Structural Basis of Acyl-Carrier Protein Interactions in Fatty Acid and Polyketide Biosynthesis

Mindrebo, Jeffrey T.; Patel, Ashay; Misson, Laëtitia; Kim, Woojoo E.; Davis, Thomas B.; Ni, Qing Zhe; Clair, James J. La; Burkart, Michael D.

doi:10.1016/b978-0-12-409547-2.14662-1

Cited by 19 publications

(33 citation statements)

References 256 publications

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“…The central player in this process is the acyl carrier protein (ACP) that shuttles thioester-linked pathway intermediates to each respective enzyme active site. 9,10 The ACP is posttranslationally modified with a prosthetic 4'-phosphopantetheine arm (PPant) at a conserved serine residue that provides the free thiol moiety to ligate pathway intermediates via a thioester bond. 11 During ACP-mediated substrate delivery, productive protein-protein interactions (PPIs) between the ACP and partner enzymes are required.…”

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confidence: 99%

“…11 During ACP-mediated substrate delivery, productive protein-protein interactions (PPIs) between the ACP and partner enzymes are required. 10,12,13 The enzymatic activities and chemical transformations in fatty acid synthases (FAS) are generally conserved, but their structural organization can be separated into two classes. Metazoans and fungi possess large megasynthases with individual domains having distinct catalytic functions (type I FAS), [14][15][16][17][18] whereas bacterial and plant plastid FASs are expressed as discrete enzymes corresponding to specific genes (type II FAS).…”

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confidence: 99%

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

et al. 2020

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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.

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

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“…In total, the AcpP-FabD interface buries 350 Å 2 of surface area, making it the smallest reported AcpP-PE interface to date. 44 Figure S3a). 52 The presence of the sulfate ion, the non-native thioether crosslinked bond, and the C-S bond length cause the carbonyl group of the probe to rotate away from the oxyanion hole formed by the backbone amides of Gln11 and Leu93 (Figures S1, S3b), which instead accepts two hydrogen bonds from the Nδ of Asn160 and the Nε of His202.…”

Section: Resultsmentioning

confidence: 99%

“…However, the AcpP-FabD crosslinked structure also departs from canonical FAS ACP binding motifs previously reported. 44 It reveals a unique set of interfacial interactions between the two proteins involving the small helix on loop 1 of AcpP (α1') and the N-terminal portion of helix II (α2) ( Figure 3c-e, S7). Additionally, unlike the dehydratases (FabA and FabZ) and elongating ketosynthases (FabB and FabF) in E. coli FAS, FabD makes no discernable contacts with helix III (α3) of AcpP ( Figure S6).…”

Section: Resultsmentioning

confidence: 99%

Structure and Dynamic Basis of Molecular Recognition Between Acyltransferase and Carrier Protein inE. coliFatty Acid Synthesis

Misson

Mindrebo

Davis

et al. 2020

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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 first 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.Significance StatementThe essential role of acyltransferases (ATs) in fatty acid synthase (FAS) and polyketide synthase (PKS) pathways, namely the selection and loading of starter and extender units onto acyl carrier proteins (ACPs), relies on catalytically productive ACP-AT interactions. Here, we describe and interrogate the first structure of a type II FAS malonyl-CoA:ACP-transacylase (MAT) in covalent complex with its cognate ACP. We combine structural, mutational, crosslinking and kinetic data with molecular dynamics simulations to describe a highly flexible and robust protein-protein interface, substrate-induced active site reorganization, and key subdomain motions that likely govern FAS function. These findings strengthen a mechanistic understanding of molecular recognitions between ACPs and partner enzymes and provide new insights for engineering AT-dependent biosynthetic pathways.

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“…19,20 ACPs must form transient, productive protein-protein interactions (PPIs) with each respective partner enzyme (PE) in order to deliver their substrates in catalytic competent forms. [21][22][23][24][25][26][27][28][29][30] KS-mediated carbon-carbon bond formation is the primary driving force of FAB. These enzymes use a two-step kinetic ping-pong bi-bi mechanism that can be viewed mechanistically as transacylation and condensation reactions ( Figure 1A).…”

Section: Introductionmentioning

confidence: 99%

Structure and mechanistic analyses of the gating mechanism of elongating ketosynthases

Mindrebo

Kim²,

Re³

et al. 2021

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Ketosynthases (KSs) catalyze carbon-carbon bond forming reactions in fatty acid synthases (FASs) and polyketide synthases (PKSs). KSs utilize a two-step ping pong kinetic mechanism to carry out an overall decarboxylative thio-Claisen condensation that can be separated into the transacylation and condensation reactions. In both steps, an acyl carrier protein (ACP) delivers thioester tethered substrates to the active sites of KSs. Therefore, protein-protein interactions (PPIs) and KS-mediated substrate recognition events are required for catalysis. Recently, crystal structures of Escherichia coli elongating type II FAS KSs, FabF and FabB, in complex with E. coli ACP, AcpP, revealed distinct conformational states of two active site KS loops. These loops were proposed to operate via a gating mechanism to coordinate substrate recognition and delivery followed by catalysis. Here we interrogate this proposed gating mechanism by solving two additional high-resolution structures of substrate engaged AcpP-FabF complexes, one of which provides the missing AcpP-FabF gate-closed conformation. Clearly defined interactions of one of these active site loops with AcpP are present in both the open and closed conformations, suggesting AcpP binding triggers or stabilizes gating transitions, further implicating PPIs in carrier protein-dependent catalysis. We functionally demonstrate the importance of gating in the overall KS condensation reaction and provide experimental evidence for its role in the transacylation reaction. Furthermore, we evaluate the catalytic importance of these loops using alanine scanning mutagenesis and also investigate chimeric FabF constructs carrying elements found in type I PKS KS domains. These findings broaden our understanding of the KS mechanism which advances future engineering efforts in both FASs and evolutionarily related PKSs.

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Structural Basis of Acyl-Carrier Protein Interactions in Fatty Acid and Polyketide Biosynthesis

Cited by 19 publications

References 256 publications

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

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

Structure and Dynamic Basis of Molecular Recognition Between Acyltransferase and Carrier Protein inE. coliFatty Acid Synthesis

Structure and mechanistic analyses of the gating mechanism of elongating ketosynthases

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