TY - JOUR
T1 - Adapting an acyl CoA ligase from Metallosphaera sedula for lactam formation by structure-guided protein engineering
AU - Capra, Nikolas
AU - Lelièvre, Chloé
AU - Touré, Océane
AU - Fossey-Jouenne, Aurélie
AU - Vergne-Vaxelaire, Carine
AU - Janssen, Dick
AU - Thunnissen, Andy-Mark W. H.
AU - Zaparucha, Anne
PY - 2024/3/4
Y1 - 2024/3/4
N2 - The CoA ligase from Metallosphaera sedula (MsACL) can be used for the chemoenzymatic synthesis of amides from carboxylic acids. In this CoA-independent conversion, the enzyme catalyzes the adenylation of a carboxylic acid with the help of ATP, followed by the uncatalyzed cleavage of acyl-AMP by a nucleophilic amine to yield an amide. With ω-amino acids as substrates this reaction may result in formation of lactams, but unfortunately the substrate preference of the wild-type enzyme is rather limited. To allow structure-based protein engineering and expand the substrate scope of the enzyme, crystal structures of MsACL were solved in the thioesterification conformational state with AMP, CoA and with the reaction intermediate acetyl-AMP bound in the active site. Using substrate docking and by comparing the crystals structures and sequence of MsACL to those of related CoA ligases, mutations were predicted which increase the affinity in the carboxylic acid binding pocket for ω-amino acids. The resulting mutations transformed a non-active enzyme into an active enzyme for ε-caprolactam synthesis, highlighting the potential of the thermophilic CoA ligase for this synthetic and biotechnologically relevant reaction.
AB - The CoA ligase from Metallosphaera sedula (MsACL) can be used for the chemoenzymatic synthesis of amides from carboxylic acids. In this CoA-independent conversion, the enzyme catalyzes the adenylation of a carboxylic acid with the help of ATP, followed by the uncatalyzed cleavage of acyl-AMP by a nucleophilic amine to yield an amide. With ω-amino acids as substrates this reaction may result in formation of lactams, but unfortunately the substrate preference of the wild-type enzyme is rather limited. To allow structure-based protein engineering and expand the substrate scope of the enzyme, crystal structures of MsACL were solved in the thioesterification conformational state with AMP, CoA and with the reaction intermediate acetyl-AMP bound in the active site. Using substrate docking and by comparing the crystals structures and sequence of MsACL to those of related CoA ligases, mutations were predicted which increase the affinity in the carboxylic acid binding pocket for ω-amino acids. The resulting mutations transformed a non-active enzyme into an active enzyme for ε-caprolactam synthesis, highlighting the potential of the thermophilic CoA ligase for this synthetic and biotechnologically relevant reaction.
KW - Caprolactam
KW - Chemo-enzymatic lactam synthesis
KW - Lactam
KW - CoA ligase
KW - Biocatalysis
KW - Protein engineering
KW - Substrate scope engineering
U2 - 10.3389/fctls.2024.1360129
DO - 10.3389/fctls.2024.1360129
M3 - Article
SN - 2673-7841
VL - 4
JO - Frontiers in Catalysis
JF - Frontiers in Catalysis
M1 - 1360129
ER -