SAXS reveals highly flexible interdomain linkers of tandem acyl carrier protein–thioesterase domains from a fungal nonreducing polyketide synthase

Bunnak, Waraporn; Winter, Ashley; Lazarus, Colin M.; Crump, Matthew P.; Race, Paul R.; Wattana-Amorn, Pakorn

doi:10.1002/1873-3468.13954

Cited by 5 publications

(8 citation statements)

References 56 publications

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“…This is coherent with what has been observed in the structure of VirA module 5, where the structurally uncharacterized 159 residue-long portion of the KS-to-ACP linker displays a rigid structure, leading to an arm-shape architecture for the KS-ACP-ACP fragment [ 28 ]. On the contrary, the Men2 ACP2-TE and ACP1-ACP2-TE fragments have turned out to be highly flexible [ 60 ]. Nonetheless, these Men2 construct contain only the ACP1-ACP2 and ACP2-TE linkers (respectively 47 and 57 residue long) whereas our fragment fACP2-TE contains a smaller ACP2-ΨACP linker (37 residues, though 23 additional residues are missing as they were unresolved in the structure of ΨACP) and a 78-residue-long portion of the linker between AT and ACP2 (lAT-ACP2 fACP2-TE ).…”

Section: Discussionmentioning

confidence: 99%

Solution structure of the type I polyketide synthase Pks13 from Mycobacterium tuberculosis

et al. 2022

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Background Type I polyketide synthases (PKSs) are multifunctional enzymes responsible for the biosynthesis of a group of diverse natural compounds with biotechnological and pharmaceutical interest called polyketides. The diversity of polyketides is impressive despite the limited set of catalytic domains used by PKSs for biosynthesis, leading to considerable interest in deciphering their structure‐function relationships, which is challenging due to high intrinsic flexibility. Among nineteen polyketide synthases encoded by the genome of Mycobacterium tuberculosis, Pks13 is the condensase required for the final condensation step of two long acyl chains in the biosynthetic pathway of mycolic acids, essential components of the cell envelope of Corynebacterineae species. It has been validated as a promising druggable target and knowledge of its structure is essential to speed up drug discovery to fight against tuberculosis. Results We report here a quasi-atomic model of Pks13 obtained using small-angle X-ray scattering of the entire protein and various molecular subspecies combined with known high-resolution structures of Pks13 domains or structural homologues. As a comparison, the low-resolution structures of two other mycobacterial polyketide synthases, Mas and PpsA from Mycobacterium bovis BCG, are also presented. This study highlights a monomeric and elongated state of the enzyme with the apo- and holo-forms being identical at the resolution probed. Catalytic domains are segregated into two parts, which correspond to the condensation reaction per se and to the release of the product, a pivot for the enzyme flexibility being at the interface. The two acyl carrier protein domains are found at opposite sides of the ketosynthase domain and display distinct characteristics in terms of flexibility. Conclusions The Pks13 model reported here provides the first structural information on the molecular mechanism of this complex enzyme and opens up new perspectives to develop inhibitors that target the interactions with its enzymatic partners or between catalytic domains within Pks13 itself.

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Section: Discussionmentioning

confidence: 99%

Solution structure of the type I polyketide synthase Pks13 from Mycobacterium tuberculosis

et al. 2022

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“…Therefore, the presence of the phosphopantetheinyl group of the holo form did not alter the shape distribution profile, as previously observed for ACPs within the menisporopsin A system. 31 For all EOM analysis, irrespective of linker length, the resultant subpopulations were consistent with a sharp R g distribution: the bent conformation (R g = 23.7−25.6 Å) and the extended dumbbell (R g = 31.7−34.3 Å) (Figure 4B and Figure S17B). Additionally, for all EOM ensembles, the average R g and D max values were larger than those of the random pool, indicating the conformations were extended in solution for both data sets as previously suggested by DATCLASS.…”

