The role of intermediate binding in Type I and Type II acyl carrier proteins

Lead Research Organisation: University of Bristol
Department Name: Chemistry

Abstract

Natural products play an enormous role in human and veterinary medicine providing a valuable source of antibiotics, antifungals and anticancer agents. The widespread use of, particularly, broad-spectrum antibiotics at doses aimed at disease prevention rather than the treatment of infections has meant that many organisms have developed resistance to these drugs. These include bacteria, which cause serious infections in hospitalised patients despite government attempts to clean up wards and improve hygiene; bacteria that cause respiratory diseases such as pneumonia and tuberculosis; food-borne pathogens and sexually transmitted organisms. A significant research priority is to develop new drugs in this constant to and fro battle, which can deal with these resistant organisms. To combat this we are studying the bacteria and fungi that provide these valuable sources of natural products. Intense research over the last 30 years has greatly increased our understanding of how these organisms make these molecules. It turns out not to be simple. It is now known that there are vastly complex arrays of enzymes (complex biological molecules) that perform a series of programmed building steps to produce the final molecule. This can be likened to a production line where each element has a particular job to do and must do it in the correct order and with very high precision. Sometimes these arrays are arranged as one large assembly, in others they are present as separate components that somehow find each other as required. Despite these different architectures, each of these assembly lines features a common component, a so called Acyl Carrier Protein or ACP. This protein is an intelligent chip that must carry the molecule being processed to each enzyme and in some cases may shield it from the surrounding environment. We want to understand how this protein works, how it recognises its partners and how it may protect the molecule it is carrying. To do this, we use a technique called Nuclear Magnetic Resonance spectroscopy (NMR) that works with aqueous solutions of the proteins and tells us their shape. We combine this technique with our ability to modify the ACP with molecules that resemble the natural molecules it carries. We wish to understand if it actively uses the molecule it carries to change its shape so it then fits correctly into the correct next enzyme in the synthetic sequence. We will look at a number of different ACPs that carry different types of molecules and which have a varying need for molecular recognition and product stabilisation. We have also discovered that some assemblies use more than one ACP at critical junctions and these may help relieve bottlenecks in the biosynthetic sequence. Our understanding of these ACPs is very limited so we wish to begin to understand how 2 or 3 ACPs might fit together and cooperate with one another.

Technical Summary

Polyketide and Fatty acids are produced by synthases (PKSs or FASs) that share many common mechanistic and structural features. In Type I synthases found in animal cells and fungi, the component enzymes are covalently linked on a single polypeptide chain. These can arranged as 'megasynthases', hugely complex enzymes with multiple domains responsible for each individual catalytic step (as in macrolide biosynthesis) or as a single set of enzymes that are used iteratively (eg Type I FAS). In Type II synthases found in prokaryotes and plants, the biosynthetic steps are mediated by transient complexes of these components. In both Type I and Type II synthases, a small protein, the acyl carrier protein (ACP) transports the nascent polyketide or fatty acid and mediates the correct protein-protein interactions along a pre-defined biosynthetic sequence. There are many published structures of ACPs and a few examples of Type II ACPs bearing fully saturated fatty acid intermediates. However equivalent examples in Type I FASs are missing and there are no examples of bacterial of fungal PKS ACPs with more challenging polyketide intermediates bound. We have used a synthetic approach to make CoAs derivatised with stabilised polyketide equivalents and a range of fatty acid intermediates from different points along the fatty acid cycle. We have combined this with solution state NMR where we have already solved the three-dimensional structures of a range of FAS and PKS ACPs and will now complete structures of these ACPs with covalently bound intermediates. We have selected a broad spectrum of examples that address Type I and Type II FASs and PKSs. In addition we want to solve the three dimensional structure of an ACP di- and tri- domain from a Type I modular PKS where preliminary data suggests their function may be uniquely defined by their location in the synthase rather than absolute enzymic activity.

