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Study On Crystal Structure And Bioactivity Of Thermosynechococcus Elongatus Sucrose Phosphate Synthase Tll1590

Posted on:2021-04-19Degree:MasterType:Thesis
Country:ChinaCandidate:Y Y LiFull Text:PDF
GTID:2370330626963737Subject:Biochemistry and Molecular Biology
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Sucrose can help green organisms antagonize abiotic stress,including freezing and drought.Sucrose phosphate synthase(SPS)is a well-known rate limiting enzyme in the synthesis of sucrose.However,the crystal structure of SPS of green organisms has not been resolved,and the catalytic mechanism of SPS has not been fully elucidated.In this paper,we used the Uniprot code tll1590(TeSPS)from Synechococcus elongatus to identify its substrate and enzyme activity,and to analysis its crystal structure.The following are specific research contents and results:Firstly,the recombinant plasmid of tll1590-pET28 a was constructed and transformed into E.coli BL21(DE3).The high purity protein was obtained by E.coli extended culture,IPTG induced expression and Ni-NTA purification.It is preliminarily speculated that tll1590 can make UDP-glu(UDPG)and fructose-6-phosphate(F6P)produce UDP and sucrose-6-phosphate(S6P)by thin layer chromatography(TLC)detection.Mass spectrometry(MS)confirmed that Synechococcus elongates Sucrose phosphate synthase tll1590 can catalyze UDPG and F6 P to produce UDP and S6 P.In this study,we report the first co-crystal structure of sucrose phosphate synthetase tll1590 with UDP and S6 P.The structure shows that tll1590 has 16 ? helix and 14 ? sheets,with UDP and S6 P being bound at the interface of two domains(A-and B-domains).Within the catalytic site,the side chains of His158 and Glu331,along with two phosphate groups from UDP,form hydrogen bonds with the four hydroxyl groups of the glucose moiety in S6 P.This association causes these four hydroxyl groups to become partially negatively charged,thus promoting formation of the C1 oxocarbenium ion.Breakage of the hydrogen bond between His158 and one of the hydroxyl groups may trigger covalent bond formation between the C1 oxocarbenium ion and the C2 hydroxyl of F6 P.His158 and Glu331 are the key amino acids to promote the reaction.Two mutants(H158A and E331A)lost all catalytic activity.In addition,A-and B-domains were prepared and the crystal structure of A-domain was analyzed.Structure shows that two loops(loop1 and loop2)in the A-domain are too active causing their structure could not be solved.Temperature factor(B-factor)analysis and the molecular dynamics stimulations of the full-length enzyme and A-domain indicated that loop1 and loop2 are crucial for binding and releasing substrate and product.In addition,the reaction time gradient of tll1590 showed that the reaction time of the enzyme was very fast,and temperature gradient analysis shows that this tll1590 exhibits its highest activity at 70 ?,suggesting that this enzyme has the potential of being used in industrial production of S6 P.In conclusion,tll1590 was proved to be a sucrose phosphate synthetase through crystal structure and enzymology experiments,which has good activity and high temperature tolerance.It is speculated that tll1590 has the potential of being used in industrial production of S6 P.
Keywords/Search Tags:Sucrose phosphate synthase, Thermosynechococcus elongatus, Sucrose-6-phosphate, Crystal structure, Catalysis mechanism
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