipitation combined with mass spectrometry revealed that AK8 and AK9 interact with the radial spoke(RS)of the axoneme.Examination of various human and mouse sperm samples with substructural damage,including the presence of multiple RS subunits,showed that the head of radial spoke 3 acts as an adapter for AK9 in the flagellar axoneme.Using an ATP probe together with metabolomic analysis,it was found that AK8 and AK9 cooperatively regulated ATP transfer in the axoneme,and were concentrated at sites associated with energy consumption in the flagellum.These findings indicate a novel function for RS beyond its structural role,namely,the regulation of ATP transfer.In conclusion,the results expand the functional spectrum of AK proteins and suggest a fresh model regarding ATP transfer within mammalian flagella.展开更多
基金supported by Natural Science Foundation of China,No.31360219 and No.30960012the Open Project Program of Key Laboratory of Functional Small Organic Molecule,Ministry of Education,Jiangxi Normal University(No.KLFS-KF-201414)
文摘Objective In Corynebacterium crenatum,the adjacent D311 and D312 of N-acetyl-L-glutamate kinase(NAGK),as a key rate-limiting enzyme of L-arginine biosynthesis under substrate regulatory control by arginine,were initially replaced with two arginine residues to investigate the L-arginine feedback inhibition for NAGK.Methods NAGK enzyme expression was evaluated using a plasmid-based method.Homologous recombination was employed to eliminate the pro B.Results The IC50 and enzyme activity of NAGK M4,in which the D311 R and D312 R amino acid substitutions were combined with the previously reported E19 R and H26 E substitutions,were 3.7-fold and 14.6% higher,respectively,than those of the wild-type NAGK.NAGK M4 was successfully introduced into the C.crenatum MT genome without any genetic markers;the L-arginine yield of C.crenatum MT-M4 was 26.2% higher than that of C.crenatum MT.To further improve upon the L-arginine yield,we constructed the mutant C.crenatum MT-M4 ?pro B.The optimum concentration of L-proline was also investigated in order to determine its contribution to L-arginine yield.After L-proline was added to the medium at 10 mmol/L,the L-arginine yield reached 16.5 g/L after 108 h of shake-flask fermentation,approximately 70.1% higher than the yield attained using C.crenatum MT.Conclusion Feedback inhibition of L-arginine on NAGK in C.crenatum is clearly alleviated by the M4 mutation of NAGK,and deletion of the pro B in C.crenatum from MT to M4 results in a significant increase in arginine production.
基金Supported by the National Science Foundation for Post-Doctoral Scientists of China(No.2013M541965)the International Postdoctoral Academic Exchange Program+2 种基金the Qingdao Postdoctoral Science Foundation Funded Projectthe Construction Program for“Taishan Scholarship”of Shandong Province of Chinathe Program for Chinese Outstanding Talents in Agricultural Scientific Research
文摘Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31(WSV340/WSSV396), an envelope protein of white spot syndrome virus(WSSV), contains an Arg-Gly-Asp(RGD) peptide domain known as a cellular attachment site. At present, the process of VP31 interacting with shrimp host cells has not been explored. Therefore, the VP31 gene was cloned into p ET30a(+), expressed in Escherichia coli strain BL21 and purifi ed with immobilized metal ion affi nity chromatography. Four gill cellular proteins of shrimp( Fenneropenaeus c hinensis) were pulled down by an affi nity column coupled with recombinant VP31(r VP31), and the amino acid sequences were identifi ed with MALDI-TOF/TOF mass spectrometry. Hemocyanin, beta-actin, arginine kinase(AK), and an unknown protein were suggested as the putative VP31 receptor proteins. SDS-PAGE showed that AK is the predominant binding protein of VP31. An i n vitro binding activity experiment indicated that recombinant AK's(r AK) binding activity with r VP31 is comparable to that with the same amount of WSSV. These results suggested that AK, as a member of the phosphagen kinase family, plays a role in WSSV infection. This is the fi rst evidence showing that AK is a binding protein of VP31. Further studies on this topic will elucidate WSSV infection mechanism in the future.