Publications

10 25 50

 
Description We have greatly furthered our knowledge of acyl carrier proteins and have established collaborations to now address whether these can be engineered to create new hybrid antibiotics.
Exploitation Route We require further funding but have interest from Industry to support similar investigation in mupirocin and kalamanticin pathways. Our findings will be used to test whether we can now create new ACPs, engineer novel pathways using these ACPs and then test new molecular entities for anti-microbial action. Update 2018: We have since gained further funding from the BBSRC to investigate mupirocin biosynthesis, a potent anti_MRSA compound. This application was also supported by Public Health England and GSK who currently market mupirocin under the trade name bactroban. We aim to engineer production of a more stable variant that might have utility as a new antibiotic.
Sectors Chemicals,Pharmaceuticals and Medical Biotechnology

 
Description Our results have informed further work with acyl carrier proteins that seek to probe their unique protein protein interaction properties so we can manipulate and assemble novel biosynthetic pathways. This work has led us to now probe questions around recognition in mixed PKS/NRPS systems with the aim of generating novel hybrid antibiotics. This will be the aim of a future application to the BBSRC.
First Year Of Impact 2013
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Economic

 
Title Chemical Shift Files and Structure codes summary 
Description The ensemble of 20 NMR structures of act apo-ACP has been deposited with the Brookhaven protein database (PDB ID: 2k0y) and NMR chemical shifts have been deposited with BioMagResBank (accession code: 15659). The ensemble of 20 NMR structures of act holo-ACP has been deposited with the Brookhaven protein database (PDB ID: 2k0x) and NMR chemical shifts have been deposited with BioMagResBank (accession code: 15658). The ensemble of 20 NMR structures of each derivatised actinorhodin ACP and associated NMR chemical shifts have been deposited with the Protein Data Bank/Biological Magnetic Resonance Data Bank with the following accession codes: acetyl ACP, 2kg6/16196; malonyl ACP, 2kg8/ 16197; 3-oxobutyl ACP, 2kgd/16202; 3,5-dioxohexyl ACP, 2kge/16203; butyryl ACP, 2kg9/16199; hexanoyl ACP, 2kga/16200; and octanoyl ACP, 2kgc/16201. The coordinate files for the structures of derivatised S. coelicolor FAS ACP over a complete cycle of fatty acid biosynthesis have been deposited with the following PDB/BMRB codes: hexanoyl-ACP, 2koo/16524; 3-oxooctanyl-ACP, 2kop/16525; 3R-hydroxyctanoyl-ACP, 2koq/16526; 2-octenoyl-ACP, 2kor/16527; octanoyl-ACP, 2kos/ 16528. Coordinates for the mupirocin di-domain Acyl Carrier Protein involved in beta-branch insertion during type I polyketide synthesis has been deposited with PDB code 2l22 and the chemical shift assignments deposited with BRMB with access code 17111 The first example of a solution structure of an Acyl Carrier Protein Domain from a Fungal Type I Polyketide Synthase has been deposited with codes 2kr5 and 16624, respectively, for the holo form of the ACP. Finally a high resolution structure of the rat Type I FAS ACP has been deposited with accession code 2png and replaces a previously deposited low resolution structure of this protein. 
Type Of Material Database/Collection of data 
Year Produced 2012 
Provided To Others? No  
Impact No actual impacts realised to date 
 
Title NMR Solution Structures of 3,5-dioxohexyl ACP (a triketide mimic) from the actinorhodin polyketide synthase in Streptomyces coelicolor 
Description Acyl carrier proteins (ACPs) are essential to both fatty acid synthase (FAS) and polyketide synthase (PKS) biosynthetic pathways, yet relatively little is known about how they function at a molecular level. This structure of ACP bound to a triketide mimic was made to probe conformational changes that may play a role in protein-protein recognition and assist the binding of polyketide intermediates. PDB code: 2KGE This latter system above all others has the profound impact for engineering synthetic biology systems as it is beginning to sugdest minimal involvement of the ACP in binding polyketide intermediates and improves the prospect of engineering a generic set of ACPs that may be a universal reagents in synthetic biology systems for use for exmaple in novel antibiotic production. RCSB Protein Data Bank 
Type Of Material Database/Collection of data 
Year Produced 2009 
Provided To Others? No  
Impact No actual impacts realised to date 
 
Title NMR Solution Structures of 3-oxo-butyl-ACP, an intermediate mimic from the actinorhodin polyketide synthase in Streptomyces coelicolor 
Description Acyl carrier proteins (ACPs) are essential to both fatty acid synthase (FAS) and polyketide synthase (PKS) biosynthetic pathways, yet relatively little is known about how they function at a molecular level. This structure of ACP bound to a 3-oxo butyl polyketide mimic was made to probe conformational changes may play a role in protein-protein recognition and assist the binding of polyketide intermediates. PDB code: 2KGD RCSB Protein Data Bank 
Type Of Material Database/Collection of data 
Year Produced 2009 
Provided To Others? No  
Impact No actual impacts realised to date 
 
