Localization of the ActIII actinorhodin polyketide ketoreductase to the cell wall

Xu, Xinping; Wang, Zhijun; Fan, Keqiang; Wang, Shenglan; Jia, Cui-Juan; Han, Hui; Ramalingam, Eswar; Yang, Keqian

doi:10.1111/j.1574-6968.2008.01289.x

Cited by 6 publications

(6 citation statements)

References 21 publications

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“…Also, an enzyme required for one of the late actinorhodin‐tailoring steps (ActVI‐ORF3, a dehydratase) is found specifically in the culture supernatant, in a form lacking the secretion signal peptide predicted from the gene sequence (Hesketh et al , 2002; Hesketh & Chater, 2003). Even more surprisingly, the NADPH‐dependent ActIII ketoreductase protein, whose enzymatic activity determines the nature of the ring closure during formation of the actinorhodin backbone, is covalently attached to the cell wall, despite the absence of any obvious secretion signal sequence (Xu et al , 2008b). In addition, two FAD‐dependent enzymes for biosynthesis of an uncharacterized polyketide, encoded by SCO6272 and SCO6281, are exported via the Tat system of S. coelicolor (Widdick et al , 2006).…”

Section: Some Antibiotic Biosynthetic Enzymes Are Extracellularmentioning

confidence: 99%

The complex extracellular biology ofStreptomyces

et al. 2010

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Streptomycetes, soil-dwelling mycelial bacteria that form sporulating aerial branches, have an exceptionally large number of predicted secreted proteins, including many exported via the twin-arginine transport system. Their use of noncatalytic substrate-binding proteins and hydrolytic enzymes to obtain soluble nutrients from carbohydrates such as chitin and cellulose enables them to interact with other organisms. Some of their numerous secreted proteases participate in developmentally significant extracellular cascades, regulated by inhibitors, which lead to cannibalization of the substrate mycelium biomass to support aerial growth and sporulation. They excrete many secondary metabolites, including important antibiotics. Some of these play roles in interactions with eukaryotes. Surprisingly, some antibiotic biosynthetic enzymes are extracellular. Antibiotic production is often regulated by extracellular signalling molecules, some of which also control morphological differentiation. Amphipathic proteins, assembled with the help of cellulose-like material, are required for both hyphal attachment to surfaces and aerial reproductive growth. Comparative genomic analysis suggests that the acquisition of genes for extracellular processes has played a huge part in speciation. The rare codon TTA, which is present in the key pleiotropic regulatory gene adpA and many pathway-specific regulatory genes for antibiotic production, has a particular influence on extracellular biology.

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Section: Some Antibiotic Biosynthetic Enzymes Are Extracellularmentioning

confidence: 99%

The complex extracellular biology ofStreptomyces

et al. 2010

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“…At the time of this discovery megacomplex formation was a complete mystery since trans -AT PKS polypeptides lack the N- and C-terminal docking domains that linearly organize cis -AT PKSs and no PKS architectural scaffolding proteins were known; however, ATd was hypothesized to help stabilize the observed structure (Figure 1) 18 . Membrane localization of PKS enzymes has been observed for the mycolactone and actinorhodin pathways, and the biosynthetic machinery for the siderophore pyoverdine is known to accrete into a dense, membrane-bound mass termed the “siderosome”; the co-localization of large enzymatic complexes at the bacterial plasma membrane is emerging as a theme in the biosynthesis of natural products 19–23 .…”

Section: Introductionmentioning

confidence: 99%

The LINKS motif zippers trans-acyltransferase polyketide synthase assembly lines into a biosynthetic megacomplex

Wagner

Meinke

Zogzas

et al. 2016

Journal of Structural Biology

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Polyketides such as the clinically-valuable antibacterial agent mupirocin are constructed by architecturally-sophisticated assembly lines known as trans-acyltransferase polyketide synthases. Organelle-sized megacomplexes composed of several copies of trans-acyltransferase polyketide synthase assembly lines have been observed by others through transmission electron microscopy to be located at the Bacillus subtilis plasma membrane, where the synthesis and export of the antibacterial polyketide bacillaene takes place. In this work we analyze ten crystal structures of trans-acyltransferase polyketide synthases ketosynthase domains, seven of which are reported here for the first time, to characterize a motif capable of zippering assembly lines into a megacomplex. While each of the three-helix LINKS (Laterally-INteracting Ketosynthase Sequence) motifs is observed to similarly dock with a spatially-reversed copy of itself through hydrophobic and ionic interactions, the amino acid sequences of this motif are not conserved. Such a code is appropriate for mediating homotypic contacts between assembly lines to ensure the ordered self-assembly of a noncovalent, yet tightly-knit, enzymatic network. LINKS-mediated lateral interactions would also have the effect of bolstering the vertical association of the polypeptides that comprise a polyketide synthase assembly line.

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“…It is interesting to speculate how these megadalton-sized molecular machines with their predicted homodimeric and interconnected structure are arranged within the mycobacterial cell wall. Only a handful of reports have uncovered PKS linked to the cell wall [33], [34]. In mycobacteria there is a proposed pathway linking an RND superfamily transporter protein (MmpL7) with the type I PKS, PpsE [34].…”

Section: Discussionmentioning

confidence: 99%

The Cell Wall-Associated Mycolactone Polyketide Synthases Are Necessary but Not Sufficient for Mycolactone Biosynthesis

et al. 2013

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Mycolactones are polyketide-derived lipid virulence factors made by the slow-growing human pathogen, Mycobacterium ulcerans. Three unusually large and homologous plasmid-borne genes (mlsA1: 51 kb, mlsB: 42 kb and mlsA2: 7 kb) encode the mycolactone type I polyketide synthases (PKS). The extreme size and low sequence diversity of these genes has posed significant barriers for exploration of the genetic and biochemical basis of mycolactone synthesis. Here, we have developed a truncated, more tractable 3-module version of the 18-module mycolactone PKS and we show that this engineered PKS functions as expected in the natural host M. ulcerans to produce an additional polyketide; a triketide lactone (TKL). Cell fractionation experiments indicated that this 3-module PKS and the putative accessory enzymes encoded by mup045 and mup038 associated with the mycobacterial cell wall, a finding supported by confocal microscopy. We then assessed the capacity of the faster growing, Mycobacterium marinum to harbor and express the 3-module Mls PKS and accessory enzymes encoded by mup045 and mup038. RT-PCR, immunoblotting, and cell fractionation experiments confirmed that the truncated Mls PKS multienzymes were expressed and also partitioned with the cell wall material in M. marinum. However, this heterologous host failed to produce TKL. The systematic deconstruction of the mycolactone PKS presented here suggests that the Mls multienzymes are necessary but not sufficient for mycolactone synthesis and that synthesis is likely to occur (at least in part) within the mycobacterial cell wall. This research is also the first proof-of-principle demonstration of the potential of this enzyme complex to produce tailored small molecules through genetically engineered rearrangements of the Mls modules.

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Localization of the ActIII actinorhodin polyketide ketoreductase to the cell wall

Cited by 6 publications

References 21 publications

The complex extracellular biology ofStreptomyces

The complex extracellular biology ofStreptomyces

The LINKS motif zippers trans-acyltransferase polyketide synthase assembly lines into a biosynthetic megacomplex

The Cell Wall-Associated Mycolactone Polyketide Synthases Are Necessary but Not Sufficient for Mycolactone Biosynthesis

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