Structure and Dynamic Basis of Molecular Recognition Between Acyltransferase and Carrier Protein in E. coli Fatty Acid Synthesis

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2009805117 ◽

2020 ◽

Vol 117 (39) ◽

pp. 24224-24233 ◽

Cited By ~ 3

Author(s):

Laetitia E. Misson ◽

Jeffrey T. Mindrebo ◽

Tony D. Davis ◽

Ashay Patel ◽

J. Andrew McCammon ◽

...

Keyword(s):

Fatty Acid ◽

De Novo ◽

Structural Data ◽

Md Simulations ◽

Type Ii ◽

Acyl Carrier Proteins ◽

Covalent Crosslinking ◽

Binding Interface ◽

Transient Interactions ◽

Covalently Linked

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.

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Engineered chimeras unveil swappable modular features of fatty acid and polyketide synthase acyl carrier proteins

10.1101/2021.12.30.471467 ◽

2021 ◽

Author(s):

Yae In Cho ◽

Claire L Armstrong ◽

Ariana Sulpizio ◽

Kofi K Acheampong ◽

Kameron N Banks ◽

...

Keyword(s):

Natural Products ◽

Fatty Acid ◽

Fatty Acid Synthase ◽

Polyketide Synthase ◽

Building Blocks ◽

Combinatorial Biosynthesis ◽

Biosynthetic Pathways ◽

Carrier Proteins ◽

Acyl Carrier Proteins ◽

E Coli

The strategic redesign of microbial biosynthetic pathways is a compelling route to access molecules of diverse structure and function in a potentially environmentally sustainable fashion. The promise of this approach hinges on an improved understanding of acyl carrier proteins (ACPs), which serve as central hubs in biosynthetic pathways. These small, flexible proteins mediate the transport of molecular building blocks and intermediates to enzymatic partners that extend and tailor the growing natural products. Past combinatorial biosynthesis efforts have failed due to incompatible ACP-enzyme pairings. Herein we report the design of chimeric ACPs with features of the actinorhodin polyketide synthase ACP (ACT) and of the E. coli fatty acid synthase (FAS) ACP (AcpP). We evaluate the ability of the chimeric ACPs to interact with the E. coli FAS ketosynthase FabF, which represents an interaction essential to building the carbon backbone of the synthase molecular output. Given that AcpP interacts with FabF but ACT does not, we sought to exchange modular features of ACT with AcpP to confer functionality with FabF. The interactions of chimeric ACPs with FabF were interrogated using sedimentation velocity experiments, surface plasmon resonance analyses, mechanism-based crosslinking assays, and molecular dynamics simulations. Results suggest that the residues guiding AcpP-FabF compatibility and ACT-FabF incompatibility may reside in the loop I, α-helix II region. These findings can inform the development of strategic secondary element swaps that expand the enzyme compatibility of ACPs across systems and therefore represent a critical step towards the strategic engineering of unnatural natural products.

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The effect of divalent cations on the thermostability of type II polyketide synthase acyl carrier proteins

AIChE Journal ◽

10.1002/aic.16402 ◽

2018 ◽

Vol 64 (12) ◽

pp. 4308-4318 ◽

Cited By ~ 3

Author(s):

Marco A. Rivas ◽

Valentine C. Courouble ◽

Miranda C. Baker ◽

David L. Cookmeyer ◽

Kristen E. Fiore ◽

...

Keyword(s):

Polyketide Synthase ◽

Divalent Cations ◽

Type Ii ◽

Carrier Proteins ◽

Acyl Carrier Proteins

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Fatty-acid synthase activity and mRNA level in hypertrophic type II cells from silica-treated rats

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1994.267.2.l128 ◽

1994 ◽

Vol 267 (2) ◽

pp. L128-L136

Author(s):

J. Rami ◽

W. Stenzel ◽

S. M. Sasic ◽

C. Puel-M'Rini ◽

J. P. Besombes ◽

...

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthase ◽

Fatty Acid Biosynthesis ◽

De Novo ◽

Mrna Level ◽

Type Ii Cells ◽

Mrna Levels ◽

Type Ii ◽

Maximum Increase

Silica instillation causes a massive increase in lung surfactant. Two populations of type II pneumocytes can be isolated from rats administered silica by intratracheal injection: type IIA cells similar to type II cells from normal rats and type IIB cells, which are larger and contain elevated levels of surfactant protein A and phospholipid. Activities of choline-phosphate cytidylyltransferase, a rate-regulatory enzyme in phosphatidylcholine biosynthesis, and fatty-acid synthase (FAS) are increased in type IIB cells isolated from rats 14 days after silica injection. In the present study, we examined the increase in FAS and cytidylyltransferase activities in type IIB cells as a function of time after silica administration. FAS activity increased rapidly, was approximately threefold elevated 1 day after silica administration and has reached close to the maximum increase by 3 days. Cytidylyltransferase activity was not increased on day 1, was significantly increased on day 3 but was not maximally increased until day 7. Inhibition of de novo fatty-acid biosynthesis, by in vivo injection of hydroxycitric acid and inclusion of agaric acid in the type II cell culture medium, abolished the increase in cytidylyltransferase activity on day 3 but not FAS and had no effect on activities of two other enzymes of phospholipid synthesis. FAS mRNA levels were not increased in type IIB cells isolated 1-14 days after silica injection. These data show that the increase in FAS activity in type IIB cells is an early response to silica, that it mediates the increase in cytidylyltransferase activity, and that it is not due to enhanced FAS gene expression.

