Succinyl-CoA reductase (SucD) is an acylating aldehyde
reductase
that catalyzes the NADPH-dependent reduction of succinyl-CoA to succinic
semialdehyde. The reaction sequence from succinate to crotonyl-CoA
is of particular interest for several new-to-nature CO2-fixation pathways, such as the crotonyl-CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA
(CETCH) cycle, in which SucD plays a key role. However, pathways like
the CETCH cycle feature several CoA-ester intermediates, which could
be potentially side substrates for this enzyme. Here, we show that
the side reaction for most CETCH cycle metabolites is relatively small
(<2%) with the exception of mesaconyl-C1-CoA (16%), which represents
a competing substrate in this pathway. We addressed this promiscuity
by solving the crystal structure of a SucD ofClostridium
kluyveriin complex with NADP+and mesaconyl-C1-CoA.
We further identified two residues (Lys70 and Ser243) that coordinate
mesaconyl-C1-CoA at the active site. We targeted those residues with
site-directed mutagenesis to improve succinyl-CoA over mesaconyl-C1-CoA
reduction. The best resulting SucD variant, K70R, showed a strongly
reduced side activity for mesaconyl-C1-CoA, but the substitution also
reduced the specific activity for succinyl-CoA by a factor of 10.
Transferring the same mutations into a SucD homologue fromClostridium difficilesimilarly decreases the side
reaction of this enzyme for mesaconyl-C1-CoA from 12 to 2%, notably
without changing the catalytic efficiency for succinyl-CoA. Overall,
our structure-based engineering efforts provided a highly specific
enzyme of interest for several applications in biocatalysis and synthetic
biology.