Interfacial plasticity facilitates high reaction rate of E. coli FAS malonyl-CoA:ACP transacylase, FabD

Abstract

Fatty acid synthases (FASs) and polyketide synthases (PKSs) iteratively elongate and often reduce two-carbon ketide units in de novo fatty acid and polyketide biosynthesis. Cycles of chain extensions in FAS and PKS are initiated by an acyltransferase (AT), which loads monomer units onto acyl carrier proteins (ACPs), small, flexible proteins that shuttle covalently linked intermediates between catalytic partners. Formation of productive ACP–AT interactions is required for catalysis and specificity within primary and secondary FAS and PKS pathways. Here, we use the Escherichia coli FAS AT, FabD, and its cognate ACP, AcpP, to interrogate type II FAS ACP–AT interactions. We utilize a covalent crosslinking probe to trap transient interactions between AcpP and FabD to elucidate the X-ray crystal structure of a type II ACP–AT complex. Our structural data are supported using a combination of mutational, crosslinking, and kinetic analyses, and long-timescale molecular dynamics (MD) simulations. Together, these complementary approaches reveal key catalytic features of FAS ACP–AT interactions. These mechanistic inferences suggest that AcpP adopts multiple, productive conformations at the AT binding interface, allowing the complex to sustain high transacylation rates. Furthermore, MD simulations support rigid body subdomain motions within the FabD structure that may play a key role in AT activity and substrate selectivity.

Acyl Carrier Protein is essential for MukBEF action in Escherichia coli chromosome organization-segregation

“…MukB KR (K1114E, R1122E), MukB RK (R1122E, K1125E) and MukB KC (K1114E, C1118E) cells were Muk + , as assessed by growth at 37 °C, indicating that the temperature-sensitivity of MukB KRK is likely due to a lack of AcpP interaction, rather than protein conformational changes induced by the mutations. Consistent with our observations, multiple substitutions in other AcpP-target protein interfaces are required to abolish AcpP binding with other AcpP binding proteins in addition to MukB 34,35 .…”

Acyl Carrier Protein is essential for MukBEF action inEscherichia colichromosome organization-segregation

“…MukB KR (K1114E, R1122E), MukB RK (R1122E, K1125E) and MukB KC (K1114E, C1118E) were Muk + , as assessed by growth at 37 °C, indicating that the temperature-sensitivity of MukB KRK is likely due to a lack of AcpP interaction, rather than protein conformational changes induced by the mutations. Consistent with our observations, multiple substitutions in the AcpP-target protein interface are required to abolish AcpP binding with other AcpP binding proteins in addition to MukB 34,35 .…”

Transacylation Kinetics in Fatty Acid and Polyketide Synthases and its Sensitivity to Point Mutations**

“…Specifically, we titrated X−CoA substrates to five different, fixed ACP concentrations and globally fitted all titration curves. This approach is robust to measurement errors as well as delivers absolute kinetic constants and is in its quality superior to other approaches titrating one substrate while keeping the other at a fixed saturated concentration [22,23] . For all ATs, absolute kinetic parameters were received, although the kinetic parameters for systems with high were determined less accurately, simply because the vast amounts of ACP needed to cover an appropriate substrate range could not always be provided.…”