Title NMR Solution Structures of butyryl-ACP (a non-polar, non pathway intermediate) from the actinorhodin polyketide synthase in Streptomyces coelicolor 
Description Acyl carrier proteins (ACPs) are essential to both fatty acid synthase (FAS) and polyketide synthase (PKS) biosynthetic pathways, yet relatively little is known about how they function at a molecular level. This structure of ACP bound to a butyryl unit was made to probe conformational changes may play a role in protein-protein recognition and assist the binding of polyketide intermediates. PDB code: 2KG9 RCSB Protein Data Bank 
Type Of Material Database/Collection of data 
Year Produced 2009 
Provided To Others? No  
Impact No actual impacts realised to date 
 
Title NMR Solution Structures of hexanoyl ACP (a non natural intermediate) from the actinorhodin polyketide synthase in Streptomyces coelicolor 
Description Acyl carrier proteins (ACPs) are essential to both fatty acid synthase (FAS) and polyketide synthase (PKS) biosynthetic pathways, yet relatively little is known about how they function at a molecular level. This structure of ACP bound to a hexanoyl unit was made to probe conformational changes may play a role in protein-protein recognition and assist the binding of polyketide intermediates. PDB code: 2KGA RCSB Protein Data Bank 
Type Of Material Database/Collection of data 
Year Produced 2009 
Provided To Others? No  
Impact No actual impacts realised to date 
 
Title NMR Solution Structures of malonyl ACP from the actinorhodin polyketide synthase in Streptomyces coelicolor 
Description Acyl carrier proteins (ACPs) are essential to both fatty acid synthase (FAS) and polyketide synthase (PKS) biosynthetic pathways, yet relatively little is known about how they function at a molecular level. This structure of ACP bound to a malonyl extender unit was made to probe conformational changes may play a role in protein-protein recognition and assist the binding of polyketide intermediates. PDB code: 2KG8 RCSB Protein Data Bank 
Type Of Material Database/Collection of data 
Year Produced 2009 
Provided To Others? No  
Impact No actual impacts realised to date 
 
Title NMR Solution Structures of octanoyl ACP (a non-natural intermediate) from the actinorhodin polyketide synthase in Streptomyces coelicolor 
Description Acyl carrier proteins (ACPs) are essential to both fatty acid synthase (FAS) and polyketide synthase (PKS) biosynthetic pathways, yet relatively little is known about how they function at a molecular level. This structure of ACP bound to a octanoyl unit was made to probe conformational changes may play a role in protein-protein recognition and assist the binding of polyketide intermediates. PDB code: 2KGC RCSB Protein Data Bank 
Type Of Material Database/Collection of data 
Year Produced 2009 
Provided To Others? No  
Impact No actual impacts realised to date 
 
Title NMR solution structures of 2-octenoyl-ACP from Streptomyces coelicolor Fatty Acid Synthase 
Description It remains unclear whether in a bacterial fatty acid synthase (FAS) acyl chain transfer is a programmed or diffusion controlled and random action. Acyl carrier protein (ACP), which delivers all intermediates and interacts with all synthase enzymes, is the key player in this process. High-resolution structures of intermediates covalently bound to an ACP representing each step in fatty acid biosynthesis have been solved by solution NMR. These include hexanoyl-, 3-oxooctanyl-, 3R-hydroxyoctanoyl-, 2-octenoyl-, and octanoyl-ACP from Streptomyces coelicolor FAS. The high-resolution structures reveal that the ACP adopts a unique conformation for each intermediate driven by changes in the internal fatty acid binding pocket. The binding of each intermediate shows conserved structural features that may ensure effective molecular recognition over subsequent rounds of fatty acid biosynthesis. RCSB Protein Data Bank PDB code: 2KOR 
Type Of Material Database/Collection of data 
Year Produced 2010 
Provided To Others? No  
Impact No actual impacts realised to date 
 
