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Functional dissection of a multimodular polypeptide of the pikromycin polyketide synthase into monomodules by using a matched pair of heterologous docking domains.

ABSTRACT: The pikromyin polyketide synthase (PKS) in Streptomyces venezulae is comprised of a loading module and six extension modules, which generate the corresponding 14-membered macrolactone product. PikAI is a multimodular component of this PKS and houses both the loading domain and the first two extension modules, joined by short intraprotein linkers. We have shown that PikAI can be separated into two proteins at either of these linkers, only when matched pairs of docking domains (DDs) from a heterologous modular phoslactomycin PKS are used in place of the intraprotein linker. In both cases the yields of pikromycin produced by the S. venezuelae mutant were 50% of that of a S. venezuelae strain expressing the native trimodular PikAI. This observation provides the first demonstration that such separations do not dramatically impact the efficiency of the entire in vivo biosynthetic process. Expression of module 2 as a monomodular protein fused to a heterologous N-terminal docking domain was also observed to give almost a tenfold improvement in the in vivo generation of pikromycin from a synthetic diketide intermediate. These results demonstrate the utility of DDs to manipulate biosynthetic processes catalyzed by modular PKSs and the quest to generate novel polyketide products.

SUBMITTER: Yan J

PROVIDER: S-EPMC4652847 | biostudies-literature | 2009 Jun

REPOSITORIES: biostudies-literature

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Functional dissection of a multimodular polypeptide of the pikromycin polyketide synthase into monomodules by using a matched pair of heterologous docking domains.

Yan John J Gupta Shuchi S Sherman David H DH Reynolds Kevin A KA

Chembiochem : a European journal of chemical biology 20090601 9

The pikromyin polyketide synthase (PKS) in Streptomyces venezulae is comprised of a loading module and six extension modules, which generate the corresponding 14-membered macrolactone product. PikAI is a multimodular component of this PKS and houses both the loading domain and the first two extension modules, joined by short intraprotein linkers. We have shown that PikAI can be separated into two proteins at either of these linkers, only when matched pairs of docking domains (DDs) from a heterol ...[more]

PMID: 19437523

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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

Functional dissection of a multimodular polypeptide of the pikromycin polyketide synthase into monomodules by using a matched pair of heterologous docking domains.

Publications

Functional dissection of a multimodular polypeptide of the pikromycin polyketide synthase into monomodules by using a matched pair of heterologous docking domains.

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