Using Modern Tools To Probe the Structure–Function Relationship of Fatty Acid Synthases

Finzel, Kara; Lee, D. John; Burkart, Michael D.

doi:10.1002/cbic.201402578

Cited by 59 publications

(64 citation statements)

References 194 publications

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“…Because of these weak and transient interactions, structural characterization of the enzyme-CP complex is difficult. Recently, several methods using synthetic probes to capture CP in functional association with a partner enzyme were developed, allowing the structural characterization of these complexes to visualize the protein-protein interactions (17,(21)(22)(23). However, no crystal 10.9 ± 0.5 −5.5 −7.9 ± 0.1 2.4 1.0 † *Values are ± SD from the fit.…”

Section: Discussionmentioning

confidence: 99%

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: Discussionmentioning

confidence: 99%

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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“…3). 1 Additionally, students found it fascinating that, experimentally, these two proteins interact and crosslink, but (panel 3 of Fig. 3) only 1 of 10 predicted ClusPro PDB outputs showed the active sites close enough for functional interaction (in silico docking has its limitations!)…”

Section: Resultsmentioning

confidence: 99%

“…1 Natural product biosynthesis represents an excellent venue to expose undergraduate students to modern convergent themes in biochemical research. Herein we present a natural product-inspired undergraduate lab experiment, which blends concepts and techniques stemming from chemistry, biology, and computational chemistry.…”

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confidence: 99%

Utilizing Mechanistic Cross-Linking Technology To Study Protein–Protein Interactions: An Experiment Designed for an Undergraduate Biochemistry Lab

et al. 2017

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Over the past decade, mechanistic crosslinking probes have been used to study protein-protein interactions in natural product biosynthetic pathways. This approach is highly interdisciplinary, combining elements of protein biochemistry, organic chemistry, and computational docking. The development of an experiment to engage undergraduate students in multidisciplinary research is described that leverages mechanistic crosslinking probes to study protein conformations and protein-protein interactions. This experiment provides students with a platform to learn chemoenzymatic synthesis, polyacrylamide gel electrophoresis, biochemical assays, and computational docking all while exploring a contemporary biochemical topic.

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“…The two catalytic domains are connected by and communicate via the 90-residue intermediate domain, which is a structural and functional homolog of acyl carrier proteins (ACP) (13). Another example of such a carrier protein in humans is the fatty acid synthase complex, where it functions in transferring of growing acyl chain (16). The distinctive feature of acyl-carrier proteins is the presence of a covalently linked 4′-phosphopantetheine (4-PP) prosthetic group which functions as a long (~20 Å), flexible arm that can reach into buried catalytic centers and carrying the reaction intermediate from one center to another (16).…”

Section: Introductionmentioning

confidence: 99%

Modeling of interactions between functional domains of ALDH1L1

Horita

Krupenko

2017

Chemico-Biological Interactions

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ALDH1L1, a member of the aldehyde dehydrogenase superfamily of enzymes, catalyzes the conversion of 10-formyltetrahydrofolate to tetrahydrofolate and CO2. The enzyme is a tetramer of identical subunits, with each subunit consisting of three functional domains that originated from unrelated genes. The N- and C-terminal domains are catalytic, while the intermediate domain transfers the reaction intermediate from the N- to the C-terminal domain. The intermediate domain is an acyl carrier protein, possessing the covalently attached 4′-phosphopantetheine (4-PP) prosthetic group. This prosthetic group is known to function as a swinging arm transferring intermediates between enzymes in complex biosynthetic reactions. Here we have applied computer modeling using available structures of the three functional domains of ALDH1L1 to evaluate the extent of flexibility within the full-length protein. This approach allowed us to define positions of the 4-PP arm within the two catalytic domains and to predict N-terminal:intermediate and intermediate:C-terminal domain interfaces. Our models further suggested high degree of flexibility within the full-length enzyme.

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Using Modern Tools To Probe the Structure–Function Relationship of Fatty Acid Synthases

Cited by 59 publications

References 194 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

Utilizing Mechanistic Cross-Linking Technology To Study Protein–Protein Interactions: An Experiment Designed for an Undergraduate Biochemistry Lab

Modeling of interactions between functional domains of ALDH1L1

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