Chemistry amp Biology Brief Communication

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Please cite this article in press as Luo et al Peptide Macrocyclization Catalyzed by a Prolyl Oligopeptidase Involved in a Amanitin Biosynthesis . Chemistry Biology 2014 http dx doi org 10 1016 j chembiol 2014 10 015. Chemistry Biology, Peptide Cyclization by Prolyl Oligopeptidase. Expression and Assay of GmPOPA and GmPOPB in Yeast. GmPOPB protein was expressed in Saccharomyces cerevisiae. and purified on an anti c myc peptide antibody affinity col . umn Figure S1D GmPOPA protein was also expressed in. S cerevisiae Figure S1D GmPOPA showed good activity. against the chromogenic substrate Z Gly Pro pNA 176 . 5 mmol p nitroaniline min mg protein which is comparable. with published values for POP from lamb kidney Koida and. Walter 1976 but GmPOPB showed much weaker activity. 10 2 mmol min mg Versions of GmPOPB expressed without. the c myc tag or with an N terminal c myc tag were also ex . pressed in yeast but they had the same poor activity data. not shown , Figure 1 Structure of a Amanitin GmPOPA and GmPOPB were also assayed using the 35 . The macrocyclic outer ring has the sequence IWGIGCNP in single letter amino amino acid a amanitin precursor GmAMA1 as substrate . acid code GmPOPB converted GmAMA1 to a series of products consistent. with cleavage at either one or both flanking Pro residues Figures. 2A and 2B Another major product had a mass and retention. The enzymological basis of macrocyclization has been eluci time identical to synthetic cyclo IWGIGCNP Figure 2C In a. dated for several other RiPPs A protease that cuts at a con time course experiment conversion of the 35 mer to the puta . served Asn residue in the precursor to the cyclotide kalata B1 tive cyclic product progressed to near completion in 50 min. is necessary for cyclization Saska et al 2007 Cyclization of Figure 3A The 25 mer intermediate accumulated transiently. patellamide a cyanobactin is catalyzed by PatG Agarwal Figure 3B This indicates that GmPOPB catalyzed two nonpro . et al 2012 Koehnke et al 2012 Lee et al 2009 Cyclization cessive reactions hydrolysis at the carboxyl side of the first Pro. of the orbitide segetalin A is catalyzed by PCY1 Barber et al and transpeptidation of the new N terminal residue Ile to the. 2013 PCY1 is like POPB a member of the S9A family of second Pro with release of the C terminal 17 amino acid pep . serine proteases Butelase 1 is a cyclizing enzyme that can tide Figure 3C . cyclize a variety of peptides of 14 to 58 residues Nguyen POPA hydrolyzed the 25 amino acid intermediate but not. et al 2014 the full length 35 amino acid propeptide Figures S2A S2D . A bisporigera and G marginata each have two POP genes In neither case was any cyclo IWGIGCNP produced Mamma . known as POPA and POPB POPB of G marginata GmPOPB lian POP obtained commercially could also neither cleave. and POPB of A bisporigera AbPOPB are present only in am GmAMA1 nor cyclize either GmAMA1 or the 25 amino acid inter . atoxin producing species in each genus whereas the POPA mediate data not shown . genes are present in all species of Amanita and Galerina as. well as other agarics Luo et al 2010 2012 GmPOPB is Proof of the Structure of the Cyclic Product by Nuclear. clustered with one of two copies of GmAMA1 Riley et al Magnetic Resonance Spectroscopy. 2014 Collectively these observations suggested that POPB A large scale reaction using purified GmPOPB and 10 mg of. is involved in and dedicated to the biosynthesis of a amanitin GmAMA1 was used to produce 1 5 mg of the putative cyclic. and that it catalyzes cleavage of the toxin propeptides to product All 1H and 13C atoms in the product were assigned. release the mature octa or heptapeptides However direct using correlation spectroscopy COSY total correlation spec . proof of a role of POPB in amatoxin and phallotoxin biosyn troscopy TOCSY heteronuclear single quantum correlation. thesis is lacking and no evidence addresses its possible role HSQC spectroscopy and heteronuclear multiple bond correla . in macrocyclization tion HMBC NMR spectroscopy Figures S3A and S3B A signal. from the free thiol proton of Cys indicated that the product did. RESULTS not contain an internal thioester Figure S3C Rotating frame. nuclear Overhauser effect correlation spectroscopy ROESY . Targeted Mutation of GmPOPB indicated that the Ile1 HN and Pro8 Ha atoms were positioned. A transformation system using Agrobacterium tumefaciens and within 5 A of each other Figure S3D and HMBC NMR. hygromycin selection was developed based on a method devel spectroscopy indicated through bond coupling of Ile 1HN to. oped for Laccaria bicolor Kemppainen and Pardo 2010 2011 Pro8 13CO i e the presence of a new amide bond Figure S3E . The POPB gene was disrupted by targeted gene replacement Taken together the structural evidence proved that the com . in a monokaryotic strain of G marginata Four independent pound produced by the action of pure GmPOPB on the 35 