Structure and Malonyl CoA-ACP Transacylase Binding of <i>Streptomyces coelicolor</i> Fatty Acid Synthase Acyl Carrier Protein

Arthur, Christopher J.; Williams, Christopher; Pottage, Katherine; Płoskoń, Eliza; Findlow, Stuart C.; Burston, Steven G.; Simpson, Thomas J.; Crump, Matthew P.; Crosby, John

doi:10.1021/cb900099e

Cited by 36 publications

(54 citation statements)

References 36 publications

(79 reference statements)

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“…The VinK wild type was shown to bind VinL with a K a value of 8.2 ± 0.8 × 10 4 M −1 (Table 1 and Fig. S4A), which is similar to the affinities of other MCATs for ACPs (19,20). The R153A, M206A, and R299A mutants showed significantly reduced affinity for VinL (Fig.…”

Section: Resultssupporting

confidence: 57%

“…In terms of the interaction of AT with ACP, Streptomyces coelicolor MCAT was suggested to recognize the helix II of FAS ACP, based on the previous docking and mutational studies (19). However, the details of the binding and binding interface remain elusive.…”

Section: Discussionmentioning

confidence: 99%

“…Some PKS ATs are predicted to bind ACP in a different manner based on docking analysis. For example, S. coelicolor MCAT was suggested to interact with the loop region between helix I and II of actinorhodin PKS ACP (19,29). Alternatively, the presence of an additional domain such as the KS-AT linker domain in modular PKS might be involved in the binding mode of ACP, as proposed in the interaction between AT DYN10 and ACP DYN involved in dynemicin biosynthesis (30).…”

Section: Discussionmentioning

confidence: 99%

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Structure-based analysis of the molecular interactions between acyltransferase and acyl carrier protein in vicenistatin biosynthesis

Miyanaga

Iwasawa

Shinohara

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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Acyltransferases (ATs) are key determinants of building block specificity in polyketide biosynthesis. Despite the importance of protein-protein interactions between AT and acyl carrier protein (ACP) during the acyltransfer reaction, the mechanism of ACP recognition by AT is not understood in detail. Herein, we report the crystal structure of AT VinK, which transfers a dipeptide group between two ACPs, VinL and VinP1LdACP, in vicenistatin biosynthesis. The isolated VinK structure showed a unique substrate-binding pocket for the dipeptide group linked to ACP. To gain greater insight into the mechanism of ACP recognition, we attempted to crystallize the VinK-ACP complexes. Because transient enzyme-ACP complexes are difficult to crystallize, we developed a covalent cross-linking strategy using a bifunctional maleimide reagent to trap the VinK-ACP complexes, allowing the determination of the crystal structure of the VinK-VinL complex. In the complex structure, Arg-153, Met-206, and Arg-299 of VinK interact with the negatively charged helix II region of VinL. The VinK-VinL complex structure allows, to our knowledge, the first visualization of the interaction between AT and ACP and provides detailed mechanistic insights into ACP recognition by AT.polyketide | acyltransferase | acyl carrier protein | protein-protein interaction | cross-linking P olyketide synthases (PKSs) are multifunctional enzymes responsible for the biosynthesis of various polyketide natural products (1). Bacterial modular PKSs comprise several catalytic modules that are each responsible for a single round of the polyketide chain elongation reaction. Each module minimally consists of a ketosynthase (KS) domain, an acyltransferase (AT) domain, and an acyl carrier protein (ACP) domain. The AT domain recognizes a specific acyl building block and catalyzes its transfer reaction onto the 4′-phosphopantetheine arm of the ACP. KS extends the polyketide chain by condensing the resulting ACP-bound building blocks with the elongated acylACPs. Although standard modular PKSs contain AT domains in their modules, some modular PKSs lack AT domains in each module and instead receive their acyl building blocks by standalone trans-acting ATs (2).The selection of the starter unit is generally governed by the substrate specificity of the AT domain in the loading module (1). In some modular PKS systems, a didomain-type loading module comprising a loading AT domain and an ACP domain selects an acyl starter building block such as an acetate unit to generate an acyl-ACP intermediate, which is transferred to the downstream extension module for polyketide chain elongation. Alternatively, in a KS Q -type loading module consisting of three domains, the AT domain selects an α-carboxyacyl substrate such as a malonyl group, and the KS Q domain subsequently catalyzes its decarboxylation to construct an acyl-ACP thioester. For polyketide chain elongation, the AT domain of the extension module generally recognizes a specific α-carboxyacyl-CoA as an extender building block (3). Malony...

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Section: Resultssupporting

confidence: 57%

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

Structure-based analysis of the molecular interactions between acyltransferase and acyl carrier protein in vicenistatin biosynthesis

Miyanaga

Iwasawa

Shinohara

et al. 2016

Proc. Natl. Acad. Sci. U.S.A.

