TY - JOUR
T1 - Probing the limits of interrupted adenylation domains by engineering a trifunctional enzyme capable of adenylation,
T2 - N -, and S -methylation
AU - Lundy, Taylor A.
AU - Mori, Shogo
AU - Garneau-Tsodikova, Sylvie
PY - 2019
Y1 - 2019
N2 - The adenylation (A) domains found in nonribosomal peptide synthetases (NRPSs) exhibit tremendous plasticity. Some A domains have been shown to display the ability to contain within them the catalytic portion of an auxiliary domain, most commonly that of a methyltransferase (M) enzyme. This unique feature of A domains interrupted by M domains allows them to possess bifunctionality, where they can both adenylate and methylate an amino acid substrate. Additionally, these types of inserted M domains are able to selectively carry out either backbone or side chain methylation of amino acids. Interruptions with M domains are naturally found to occur either between the a2-a3 or the a8-a9 of the ten conserved motifs of A domains. Herein, we set out to answer the following question: Can one A domain support two different M domain interruptions occurring in two different locations (a2-a3 and a8-a9) of the A domain and possess the ability to adenylate an amino acid and methylate it on both its side chain and backbone? To answer this question we added a backbone methylating M 3S domain from TioS(A 3a M 3S A 3b ) between the a8-a9 region of a mono-interrupted A domain, TioN(A a M N A b ), that already contained a side chain methylating M N domain between its a2-a3 region. We evaluated the di-interrupted A domain TioN(AM N AM 3S A) with a series of radiometric and mass spectrometry assays and found that this engineered enzyme was indeed capable of all three activities. These findings show that production of an active trifunctional di-interrupted A domain is possible and represents an exciting new avenue for future nonribosomal peptide (NRP) derivatization.
AB - The adenylation (A) domains found in nonribosomal peptide synthetases (NRPSs) exhibit tremendous plasticity. Some A domains have been shown to display the ability to contain within them the catalytic portion of an auxiliary domain, most commonly that of a methyltransferase (M) enzyme. This unique feature of A domains interrupted by M domains allows them to possess bifunctionality, where they can both adenylate and methylate an amino acid substrate. Additionally, these types of inserted M domains are able to selectively carry out either backbone or side chain methylation of amino acids. Interruptions with M domains are naturally found to occur either between the a2-a3 or the a8-a9 of the ten conserved motifs of A domains. Herein, we set out to answer the following question: Can one A domain support two different M domain interruptions occurring in two different locations (a2-a3 and a8-a9) of the A domain and possess the ability to adenylate an amino acid and methylate it on both its side chain and backbone? To answer this question we added a backbone methylating M 3S domain from TioS(A 3a M 3S A 3b ) between the a8-a9 region of a mono-interrupted A domain, TioN(A a M N A b ), that already contained a side chain methylating M N domain between its a2-a3 region. We evaluated the di-interrupted A domain TioN(AM N AM 3S A) with a series of radiometric and mass spectrometry assays and found that this engineered enzyme was indeed capable of all three activities. These findings show that production of an active trifunctional di-interrupted A domain is possible and represents an exciting new avenue for future nonribosomal peptide (NRP) derivatization.
UR - http://www.scopus.com/inward/record.url?scp=85060948647&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85060948647&partnerID=8YFLogxK
U2 - 10.1039/c8ob02996b
DO - 10.1039/c8ob02996b
M3 - Article
C2 - 30644493
AN - SCOPUS:85060948647
VL - 17
SP - 1169
EP - 1175
JO - Organic and Biomolecular Chemistry
JF - Organic and Biomolecular Chemistry
SN - 1477-0520
IS - 5
ER -