amino. POPB knockout mutants generated by homologous integration acid GmAMA1 propeptide was cyclo IWGIGCNP . of the transforming DNA were shown by Southern blotting to. be deleted for GmPOPB Figures S1A and S1B available online Substrate Requirements of GmPOPB. All four mutants lost the ability to produce amanitin as shown for Synthetic peptides were tested with pure recombinant GmPOPB. transformant 1 Figure S1C The GmPOPB mutant had no other to explore its substrate structural requirements Table 1 Consis . discernible phenotype such as a change in growth rate or tent with the fact that GmPOPB also catalyzes removal of the. pigmentation N terminal 10 amino acids in a nonprocessive manner the. 2 Chemistry Biology 21 1 8 December 18 2014 2014 Elsevier Ltd All rights reserved. CHBIOL 2957, Please cite this article in press as Luo et al Peptide Macrocyclization Catalyzed by a Prolyl Oligopeptidase Involved in a Amanitin Biosynthesis . Chemistry Biology 2014 http dx doi org 10 1016 j chembiol 2014 10 015. Chemistry Biology,Peptide Cyclization by Prolyl Oligopeptidase. Figure 2 HPLC Fractionation of Reaction, Products Produced by GmPOPB from.
GmAMA1, The reaction was stopped before completion to. show the intermediates , A Mass spectrometric identification of peaks . B UV trace optical density at 280 nm OD280 , C Chromatogram of chemically synthesized. cyclo IWGIGCNP , The compound eluting at 8 7 min in B is unknown . S4B Consistent with secondary struc , ture rather than primary structure being.
important GmPOPB efficiently cyclized, AbAMA1 indicating that the critical fea . tures that are necessary for cyclization, are conserved in AbAMA1 Table 1 sub . strate 12 Figures S2E and S2F To test, specifically whether the putative a helix. is critical selected residues within the he , lix region were changed to Gly an amino. acid that tends to disrupt a helix forma , tion Table 1 substrates 8 10 All three.
modifications i e introduction of one , two or three Gly residues resulted in. drastic reduction of cyclization efficiency , The terminal Cys is conserved in. GmAMA1 and AbAMA1 Figure S4A , Changing this amino acid to Ala resulted. in a product that was cleaved at the first, Pro but not at the second Pro or cyclized. Table 1 substrate 11 , A bisporigera but not G marginata .
also biosynthesizes phallotoxins from, genes in the MSDIN family GmPOPB. did not efficiently cleave the phallacidin, propeptide from A bisporigera even at. the first Pro Table 1 substrate 13 This, resulting 25 amino acid product of this first reaction Table 1 suggests that the toxin region i e the region between the. substrate 2 was cyclized to completion two Pro residues might be involved in binding to the active. Truncating the N terminus by four amino acids led to a sharp site of GmPOPB but more peptides will need to be tested to. decrease in efficiency of cyclization due to failure to cut at confirm this A carboxy truncated version of AbPHA1 was cut. the first Pro Table 1 substrate 3 Therefore the sequence effectively at both Pro residues but no cyclization occurred. and or length of the N terminus is important for recognition by Table 1 substrate 14 This result was unexpected because. GmPOPB Five amino acids ATRLP immediately upstream of GmPOPB did not cut two carboxy truncated versions of. the toxin region are shared in common between AbAMA1 and GmAMA1 substrates 6 and 7 at the second Pro Perhaps the. GmAMA1 Figure S4A Changing this region to either AAAAP precise length of the C terminal overhang is critical for the pro . or ATAAP was sufficient to reduce but not completely abolish teolytic cleavage and or cyclization at the second Pro . cyclization Table 1 substrates 4 and 5 , To investigate the role of the C terminal region in macrocycli GmPOPB Enzyme Kinetics. zation two peptides truncated in the C terminus were synthe The enzyme kinetic constants of GmPOPB with both the 35 mer. sized They were both cut by GmPOPB at the first Pro but and the 25 mer as substrates were determined by measuring the. neither was cut at the second Pro or cyclized indicating that rate of cyclic product formation by high performance liquid chro . the C terminus is important for complete processing Table 1 matography HPLC Figures S4C and S4D The Km and kcat for. substrates 6 and 7 Because the primary sequences of the the 35 mer were 25 5 mM and 5 6 s 1 respectively The Km and. C terminal regions of GmAMA1 and AbAMA1 are quite different kcat for the 25 mer were 23 2 mM and 5 7 s 1 The similarity of the. Figure S4A we investigated their secondary structures Both values of the kinetic constants for the two substrates implies that. C domains are predicted to form an a helix Figures S4A and the overall conversion of the 35 mer to the cyclic octapeptide is. Chemistry Biology 21 1 8 December 18 2014 2014 Elsevier Ltd All rights reserved 3. CHBIOL 2957, Please cite this article in press as Luo et al Peptide Macrocyclization Catalyzed by a Prolyl Oligopeptidase Involved in a Amanitin Biosynthesis .
