The structural role of the carrier protein – active controller or passive carrier

Crosby, John; Crump, Matthew P.

doi:10.1039/c2np20062g

Cited by 151 publications

(208 citation statements)

References 347 publications

(433 reference statements)

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“…16). VinL has a helix bundle fold with three major helices (helices I, II, and III) and one minor distorted helix III′, as observed in most of the known ACP structures (9). A 4′-phosphopantetheine arm is covalently attached to Ser-36, which is located at the beginning of helix II (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The binding affinities of CP for its partner enzymes are weak, enabling it to interact reversibly with each enzyme in the biosynthetic pathway (9). Because of these weak and transient interactions, structural characterization of the enzyme-CP complex is difficult.…”

Section: Discussionmentioning

confidence: 99%

“…Proper AT-ACP interactions are believed to be essential for kinetically efficient polyketide chain elongation. However, the mechanism of ACP recognition is not well understood because isolated AT structures provide no detailed information on the AT-ACP interactions (9). Structural determination of the AT-ACP complex is necessary for the complete understanding of the basis of ACP recognition for the acyltransfer reaction.…”

mentioning

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: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

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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“…Analogous carrier proteins from polyketide synthetases can sequester their molecular cargo, although the biochemical relevance of this mechanism is not known 31, 32, 33. In the case of PCPs, the Ppant arm does not appear to interact appreciably with the protein core nor to alter the tertiary structure of the PCP in a significant way 21, 34.…”

Section: The Peptidyl Carrier Proteinmentioning

confidence: 99%

New Structural Data Reveal the Motion of Carrier Proteins in Nonribosomal Peptide Synthesis

et al. 2016

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The nonribosomal peptide synthetases (NRPSs) are one of the most promising resources for the production of new bioactive molecules. The mechanism of NRPS catalysis is based around sequential catalytic domains: these are organized into modules, where each module selects, modifies, and incorporates an amino acid into the growing peptide. The intermediates formed during NRPS catalysis are delivered between enzyme centers by peptidyl carrier protein (PCP) domains, which makes PCP interactions and movements crucial to NRPS mechanism. PCP movement has been linked to the domain alternation cycle of adenylation (A) domains, and recent complete NRPS module structures provide support for this hypothesis. However, it appears as though the A domain alternation alone is insufficient to account for the complete NRPS catalytic cycle and that the loaded state of the PCP must also play a role in choreographing catalysis in these complex and fascinating molecular machines.

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“…In other cases, the carrier protein and its partner enzymes are separate entities that form transient complexes to effect catalysis. Examples include the acyl carrier proteins interacting with enzymes involved in type II fatty acid synthesis and polyketide synthesis (5,6). In reality, these two types of carrier protein pathways are mechanistically similar because the carrier protein domain in the permanent complexes is normally itself flexibly tethered to the rest of the complex, allowing significant freedom of movement between partner enzyme domains (1,2).…”

mentioning

confidence: 99%

Structural basis for specificity and promiscuity in a carrier protein/enzyme system from the sulfur cycle

Grabarczyk

Chappell

Johnson

et al. 2015

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

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The bacterial Sox (sulfur oxidation) pathway is an important route for the oxidation of inorganic sulfur compounds. Intermediates in the Sox pathway are covalently attached to the heterodimeric carrier protein SoxYZ through conjugation to a cysteine on a protein swinging arm. We have investigated how the carrier protein shuttles intermediates between the enzymes of the Sox pathway using the interaction between SoxYZ and the enzyme SoxB as our model. The carrier protein and enzyme interact only weakly, but we have trapped their complex by using a "suicide enzyme" strategy in which an engineered cysteine in the SoxB active site forms a disulfide bond with the incoming carrier arm cysteine. The structure of this trapped complex, together with calorimetric data, identifies sites of protein-protein interaction both at the entrance to the enzyme active site tunnel and at a second, distal, site. We find that the enzyme distinguishes between the substrate and product forms of the carrier protein through differences in their interaction kinetics and deduce that this behavior arises from substrate-specific stabilization of a conformational change in the enzyme active site. Our analysis also suggests how the carrier arm-bound substrate group is able to outcompete the adjacent C-terminal carboxylate of the carrier arm for binding to the active site metal ions. We infer that similar principles underlie carrier protein interactions with other enzymes of the Sox pathway.swinging arm | thiosulfate oxidation | Sox system

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The structural role of the carrier protein – active controller or passive carrier

Cited by 151 publications

References 347 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

New Structural Data Reveal the Motion of Carrier Proteins in Nonribosomal Peptide Synthesis

Structural basis for specificity and promiscuity in a carrier protein/enzyme system from the sulfur cycle

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