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“…It has been reported that the Glu and Asp residues in helix II of Ec-ACP are important to recognize FabA, FabB, FabD, FabG, MCAT, AcpS, and FabH (61,(65)(66)(67)(68) Fig. 4A.…”

Section: Discussionmentioning

confidence: 99%

Novel Structural Components Contribute to the High Thermal Stability of Acyl Carrier Protein from Enterococcus faecalis

Park

Jung

Song

et al. 2016

Journal of Biological Chemistry

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Enterococcus faecalis is a Gram-positive, commensal bacterium that lives in the gastrointestinal tracts of humans and other mammals. It causes severe infections because of high antibiotic resistance. E. faecalis can endure extremes of temperature and pH. Acyl carrier protein (ACP) is a key element in the biosynthesis of fatty acids responsible for acyl group shuttling and delivery. In this study, to understand the origin of high thermal stabilities of E. faecalis ACP (Ef-ACP), its solution structure was investigated for the first time. CD experiments showed that the melting temperature of Ef-ACP is 78.8°C, which is much higher than that of Escherichia coli ACP (67.2°C). The overall structure of Ef-ACP shows the common ACP folding pattern consisting of four ␣-helices (helix I (residues 3-17), helix II (residues 39 -53), helix III (residues 60 -64), and helix IV (residues 68 -78)) connected by three loops. Unique Ef-ACP structural features include a hydrophobic interaction between Phe 45 in helix II and Phe 18 in the ␣ 1 ␣ 2 loop and a hydrogen bonding between Ser 15 in helix I and Ile 20 in the ␣ 1 ␣ 2 loop, resulting in its high thermal stability. Phe 45 -mediated hydrophobic packing may block acyl chain binding subpocket II entry. Furthermore, Ser 58 in the ␣ 2 ␣ 3 loop in Ef-ACP, which usually constitutes a proline in other ACPs, exhibited slow conformational exchanges, resulting in the movement of the helix III outside the structure to accommodate a longer acyl chain in the acyl binding cavity. These results might provide insights into the development of antibiotics against pathogenic drug-resistant E. faecalis strains.

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“…Most studies of streptomycete fatty acid synthesis (FAS)-which is closely linked to polyketide synthesis (PKS)-were conducted with S. coelicolor or S. glaucescens (16)(17)(18)(19)(20)(21)(22)(23)(24)(25). These bacteria utilize the dissociated FAS II system and have a highly conserved fab gene cluster consisting of fabD, fabH, acpP, and fabF.…”

mentioning

confidence: 99%

Role of Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase in Oleaginous Streptomyces sp. Strain G25

Röttig

Strittmatter

Schauer

et al. 2016

Appl Environ Microbiol

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Recently, we isolated a novel Streptomyces strain which can accumulate extraordinarily large amounts of triacylglycerol (TAG) and consists of 64% fatty acids (dry weight) when cultivated with glucose and 50% fatty acids (dry weight) when cultivated with cellobiose. To identify putative gene products responsible for lipid storage and cellobiose utilization, we analyzed its draft genome sequence. A single gene encoding a wax ester synthase/acyl coenzyme A (CoA):diacylglycerol acyltransferase (WS/DGAT) was identified and heterologously expressed in Escherichia coli. The purified enzyme Atf G25 showed acyltransferase activity with C 12 -or C 16 -acyl-CoA, C 12 to C 18 alcohols, or dipalmitoyl glycerol. This acyltransferase exhibits 24% amino acid identity to the model enzyme AtfA from Acinetobacter baylyi but has high sequence similarities to WS/DGATs from other Streptomyces species. To investigate the impact of Atf G25 on lipid accumulation, the respective gene, atf G25 , was inactivated in Streptomyces sp. strain G25. However, cells of the insertion mutant still exhibited DGAT activity and were able to store TAG, albeit in lower quantities and at lower rates than the wild-type strain. These findings clearly indicate that Atf G25 has an important, but not exclusive, role in TAG biosynthesis in the novel Streptomyces isolate and suggest the presence of alternative metabolic pathways for lipid accumulation which are discussed in the present study. IMPORTANCEA novel Streptomyces strain was isolated from desert soil, which represents an extreme environment with high temperatures, frequent drought, and nutrient scarcity. We believe that these harsh conditions promoted the development of the capacity for this strain to accumulate extraordinarily large amounts of lipids. In this study, we present the analysis of its draft genome sequence with a special focus on enzymes potentially involved in its lipid storage. Furthermore, the activity and importance of the detected acyltransferase were studied. As discussed in this paper, and in contrast to many other bacteria, streptomycetes seem to possess a complex metabolic network to synthesize lipids, whereof crucial steps are still largely unknown. This paper therefore provides insights into a range of topics, including extremophile bacteria, the physiology of lipid accumulation, and the biotechnological production of bacterial lipids. Even extreme environments, such as arid, desert-like regions, are populated by prokaryotes. These mostly Gram-positive bacteria are subjected to extreme fluctuations of temperature and water supply. In addition to desiccation and the resulting cellular damage, the bacteria frequently encounter nutrient limitation. To withstand these harsh environmental conditions, the majority of bacteria have evolved various strategies and are able to store lipophilic compounds such as polyhydroxyalkanoates (PHA), triacylglycerols (TAG), or wax esters (WE) (1-3). TAG are synthesized as primary storage compounds by many actinomycetes-which represent the pre...

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Structure and Malonyl CoA-ACP Transacylase Binding of Streptomyces coelicolor Fatty Acid Synthase Acyl Carrier Protein

Cited by 36 publications

References 36 publications

Structure-based analysis of the molecular interactions between acyltransferase and acyl carrier protein in vicenistatin biosynthesis

Structure-based analysis of the molecular interactions between acyltransferase and acyl carrier protein in vicenistatin biosynthesis

Novel Structural Components Contribute to the High Thermal Stability of Acyl Carrier Protein from Enterococcus faecalis

Role of Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase in Oleaginous Streptomyces sp. Strain G25

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