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Recognition sequences and substrate evolution in cyanobactin biosynthesis.

Sardar D, Pierce E, McIntosh JA, Schmidt EW - ACS Synth Biol (2014)

Bottom Line: In addition to the previously assigned N- and C-terminal proteolysis RSs, here we assign the RS for heterocyclization modification.We show that substrate elements can be swapped in vivo leading to successful production of natural products in E. coli.The exchangeability of these elements holds promise in synthetic biology approaches to tailor peptide products in vivo and in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicinal Chemistry, University of Utah , Salt Lake City, Utah 84112, United States.

ABSTRACT
Ribosomally synthesized and posttranslationally modified peptide (RiPP) natural products are of broad interest because of their intrinsic bioactivities and potential for synthetic biology. The RiPP cyanobactin pathways pat and tru have been experimentally shown to be extremely tolerant of mutations. In nature, the pathways exhibit "substrate evolution", where enzymes remain constant while the substrates of those enzymes are hypervariable and readily evolvable. Here, we sought to determine the mechanism behind this promiscuity. Analysis of a series of different enzyme-substrate combinations from five different cyanobactin gene clusters, in addition to engineered substrates, led us to define short discrete recognition elements within substrates that are responsible for directing enzymes. We show that these recognition sequences (RSs) are portable and can be interchanged to control which functional groups are added to the final natural product. In addition to the previously assigned N- and C-terminal proteolysis RSs, here we assign the RS for heterocyclization modification. We show that substrate elements can be swapped in vivo leading to successful production of natural products in E. coli. The exchangeability of these elements holds promise in synthetic biology approaches to tailor peptide products in vivo and in vitro.

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Biochemical characterizationof thc pathway enzymes.(A) ThcE4/ThcD: FTMS spectra of chymotryptic digests ASSCDCSLY andGGCESCSYEGDEAE of ThcE4 modified by ThcD. Each [M+2H]2+ mass peak corresponds to the peptide sequence given in a gray box,and a heterocycle PTM is indicated by a C (inred) within the sequence. (B) ThcE4/ThcA: Deconvoluted ESI-MS spectrumof the ThcE4/ThcA reaction. The [M-H]− mass peak7098.0 Da is unmodified ThcE4 (His-tag removed) and 4915.0 Da correspondsto the leader after ThcA proteolysis at the AVLAS RSII site. The insetshows SDS-PAGE visualization of the same reaction, where the leftlane is ThcE4 only and the right lane is ThcE4 in presence of ThcA.The smaller band in the right lane indicates the ThcA cleaved product.A schematic representation of results is shown where (X38) represents the 38-residue leader sequence before RSI, RSII (red)and core (blue) sequences.
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fig2: Biochemical characterizationof thc pathway enzymes.(A) ThcE4/ThcD: FTMS spectra of chymotryptic digests ASSCDCSLY andGGCESCSYEGDEAE of ThcE4 modified by ThcD. Each [M+2H]2+ mass peak corresponds to the peptide sequence given in a gray box,and a heterocycle PTM is indicated by a C (inred) within the sequence. (B) ThcE4/ThcA: Deconvoluted ESI-MS spectrumof the ThcE4/ThcA reaction. The [M-H]− mass peak7098.0 Da is unmodified ThcE4 (His-tag removed) and 4915.0 Da correspondsto the leader after ThcA proteolysis at the AVLAS RSII site. The insetshows SDS-PAGE visualization of the same reaction, where the leftlane is ThcE4 only and the right lane is ThcE4 in presence of ThcA.The smaller band in the right lane indicates the ThcA cleaved product.A schematic representation of results is shown where (X38) represents the 38-residue leader sequence before RSI, RSII (red)and core (blue) sequences.

Mentions: The thc pathway was characterized to add to therepertoire of the previously studied pat and tru pathways. ThcE4 (1) was chosen as the precursorpeptide substrate. Putative heterocyclase ThcD and putative N-terminalprotease ThcA were used in enzymatic assays. Using optimized reactionconditions, ThcD was incubated with ThcE4 (1) and thereaction was analyzed by MS methods. High-resolution FT-ICR ESI MS/MS(FTMS) has previously been used to localize the position of heterocyclization.16,23,24 The method identifies sites ofdehydration via the 18 Da mass difference, and further, it is possibleto determine the type of dehydration (whether reverse-Michael or cyclodehydration)by fragmentation pattern.25 The ThcD/ThcE4reaction was digested with chymotrypsin to yield easily detectablefragments. MS/MS analysis showed that all heterocycles were thiazolinesderived from Cys and clearly localized them to SCDCSLYGGCESC, where C stands for thiazoline ring modification(Figures 2A and SupportingInformation S3). Detailed analysis of MS spectra is given in Supporting Information.


