NDLI: <i>In vitro</i> reconstitution of α-pyrone ring formation in myxopyronin biosynthesis

Content Provider	Royal Society of Chemistry (RSC)
Author	Sahner, J. H. Hartmann, R. W. Koehnke, J. Prusov, E. Müller, R. Sucipto, H. Wenzel, S. C.
Copyright Year	2015
Abstract	Myxopyronins are α-pyrone antibiotics produced by the terrestrial bacterium Myxococcus fulvus Mx f50 and possess antibacterial activity against Gram-positive and Gram-negative pathogens. They target the bacterial RNA polymerase (RNAP) “switch region” as non-competitive inhibitors and display no cross-resistance to the established RNAP inhibitor rifampicin. Recent analysis of the myxopyronin biosynthetic pathway led to the hypothesis that this secondary metabolite is produced from two separate polyketide parts, which are condensed by the stand-alone ketosynthase MxnB. Using in vitro assays we show that MxnB catalyzes a unique condensation reaction forming the α-pyrone ring of myxopyronins from two activated acyl chains in form of their β-keto intermediates. MxnB is able to accept thioester substrates coupled to either N-acetylcysteamine (NAC) or a specific carrier protein (CP). The turnover rate of MxnB for substrates bound to CP was 12-fold higher than for NAC substrates, demonstrating the importance of protein–protein interactions in polyketide synthase (PKS) systems. The crystal structure of MxnB reveals the enzyme to be an unusual member of the ketosynthase group capable of binding and condensing two long alkyl chains bound to carrier proteins. The geometry of the two binding tunnels supports the biochemical data and allows us to propose an order of reaction, which is supported by the identification of novel myxopyronin congeners in the extract of the producer strain. Insights into the mechanism of this unique condensation reaction do not only expand our knowledge regarding the thiolase enzyme family but also opens up opportunities for PKS bioengineering to achieve directed structural modifications.
Starting Page	5076
Ending Page	5085
Page Count	10
File Format	HTM / HTML PDF
ISSN	20416520
Volume Number	6
Issue Number	8
Journal	Chemical Science
DOI	10.1039/c5sc01013f
Language	English
Publisher	Royal Society of Chemistry
Access Restriction	Subscribed
Subject Keyword	Condensation reaction DNA polymerase Secondary metabolite Enzyme Myxococcus Thiolase Gram-positive bacteria Enzyme inhibitor RNAP Protein Molecular geometry Polyketide Polyketide synthase NAC Rifampicin Thioester Alkane PKS Gram-negative bacteria Catalysis Biological engineering
Content Type	Text
Resource Type	Article
Subject	Chemistry

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