Activity Mapping the Acyl Carrier Protein: Elongating Ketosynthase Interaction in Fatty Acid Biosynthesis

Abstract

Elongating ketosynthases (KSs) catalyze carbon–carbon bond-forming reactions during the committed step for each round of chain extension in both fatty acid synthases (FASs) and polyketide synthases (PKSs). A small α-helical acyl carrier protein (ACP) shuttles fatty acyl intermediates between enzyme active sites. To accomplish this task, the ACP relies on a series of dynamic interactions with multiple partner enzymes of FAS and associated FAS-dependent pathways. Recent structures of the Escherichia coli FAS ACP, AcpP, in covalent complexes with its two cognate elongating KSs, FabF and FabB, provide high-resolution details of these interfaces, but a systematic analysis of specific interfacial interactions responsible for stabilizing these complexes has not yet been undertaken. Here, we use site-directed mutagenesis with both in vitro and in vivo activity analyses to quantitatively evaluate these contacting surfaces between AcpP and FabF. We delineate the FabF interface into three interacting regions and demonstrate the effects of point mutants, double mutants, and region deletion variants. Results from these analyses reveal a robust and modular FabF interface capable of tolerating seemingly critical interface mutations with only the deletion of an entire region significantly compromising activity. Structure and sequence analyses of FabF orthologs from related type II FAS pathways indicate significant conservation of type II FAS KS interface residues and, overall, support its delineation into interaction regions. These findings strengthen our mechanistic understanding of molecular recognition events between ACPs and FAS enzymes and provide a blueprint for engineering ACP-dependent biosynthetic pathways.

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 .…”

Enzymology of standalone elongating ketosynthases

“…Alanine scan of interacting protein–protein interface residues of FabF shows that triple mutation removing all interactions in region 1 or region 2 has a greater impact on activity than a triple mutant containing a mix of residues from both regions, indicating that anchoring ACP in both regions is essential. 94 IgaKS–CLF was also subjected to the interface residue alanine scan, and all of them show some degree of decline in crosslinking activity. Among them, the R210A mutant stands out to totally abolish crosslinking activity because of the profound interactions R210 has with ACP helix 2, forming polar contacts with the side chains of S49 and E52.…”

“…Mutational study on R206 reveals that mutating the residue to an alanine largely decreases the k cat value of FabF while the K M value remains the same. 94 This indicates that interaction with ACP helix 3 serves more of a catalytic role rather than strictly stabilizing the protein–protein interface. Interestingly, such an interaction is quite unique across the four KSs that have the KS–ACP structure solved ( Fig.…”