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Identification of a Novel Mycobacterial 3-Hydroxyacyl-Thioester Dehydratase, HtdZ (Rv0130), by Functional Complementation in Yeast

Journal of Bacteriology ◽

10.1128/jb.00016-08 ◽

2008 ◽

Vol 190 (11) ◽

pp. 4088-4090 ◽

Cited By ~ 6

Author(s):

Aner Gurvitz ◽

J. Kalervo Hiltunen ◽

Alexander J. Kastaniotis

Keyword(s):

Saccharomyces Cerevisiae ◽

Mycobacterium Tuberculosis ◽

Fatty Acid ◽

Fatty Acid Synthase ◽

De Novo ◽

Acid Synthesis ◽

Functional Complementation ◽

Type Ii ◽

Mutant Cells ◽

Dehydratase Activity

ABSTRACT We report on the identification of Mycobacterium tuberculosis HtdZ (Rv0130), representing a novel 3-hydroxyacyl-thioester dehydratase. HtdZ was picked up by the functional complementation of Saccharomyces cerevisiae htd2Δ cells lacking the dehydratase of mitochondrial type II fatty acid synthase. Mutant cells expressing HtdZ contained dehydratase activity, recovered their respiratory ability, and partially restored de novo lipoic acid synthesis.

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Type II fatty acid and polyketide synthases: deciphering protein–protein and protein–substrate interactions

Natural Product Reports ◽

10.1039/c8np00040a ◽

2018 ◽

Vol 35 (10) ◽

pp. 1029-1045 ◽

Cited By ~ 19

Author(s):

Aochiu Chen ◽

Rebecca N. Re ◽

Michael D. Burkart

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthase ◽

Polyketide Synthase ◽

Polyketide Synthases ◽

Type Ii ◽

Substrate Interactions ◽

Protein Substrate ◽

Functional Roles

Metabolites from type II fatty acid synthase (FAS) and polyketide synthase (PKS) pathways differ broadly in their identities and functional roles.

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Enhanced fatty acid biosynthesis and normal surfactant secretion in hypertrophic rat type II cells

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.1991.260.6.l577 ◽

1991 ◽

Vol 260 (6) ◽

pp. L577-L585 ◽

Cited By ~ 3

Author(s):

J. Rami ◽

S. M. Sasic ◽

S. A. Rooney

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthase ◽

Fatty Acid Biosynthesis ◽

De Novo ◽

Enzyme Activation ◽

Type Ii Cells ◽

Type Ii ◽

Acid Biosynthesis ◽

Surfactant Secretion ◽

Phosphatidylcholine Secretion

Silica instillation causes lung surfactant accumulation as well as hyperplasia and hypertrophy of type II pneumocytes. Two populations of type II cells can be isolated from silica-treated rats: type IIA, which are similar to type II cells from normal animals and type IIB, which are larger and have a higher rate of phosphatidylcholine biosynthesis. We have compared fatty acid biosynthesis and phosphatidylcholine secretion in types IIA and IIB cells and in type II cells from control rats. The cells were isolated by elastase digestion and panning on immunoglobulin G-coated plates and fractionated into types IIA and IIB by centrifugal elutriation. Type IIB cells contained more phospholipid and had an enhanced rate of [3H]choline incorporation into phosphatidylcholine. The activity of choline-phosphate cytidylyltransferase was elevated in the type IIB cells and the extent of the increase was diminished when phosphatidylglycerol was included in the assay, suggesting that the enhanced activity was due to enzyme activation rather than protein synthesis. The basal rate of phosphatidylcholine secretion was the same in all three groups as was the response to a variety of secretagogues. Incorporation of [3H]acetate into fatty acids was elevated in type IIB cells and the activity of fatty acid synthase was eightfold greater than in control cells. These data show that de novo fatty acid biosynthesis is increased in hypertrophic type II cells and that surfactant secretion is not elevated.

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Novel inhibitors of the condensing enzymes of the Type II fatty acid synthase of pea (Pisum sativum)

Biochemical Journal ◽

10.1042/bj3470205 ◽

2000 ◽

Vol 347 (1) ◽

pp. 205-209 ◽

Cited By ~ 11

Author(s):

A. Lesley JONES ◽

Derek HERBERT ◽

Andrew J. RUTTER ◽

Jane E. DANCER ◽

John L. HARWOOD

Keyword(s):

Fatty Acid ◽

Fatty Acid Synthase ◽

Fatty Acid Biosynthesis ◽

De Novo ◽

Higher Plants ◽

Acyl Carrier Protein ◽

Type Ii ◽

General Activity ◽

The Individual ◽

Derivatives Of

The type II fatty acid synthases (FASs) of higher plants (and Escherichia coli) contain three condensing enzymes called β-ketoacyl-ACP synthases (KAS), where ACP is acyl-carrier-protein. We have used novel derivatives of the antibiotic thiolactomycin to inhibit these enzymes. Overall de novo fatty acid biosynthesis was measured using [1-14C]acetate substrate and chloroplast preparations from pea leaves, and [1-14C]laurate was used to distinguish between the effects of the inhibitors on KAS I from those on KAS II. In addition, the activities of these enzymes, together with the short-chain condensing enzyme, KAS III, were measured directly. Six analogues were tested and two, both with extended hydrocarbon side chains, were found to be more effective inhibitors than thiolactomycin. Incubations with chloroplasts and direct assay of the individual condensing enzymes showed that all three compounds inhibited the pea FAS condensing enzymes in the order KAS II > KAS I > KAS III. These results demonstrate the general activity of thiolactomycin and its derivatives against these FAS condensation reactions, and suggest that such compounds will be useful for further detailed studies of inhibition and for use as pharmaceuticals against Type II FASs of pathogens.

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