Title NMR solution structures of 3-hydroxyoctanoyl-ACP from Streptomyces coelicolor Fatty Acid Synthase 
Description It remains unclear whether in a bacterial fatty acid synthase (FAS) acyl chain transfer is a programmed or diffusion controlled and random action. Acyl carrier protein (ACP), which delivers all intermediates and interacts with all synthase enzymes, is the key player in this process. High-resolution structures of intermediates covalently bound to an ACP representing each step in fatty acid biosynthesis have been solved by solution NMR. These include hexanoyl-, 3-oxooctanyl-, 3R-hydroxyoctanoyl-, 2-octenoyl-, and octanoyl-ACP from Streptomyces coelicolor FAS. The high-resolution structures reveal that the ACP adopts a unique conformation for each intermediate driven by changes in the internal fatty acid binding pocket. The binding of each intermediate shows conserved structural features that may ensure effective molecular recognition over subsequent rounds of fatty acid biosynthesis. RCSB Protein Data Bank PDB code: 2KOQ 
Type Of Material Database/Collection of data 
Year Produced 2010 
Provided To Others? No  
Impact No actual impacts realised to date 
 
Title NMR solution structures of 3-oxooctanyl-ACP from Streptomyces coelicolor Fatty Acid Synthase 
Description It remains unclear whether in a bacterial fatty acid synthase (FAS) acyl chain transfer is a programmed or diffusion controlled and random action. Acyl carrier protein (ACP), which delivers all intermediates and interacts with all synthase enzymes, is the key player in this process. High-resolution structures of intermediates covalently bound to an ACP representing each step in fatty acid biosynthesis have been solved by solution NMR. These include hexanoyl-, 3-oxooctanyl-, 3R-hydroxyoctanoyl-, 2-octenoyl-, and octanoyl-ACP from Streptomyces coelicolor FAS. The high-resolution structures reveal that the ACP adopts a unique conformation for each intermediate driven by changes in the internal fatty acid binding pocket. The binding of each intermediate shows conserved structural features that may ensure effective molecular recognition over subsequent rounds of fatty acid biosynthesis. RCSB Protein Data Bank PDB code: 2KOP 
Type Of Material Database/Collection of data 
Year Produced 2010 
Provided To Others? No  
Impact No actual impacts realised to date 
 
Title NMR solution structures of hexanoyl-ACP from the Streptomyces coelicolor Fatty Acid Synthase 
Description It remains unclear whether in a bacterial fatty acid synthase (FAS) acyl chain transfer is a programmed or diffusion controlled and random action. Acyl carrier protein (ACP), which delivers all intermediates and interacts with all synthase enzymes, is the key player in this process. High-resolution structures of intermediates covalently bound to an ACP representing each step in fatty acid biosynthesis have been solved by solution NMR. These include hexanoyl-, 3-oxooctanyl-, 3R-hydroxyoctanoyl-, 2-octenoyl-, and octanoyl-ACP from Streptomyces coelicolor FAS. The high-resolution structures reveal that the ACP adopts a unique conformation for each intermediate driven by changes in the internal fatty acid binding pocket. The binding of each intermediate shows conserved structural features that may ensure effective molecular recognition over subsequent rounds of fatty acid biosynthesis. RCSB Protein Data Bank PDB code: 2KOO 
Type Of Material Database/Collection of data 
Year Produced 2010 
Provided To Others? No  
Impact No actual impacts realised to date 
 
Title NMR solution structures of octanoyl-ACP from Streptomyces coelicolor Fatty Acid Synthase 
Description It remains unclear whether in a bacterial fatty acid synthase (FAS) acyl chain transfer is a programmed or diffusion controlled and random action. Acyl carrier protein (ACP), which delivers all intermediates and interacts with all synthase enzymes, is the key player in this process. High-resolution structures of intermediates covalently bound to an ACP representing each step in fatty acid biosynthesis have been solved by solution NMR. These include hexanoyl-, 3-oxooctanyl-, 3R-hydroxyoctanoyl-, 2-octenoyl-, and octanoyl-ACP from Streptomyces coelicolor FAS. The high-resolution structures reveal that the ACP adopts a unique conformation for each intermediate driven by changes in the internal fatty acid binding pocket. The binding of each intermediate shows conserved structural features that may ensure effective molecular recognition over subsequent rounds of fatty acid biosynthesis. RCSB Protein Data Bank PDB code: 2KOS 
Type Of Material Database/Collection of data 
Year Produced 2010 
Provided To Others? No  
Impact No actual impacts realised to date 
 
