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Crystal structure of the putative cyclase IdmH from the indanomycin nonribosomal peptide synthase/polyketide synthase.

ABSTRACT: Indanomycin is biosynthesized by a hybrid nonribosomal peptide synthase/polyketide synthase (NRPS/PKS) followed by a number of 'tailoring' steps to form the two ring systems that are present in the mature product. It had previously been hypothesized that the indane ring of indanomycin was formed by the action of IdmH using a Diels-Alder reaction. Here, the crystal structure of a selenomethionine-labelled truncated form of IdmH (IdmH-?99-107) was solved using single-wavelength anomalous dispersion (SAD) phasing. This truncated variant allows consistent and easy crystallization, but importantly the structure was used as a search model in molecular replacement, allowing the full-length IdmH structure to be determined to 2.7?Å resolution. IdmH is a homodimer, with the individual protomers consisting of an ?+? barrel. Each protomer contains a deep hydrophobic pocket which is proposed to constitute the active site of the enzyme. To investigate the reaction catalysed by IdmH, 88% of the backbone NMR resonances were assigned, and using chemical shift perturbation of [15N]-labelled IdmH it was demonstrated that indanomycin binds in the active-site pocket. Finally, combined quantum mechanical/molecular mechanical (QM/MM) modelling of the IdmH reaction shows that the active site of the enzyme provides an appropriate environment to promote indane-ring formation, supporting the assignment of IdmH as the key Diels-Alderase catalysing the final step in the biosynthesis of indanomycin through a similar mechanism to other recently characterized Diels-Alderases involved in polyketide-tailoring reactions. An animated Interactive 3D Complement (I3DC) is available in Proteopedia at https://proteopedia.org/w/Journal:IUCrJ:S2052252519012399.

SUBMITTER: Drulyte I

PROVIDER: S-EPMC6830212 | biostudies-literature | 2019 Nov

REPOSITORIES: biostudies-literature

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Crystal structure of the putative cyclase IdmH from the indanomycin nonribosomal peptide synthase/polyketide synthase.

Drulyte Ieva I Obajdin Jana J Trinh Chi H CH Kalverda Arnout P AP van der Kamp Marc W MW Hemsworth Glyn R GR Berry Alan A

IUCrJ 20191024 Pt 6

Indanomycin is biosynthesized by a hybrid nonribosomal peptide synthase/polyketide synthase (NRPS/PKS) followed by a number of 'tailoring' steps to form the two ring systems that are present in the mature product. It had previously been hypothesized that the indane ring of indanomycin was formed by the action of IdmH using a Diels-Alder reaction. Here, the crystal structure of a selenomethionine-labelled truncated form of IdmH (IdmH-Δ99-107) was solved using single-wavelength anomalous dispersio ...[more]

PMID: 31709067

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Project description:BackgroundPyridoxal 5'-phosphate (PLP)-dependent enzymes of fold type I, the most studied structural class of the PLP-dependent enzyme superfamily, are known to exist as stand-alone homodimers or homotetramers. These enzymes have been found also embedded in multimodular and multidomain assembly lines involved in the biosynthesis of polyketides (PKS) and nonribosomal peptides (NRPS). The aim of this work is to provide a proteome-wide view of the distribution and characteristics of type I domains covalently integrated in these assemblies in prokaryotes.ResultsAn ad-hoc Hidden Markov profile was calculated using a sequence alignment derived from a multiple structural superposition of distantly related PLP-enzymes of fold type I. The profile was utilized to scan the sequence databank and to collect the proteins containing at least one type I domain linked to a component of an assembly line in bacterial genomes. The domains adjacent to a carrier protein were further investigated. Phylogenetic analysis suggested the presence of four PLP-dependent families: Aminotran_3, Beta_elim_lyase and Pyridoxal_deC, occurring mainly within mixed NRPS/PKS clusters, and Aminotran_1_2 found mainly in PKS clusters. Sequence similarity to the reference PLP enzymes with solved structures ranged from 24 to 42% identity. Homology models were built for each representative type I domain and molecular docking simulations with putative substrates were carried out. Prediction of the protein-protein interaction sites evidenced that the surface regions of the type I domains embedded within multienzyme assemblies were different from those of the self-standing enzymes; these structural features appear to be required for productive interactions with the adjacent domains in a multidomain context.ConclusionsThis work provides a systematic view of the occurrence of type I domain within NRPS and PKS assembly lines and it predicts their structural characteristics using computational methods. Comparison with the corresponding stand-alone enzymes highlighted the common and different traits related to various aspects of their structure-function relationship. Therefore, the results of this work, on one hand contribute to the understanding of the functional and structural diversity of the PLP-dependent type I enzymes and, on the other, pave the way to further studies aimed at their applications in combinatorial biosynthesis.

Dataset Information

Crystal structure of the putative cyclase IdmH from the indanomycin nonribosomal peptide synthase/polyketide synthase.

Publications

Crystal structure of the putative cyclase IdmH from the indanomycin nonribosomal peptide synthase/polyketide synthase.

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