Section: ■ Materials and Methodsmentioning

confidence: 58%

“…The shorter and partially restricted linker in PigH ACP 1 -ACP 2 therefore decreases the separation between the two ACPs. In addition, conformational averaging in the linker region has not been observed previously and these connecting stretches of amino acids have been shown to be flexible in nature, − leading to the generally accepted view of structurally uncoupled ACP domains.…”

Section: Resultsmentioning

confidence: 98%

“…30 Our recent EOM results of the doublet ACP fused with the TE domain (Men2 ACP 1 -ACP 2 -TE) from a fungal nonreducing PKS involved in menisporopsin A biosynthesis also showed multiple conformations with no specific orientation between this doublet ACP or distinct subpopulations of conformers. 31 It is likely that the difference in linker length between the individual ACP domains, 48 residues for Men2 ACP 1 -ACP 2 and 17 residues for PigH ACP 1 -ACP 2 , may influence the degree of linker flexibility observed in proteins containing multiple ACP domains. Typically, multiple ACP domains are a part of complex megasynthases such as type I PKSs or NRPSs where the transfer of intermediates between several domains may require longer and flexible interdomain linkers.…”

Section: ■ Materials and Methodsmentioning

confidence: 99%

“…Despite their widespread occurrence, previous structural work is limited to low-resolution small-angle X-ray scattering (SAXS) models − and a single nuclear magnetic resonance (NMR) solution didomain ACP structure . Consequently, the structure and function of these diverse structural motifs are poorly understood.…”

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Solution Structure and Conformational Dynamics of a Doublet Acyl Carrier Protein from Prodigiosin Biosynthesis

et al. 2021

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The acyl carrier protein (ACP) is an indispensable component of both fatty acid and polyketide synthases and is primarily responsible for delivering acyl intermediates to enzymatic partners. At present, increasing numbers of multidomain ACPs have been discovered with roles in molecular recognition of trans-acting enzymatic partners as well as increasing metabolic flux. Further structural information is required to provide insight into their function, yet to date, the only high-resolution structure of this class to be determined is that of the doublet ACP (two continuous ACP domains) from mupirocin synthase. Here we report the solution nuclear magnetic resonance (NMR) structure of the doublet ACP domains from PigH (PigH ACP1-ACP2), which is an enzyme that catalyzes the formation of the bipyrrolic intermediate of prodigiosin, a potent anticancer compound with a variety of biological activities. The PigH ACP1-ACP2 structure shows each ACP domain consists of three conserved helices connected by a linker that is partially restricted by interactions with the ACP1 domain. Analysis of the holo (4′-phosphopantetheine, 4′-PP) form of PigH ACP1-ACP2 by NMR revealed conformational exchange found predominantly in the ACP2 domain reflecting the inherent plasticity of this ACP. Furthermore, ensemble models obtained from SAXS data reveal two distinct conformers, bent and extended, of both apo (unmodified) and holo PigH ACP1-ACP2 mediated by the central linker. The bent conformer appears to be a result of linker–ACP1 interactions detected by NMR and might be important for intradomain communication during the biosynthesis. These results provide new insights into the behavior of the interdomain linker of multiple ACP domains that may modulate protein–protein interactions. This is likely to become an increasingly important consideration for metabolic engineering in prodigiosin and other related biosynthetic pathways.

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Small-angle X-ray scattering studies of enzymes

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Pei

Patterson

et al. 2023

Current Opinion in Chemical Biology

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SAXS reveals highly flexible interdomain linkers of tandem acyl carrier protein–thioesterase domains from a fungal nonreducing polyketide synthase

Cited by 5 publications

References 56 publications

Solution structure of the type I polyketide synthase Pks13 from Mycobacterium tuberculosis

Solution structure of the type I polyketide synthase Pks13 from Mycobacterium tuberculosis

Solution Structure and Conformational Dynamics of a Doublet Acyl Carrier Protein from Prodigiosin Biosynthesis

Small-angle X-ray scattering studies of enzymes

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