Chemistry Biology 2014 http dx doi org 10 1016 j chembiol 2014 10 015. Chemistry Biology, Peptide Cyclization by Prolyl Oligopeptidase. Figure 3 Time Course of Conversion of GmAMA1 Propeptide to cyclo IWGIGCNP . A HPLC analysis of products at different time points . B Graph of time course results The quantitation based on OD280 was normalized to the Trp content of the products . C Diagram of the reactions catalyzed by GmPOPB , limited by the second cyclization reaction consistent with the 36 identical to PCY1 and 37 identical to porcine POP Fu lo p. transient accumulation of the 25 mer intermediate Figure 3 et al 2001 All four of the mushroom POP proteins have the b. propeller domain and the peptidase domain with an a b hydro . Sequence Comparisons of POPA and POPB from lase fold typical of POPs On the basis of alignment with porcine. G marginata and A bisporigera POP the catalytic domain of GmPOPB is predicted to comprise. The POPB enzymes of G marginata and A bisporigera are amino acids 1 to 80 and 451 to 730 POPA and POPB of. 75 5 identical the two POPAs are 65 7 identical and the A bisporigera and G marginata have the conserved catalytic. average identity between the POPAs and the POPBs of the triad typical of serine proteases in family S9A in GmPOPB these. two fungi is 57 6 Figure S4E The homology among the four are Ser577 Asp661 and His698 Both POPA and POPB also. POPs is roughly distributed throughout the proteins as are the have the Trp residue Trp619 in GmPOPB that stacks with the. differences between the POPAs and the POPBs GmPOPB is Pro of the substrate Fu lo p et al 2001 Li et al 2010 Unlike. 4 Chemistry Biology 21 1 8 December 18 2014 2014 Elsevier Ltd All rights reserved. CHBIOL 2957, Please cite this article in press as Luo et al Peptide Macrocyclization Catalyzed by a Prolyl Oligopeptidase Involved in a Amanitin Biosynthesis . Chemistry Biology 2014 http dx doi org 10 1016 j chembiol 2014 10 015. Chemistry Biology,Peptide Cyclization by Prolyl Oligopeptidase. Table 1 Synthetic Peptides Tested with GmPOPB,Substrate No Substrate Major Product Conclusion.
1 MFDTNATRLPIWGIGCNPWTAEHV cyclo IWGIGCNP efficiently cyclized. DQTLASGNDIC full length GmAMA1 , 2 IWGIGCNPWTAEHVDQTLASGNDIC cyclo IWGIGCNP cyclized as efficiently as full length. N truncated 25 mer equivalent to, native intermediate . 3 NATRLPIWGIGCNPWTAEHVDQ none N terminal four amino acids. TLASGNDIC N truncated 31 mer important for first cut. 4 MFDTAAAAAPIWGIGCNPWTAEHVDQT cyclo IWGIGCNP 2 N terminal amino acid composition. LASGNDIC modification of amino acids important for efficient processing. conserved in the N termini of GmAMA1, and AbAMA1 . 5 MFDTNATAAPIWGIGCNPWTAEHVDQTL cyclo IWGIGCNP 2 N terminal amino acid composition. ASGNDIC modification of amino acids important for efficient processing. conserved in the N termini of GmAMA1, and AbAMA1 . 6 MFDTNATRLPIWGIGCNPWTAEHVD IWGIGCNPWTAEHVD C terminus important for cyclization. C truncated 25 mer , 7 MFDTNATRLPIWGIGCNPWTAEHVDQ IWGIGCNPWTAEHVDQTLAS C terminus important for cyclization.
TLAS C truncated 30 mer , 8 IWGIGCNPWTAEGVGQGLASGNDIC cyclo IWIGCNP 1 C terminal a helix necessary. Native 25 mer intermediate altered for efficient cyclization. in a helix region , 9 IWGIGCNPWTAEHGDQGLASGNDIC cyclo IWIGCNP 2 C terminal a helix necessary. Native 25 mer intermediate altered for efficient cyclization. Chemistry amp Biology Brief Communication Peptide Macrocyclization Catalyzed by a Prolyl Oligopeptidase Involved in a Amanitin Biosynthesis Hong Luo 1 3 Sung Yong Hong 1 3 R Michael Sgambelluri 1 2 Evan Angelos 1 Xuan Li 1 4 and Jonathan D Walton1 1Department of Energy Plant Research Laboratory 2Department of Biochemistry and Molecular Biology Michigan State University East Lansing MI

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