Recognition sequences and substrate evolution in cyanobactin biosynthesis.

Sardar D, Pierce E, McIntosh JA, Schmidt EW - ACS Synth Biol (2014)

Biochemical characterizationof thc pathway enzymes.(A) ThcE4/ThcD: FTMS spectra of chymotryptic digests ASSCDCSLY andGGCESCSYEGDEAE of ThcE4 modified by ThcD. Each [M+2H]2+ mass peak corresponds to the peptide sequence given in a gray box,and a heterocycle PTM is indicated by a C (inred) within the sequence. (B) ThcE4/ThcA: Deconvoluted ESI-MS spectrumof the ThcE4/ThcA reaction. The [M-H]− mass peak7098.0 Da is unmodified ThcE4 (His-tag removed) and 4915.0 Da correspondsto the leader after ThcA proteolysis at the AVLAS RSII site. The insetshows SDS-PAGE visualization of the same reaction, where the leftlane is ThcE4 only and the right lane is ThcE4 in presence of ThcA.The smaller band in the right lane indicates the ThcA cleaved product.A schematic representation of results is shown where (X38) represents the 38-residue leader sequence before RSI, RSII (red)and core (blue) sequences.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4384831&req=5

fig2: Biochemical characterizationof thc pathway enzymes.(A) ThcE4/ThcD: FTMS spectra of chymotryptic digests ASSCDCSLY andGGCESCSYEGDEAE of ThcE4 modified by ThcD. Each [M+2H]2+ mass peak corresponds to the peptide sequence given in a gray box,and a heterocycle PTM is indicated by a C (inred) within the sequence. (B) ThcE4/ThcA: Deconvoluted ESI-MS spectrumof the ThcE4/ThcA reaction. The [M-H]− mass peak7098.0 Da is unmodified ThcE4 (His-tag removed) and 4915.0 Da correspondsto the leader after ThcA proteolysis at the AVLAS RSII site. The insetshows SDS-PAGE visualization of the same reaction, where the leftlane is ThcE4 only and the right lane is ThcE4 in presence of ThcA.The smaller band in the right lane indicates the ThcA cleaved product.A schematic representation of results is shown where (X38) represents the 38-residue leader sequence before RSI, RSII (red)and core (blue) sequences.
Mentions: The thc pathway was characterized to add to therepertoire of the previously studied pat and tru pathways. ThcE4 (1) was chosen as the precursorpeptide substrate. Putative heterocyclase ThcD and putative N-terminalprotease ThcA were used in enzymatic assays. Using optimized reactionconditions, ThcD was incubated with ThcE4 (1) and thereaction was analyzed by MS methods. High-resolution FT-ICR ESI MS/MS(FTMS) has previously been used to localize the position of heterocyclization.16,23,24 The method identifies sites ofdehydration via the 18 Da mass difference, and further, it is possibleto determine the type of dehydration (whether reverse-Michael or cyclodehydration)by fragmentation pattern.25 The ThcD/ThcE4reaction was digested with chymotrypsin to yield easily detectablefragments. MS/MS analysis showed that all heterocycles were thiazolinesderived from Cys and clearly localized them to SCDCSLYGGCESC, where C stands for thiazoline ring modification(Figures 2A and SupportingInformation S3). Detailed analysis of MS spectra is given in Supporting Information.

Bottom Line: In addition to the previously assigned N- and C-terminal proteolysis RSs, here we assign the RS for heterocyclization modification.We show that substrate elements can be swapped in vivo leading to successful production of natural products in E. coli.The exchangeability of these elements holds promise in synthetic biology approaches to tailor peptide products in vivo and in vitro.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicinal Chemistry, University of Utah , Salt Lake City, Utah 84112, United States.

ABSTRACT
Ribosomally synthesized and posttranslationally modified peptide (RiPP) natural products are of broad interest because of their intrinsic bioactivities and potential for synthetic biology. The RiPP cyanobactin pathways pat and tru have been experimentally shown to be extremely tolerant of mutations. In nature, the pathways exhibit "substrate evolution", where enzymes remain constant while the substrates of those enzymes are hypervariable and readily evolvable. Here, we sought to determine the mechanism behind this promiscuity. Analysis of a series of different enzyme-substrate combinations from five different cyanobactin gene clusters, in addition to engineered substrates, led us to define short discrete recognition elements within substrates that are responsible for directing enzymes. We show that these recognition sequences (RSs) are portable and can be interchanged to control which functional groups are added to the final natural product. In addition to the previously assigned N- and C-terminal proteolysis RSs, here we assign the RS for heterocyclization modification. We show that substrate elements can be swapped in vivo leading to successful production of natural products in E. coli. The exchangeability of these elements holds promise in synthetic biology approaches to tailor peptide products in vivo and in vitro.

Show MeSH