文摘Objective: To determine exon/intron organization of the Toxocara canis(T. canis) AK(TCAK) and to test green and black tea and several other chemicals against the activity of recombinant TCAK in the guanidino-specific region by site-directed mutants. Methods: Amplification of genomic DNA fragments containing introns was carried out by PCRs. The open-reading frame(1 200 bp) of TCAK(wild type) was cloned into the BamH 1/SalI site of pM AL-c2X. The maltose-binding protein-TCAK fusion protein was expressed in Escherichia coli TB1 cells. The purity of the expressed enzyme was verified by SDS-PAGE. Mutations were introduced into the guanidino-specific region and other areas of pM AL/TCAK by PCR. Enzyme activity was measured with an NADH-linked assay at 25℃ for the forward reaction(phosphagen synthesis). Results: Arginine kinase in T. canis has a seven-exon/six-intron gene structure. The lengths of the introns ranged from 542 bp to 2 500 bp. All introns begin with gt and end with ag. Furthermore, we measured the enzyme activity of site-directed mutants of the recombinant TCAK. The K_m value of the mutant(Alanine to Serine) decreased indicating a higher affinity for substrate arginine than the wild-type. The K_m value of the mutant(Serine to Glycine) increased to 0.19 mM. The Km value(0.19 mM) of the double mutant(Alanine-Serine to Serine-Glycine) was slightly greater than in the wild-type(0.12 mM). In addition, several other chemicals were tested; including plant extract Azadiracta indica(A. indica), an aminoglycoside antibiotic(aminosidine), a citrus flavonoid glycoside(rutin) and a commercially available catechin mixture against TCAK. Green and black tea(1:10 dilution) produced 15% and 25% inhibition of TCAK, respectively. The extract of A. indica produced 5% inhibition of TCAK. Moreover, green and black tea produced a non-competitive type of inhibition and A. indica produced a mixed-type of inhibition on TCAK. Conclusions: Arginine kinase in T. canis has a seven-exon/six-intron gene structure. However, further studies are needed to identify a specific compound within the extract causing the inhibitory effect and also to determine the molecular mechanisms behind inhibition of arginine kinase in T. canis.
基金supported by National Key Research and Development Program of China(2022YFC2702702,2021YFC2700901)the National Natural Science Foundation of China(81971441,82171607,32000584)+3 种基金the University Outstanding Young Talents Support Program(gxyq2021174)Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences(2019PT310002)Anhui Provincial Natural Science Foundation(2208085Y31)the Natural Science Foundation of Jiangsu Province(BK20230004).
文摘The complexities of energy transfer mechanisms in the flagella of mammalian sperm flagella have been intensively investigated and demonstrate significant diversity across species.Enzymatic shuttles,particularly adenylate kinase(AK)and creatine kinase(CK),are pivotal in the efficient transfer of intracellular ATP,showing distinct tissue-and species-specificity.Here,the expression profiles of AK and CK were investigated in mice and found to fall into four subgroups,of which Subgroup III AKs were observed to be unique to the male reproductive system and conserved across chordates.Both AK8 and AK9 were found to be indispensable to male reproduction after analysis of an infertile male cohort.Knockout mouse models showed that AK8 and AK9 were central to promoting sperm motility.Immunoprecipitation combined with mass spectrometry revealed that AK8 and AK9 interact with the radial spoke(RS)of the axoneme.Examination of various human and mouse sperm samples with substructural damage,including the presence of multiple RS subunits,showed that the head of radial spoke 3 acts as an adapter for AK9 in the flagellar axoneme.Using an ATP probe together with metabolomic analysis,it was found that AK8 and AK9 cooperatively regulated ATP transfer in the axoneme,and were concentrated at sites associated with energy consumption in the flagellum.These findings indicate a novel function for RS beyond its structural role,namely,the regulation of ATP transfer.In conclusion,the results expand the functional spectrum of AK proteins and suggest a fresh model regarding ATP transfer within mammalian flagella.
