Dataset Information

Analysis of intact and dissected fungal polyketide synthase-nonribosomal peptide synthetase in vitro and in Saccharomyces cerevisiae.

ABSTRACT: The widely found fungal iterative PKS-NRPS hybrid megasynthetases are highly programmed biosynthetic machines involved in the synthesis of 3-acyltetramic acids and related natural products. In vitro analysis of iterative PKS-NRPS has been hampered by the difficulties associated with obtaining pure and functional forms of these large enzymes (>400 kDa). We successfully expressed Aspergillus nidulans aspyridone synthetase (ApdA) from an engineered Saccharomyces cerevisiae strain. The complete functions of ApdA and its enoylreductase partner ApdC are reconstituted in vitro and in S. cerevisiae with the production of preaspyridone 7. The programming rules of both the PKS and NRPS modules were then examined in vitro. The key interaction between the PKS and the NRPS was dissected and reconstituted in trans by using stand-alone modules. Analogs of 7 were synthesized through heterologous combinations of PKS and NRPS modules from different sources. Our results represent one of the largest, multidomain enzyme reconstituted to date and offer new opportunities for engineered biosynthesis of fungal natural products.

SUBMITTER: Xu W

PROVIDER: S-EPMC2950873 | biostudies-literature | 2010 Oct

REPOSITORIES: biostudies-literature

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Analysis of intact and dissected fungal polyketide synthase-nonribosomal peptide synthetase in vitro and in Saccharomyces cerevisiae.

Xu Wei W Cai Xiaolu X Jung Michael E ME Tang Yi Y

Journal of the American Chemical Society 20101001 39

The widely found fungal iterative PKS-NRPS hybrid megasynthetases are highly programmed biosynthetic machines involved in the synthesis of 3-acyltetramic acids and related natural products. In vitro analysis of iterative PKS-NRPS has been hampered by the difficulties associated with obtaining pure and functional forms of these large enzymes (>400 kDa). We successfully expressed Aspergillus nidulans aspyridone synthetase (ApdA) from an engineered Saccharomyces cerevisiae strain. The complete func ...[more]

PMID: 20828130

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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

Analysis of intact and dissected fungal polyketide synthase-nonribosomal peptide synthetase in vitro and in Saccharomyces cerevisiae.

Publications

Analysis of intact and dissected fungal polyketide synthase-nonribosomal peptide synthetase in vitro and in Saccharomyces cerevisiae.

Similar Datasets

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