Title Solution Structure of an Acyl Carrier Protein Domain from Fungal Type I Polyketide Synthase 
Description Acyl (peptidyl) carrier protein (ACP or PCP) is a crucial component involved in the transfer of thiol ester-bound intermediates during the biosynthesis of primary and secondary metabolites such as fatty acids, polyketides, and nonribosomal peptides. Although many carrier protein three-dimensional structures have been determined, to date there is no model available for a fungal type I polyketide synthase ACP. Here we report the solution structure of the norsolorinic acid synthase (NSAS) holo ACP domain that has been excised from the full-length multifunctional enzyme. NSAS ACP shows similarities in three-dimensional structure with other type I and type II ACPs, consisting of a four-helix bundle with helices I, II, and IV arranged in parallel. The N-terminus of helix III, however, is unusually hydrophobic, and Phe1768 and Leu1770 pack well with the core of the protein. The result is that unlike other carrier proteins, helix III lies almost perpendicular to the three major helices. Helix III is well-defined by numerous NMR-derived distance restraints and may be less flexible than counterparts in type II FAS and PKS ACPs. When the holo ACP is derivatized with a hexanoyl group, only minor changes are observed between the HSQC spectra of the two ACP species and no NOEs are observed for this hydrophobic acyl group. Along with the mammalian type I FAS, this further strengthens the view that type I ACPs do not show any significant affinity for hydrophobic (nonpolar) chain assembly intermediates attached via the 4'-phosphopantetheine prosthetic group. RCSB Protein Data Bank 
Type Of Material Database/Collection of data 
Year Produced 2010 
Provided To Others? No  
Impact No actual impacts realised to date 
 
Title Solution Structure of the acetyl Actinorhodin Acyl Carrier Protein from Streptomyces coelicolor 
Description Acyl carrier proteins (ACPs) are essential to both fatty acid synthase (FAS) and polyketide synthase (PKS) biosynthetic pathways, yet relatively little is known about how they function at a molecular level. This structure of ACP bound to a acetyl starter unit was made to probe conformational changes may play a role in protein-protein recognition and assist the binding of polyketide intermediates. PDB code: 2KG6 RCSB Protein Data Bank 
Type Of Material Database/Collection of data 
Year Produced 2009 
Provided To Others? No  
Impact No actual impacts realised to date 
 
Title The actinorhodin apo acyl carrier protein from S. coelicolor 
Description PDB code: 2K0Y 
Type Of Material Database/Collection of data 
Year Produced 2009 
Provided To Others? No  
Impact No actual impacts realised to date 
 
Title The actinorhodin holo acyl carrier protein from S. coelicolor 
Description High resolution three-dimensional structure of the actinorhodin holo ACP RCSB Protein Data Bank PDB code: 2KOX 
Type Of Material Database/Collection of data 
Year Produced 2009 
Provided To Others? No  
Impact No actual impacts realised to date 
 
Title Type I rat fatty acid synthase acyl carrier protein (ACP) domain 
Description PDB code: 2PNG 
Type Of Material Database/Collection of data 
Year Produced 2009 
Provided To Others? No  
Impact No actual impacts realised to date 
 
Description Colaboration with the University of Birmingham for further investigate protein engineering of acyl carrier proteins 
Organisation University of Birmingham
Country United Kingdom 
Sector Academic/University 
PI Contribution We have had structural input into the design of acyl carrier proteins to be used in the assembly of biosynthetic pathways to produce novel anti-microbial compounds.
Collaborator Contribution They have provided expertise in assembling designed components into the full pathways.
Impact One main publication which is leading to applications for follow on funding from the BBSRC and partnership with Industry. Haines AS, Dong X, Song Z, Farmer R, Williams C, Hothersall J, Ploskon E, Wattana-Amorn P, Stephens ER, Yamada E, Gurney R, Takabayashi Y, Masschelein J, Lavigne R, Crosby J, Simpson TJ, Winn PJ, Thomas CM, Crump MP. A conserved mofit flags acyl carrier proteins for ß-branching in polyketide synthesis (2013) Nat. Chem. Biol. 9, 685-692.
Start Year 2011
 
Description 2009 Sanger lecture 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach local
Primary Audience Public/other audiences
Results and Impact Public engagement: Professor Simpson gave the 2009 Sanger lecture to the science club at Charterhouse School.

no actual impacts realised to date
Year(s) Of Engagement Activity 2009