In vitro reconstitution of α-pyrone ring formation in myxopyronin biosynthesis

Sucipto, Hilda; Sahner, J. Henning; Prusov, Evgeny V.; Wenzel, Silke C.; Hartmann, Rolf W.; Koehnke, Jesko; Müller, Rolf

doi:10.1039/c5sc01013f

Cited by 47 publications

(52 citation statements)

References 35 publications

(59 reference statements)

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“…In particular, some of the ketosynthases involved in bacterial pyrone biosynthesis create an ␣-pyrone ring with large orthogonal substituents, including the antibiotics myxopyronin, corallopyronin, and pseudopyronin (17,24,25). The orthogonal nature of the substituents matches well with the orthogonal alkyl channels observed in OleA, as was noted in the recent crystal structure of Myxococcus fulvus Mx f50 MxnB, the stand-alone ketosynthase that completes myxopyronin biosynthesis (26). The alkyl chains in both OleA alkyl channels A and B are not linear but curled, explaining how different lengths of alkyl chain can be accommodated in the active site and why X. campestris OleA is promiscuous (3).…”

Section: Discussionsupporting

confidence: 52%

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Substrate Trapping in Crystals of the Thiolase OleA Identifies Three Channels That Enable Long Chain Olefin Biosynthesis

Goblirsch

Jensen

Mohamed

et al. 2016

Journal of Biological Chemistry

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Edited by Norma AllewellPhylogenetically diverse microbes that produce long chain, olefinic hydrocarbons have received much attention as possible sources of renewable energy biocatalysts. One enzyme that is critical for this process is OleA, a thiolase superfamily enzyme that condenses two fatty acyl-CoA substrates to produce a ␤-ketoacid product and initiates the biosynthesis of long chain olefins in bacteria. Thiolases typically utilize a ping-pong mechanism centered on an active site cysteine residue. Reaction with the first substrate produces a covalent cysteine-thioester tethered acyl group that is transferred to the second substrate through formation of a carbon-carbon bond. Although the basics of thiolase chemistry are precedented, the mechanism by which OleA accommodates two substrates with extended carbon chains and a coenzyme moiety-unusual for a thiolaseare unknown. Gaining insights into this process could enable manipulation of the system for large scale olefin production with hydrocarbon chains lengths equivalent to those of fossil fuels. In this study, mutagenesis of the active site cysteine in Xanthomonas campestris OleA (Cys 143 ) enabled trapping of two catalytically relevant species in crystals. In the resulting structures, long chain alkyl groups (C 12 and C 14 ) and phosphopantetheinate define three substrate channels in a T-shaped configuration, explaining how OleA coordinates its two substrates and product. The C143A OleA co-crystal structure possesses a single bound acyl-CoA representing the Michaelis complex with the first substrate, whereas the C143S co-crystal structure contains both acyl-CoA and fatty acid, defining how a second substrate binds to the acyl-enzyme intermediate. An active site glutamate (Glu␤ 117 ) is positioned to deprotonate bound acyl-CoA and initiate carbon-carbon bond formation.

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Section: Discussionsupporting

confidence: 52%

“…2A), consistent with this binding the first substrate and thus the covalent acyl-enzyme intermediate prior to condensation. Interestingly, Sucipto et al (26) suggested that in MxnB the first substrate would bind in alkyl channel B based on the steric constraints required for ␣-pyrone ring formation.…”

Section: Discussionmentioning

confidence: 99%

Substrate Trapping in Crystals of the Thiolase OleA Identifies Three Channels That Enable Long Chain Olefin Biosynthesis

Goblirsch

Jensen

Mohamed

et al. 2016

Journal of Biological Chemistry

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“…One central aim is to elucidate unknown biosynthetic steps and to identify and study the corresponding biocatalysts. Important examples include the identification of enzymes responsible for cyclocondensations to yield tetramate ring systems from one thioester-activated polyketide chain (Gui et al, 2015;Sims and Schmidt, 2008), or pyrone ring systems from two chains (Sucipto et al, 2015;Zocher et al, 2015). A tandem mass spectrometrybased analysis granted insight into a double-bond shift during corallopyronin biosynthesis (Lohr et al, 2013).…”

Section: Applications Of Nac Thioestersmentioning

confidence: 99%

Biomimetic Thioesters as Probes for Enzymatic Assembly Lines: Synthesis, Applications, and Challenges

Franke

Hertweck

2016

Cell Chemical Biology

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Thioesters play essential roles in many biosynthetic pathways to fatty acids, esters, polyketides, and non-ribosomal peptides. Coenzyme A (CoA) and related phosphopantetheine thioesters are typically employed as activated acyl units for diverse C-C, C-O, and C-N coupling reactions. To study and control these enzymatic assembly lines in vitro and in vivo structurally simplified analogs such as N-acetylcysteamine (NAC) thioesters have been developed. This review gives an overview on experimental strategies enabled by synthetic NAC thioesters, such as the elucidation of complex biosynthetic pathways and enzyme mechanisms as well as precursor-directed biosynthesis and mutasynthesis. The review also summarizes synthetic protocols and protection group strategies to access these versatile synthetic tools, which are reactive and often unstable compounds. In addition, alternative phosphopantetheine thioester mimics are presented that can be used as protein tags or suicide inhibitors for protein crosslinking and off-loading probes to elucidate polyketide intermediates.

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“…Other structurally characterized thiolase superfamily enzymes, such as FabH, PKS type II, and HMG-CoA synthase condense smaller substrates and only require two (FabH and PKS type II) or one (HMG-CoA synthase) substrate binding channels, respectively [14, 16, 18, 19]. The only thiolase superfamily enzymes known to require three distinct channels are OleA and pyrone and pseudopyrone ketosynthases [17, 20]. It has been proposed that these enzymes also utilize an active site general base, a glutamate, that comes from the second subunit of the dimer to initiate Claisen condensation, but this has never been demonstrated [17, 21].…”

Section: Introductionmentioning

confidence: 99%

OleA Glu117 is key to condensation of two fatty-acyl coenzyme A substrates in long-chain olefin biosynthesis

Jensen

Goblirsch

Christenson

et al. 2017

Biochemical Journal

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In the interest of decreasing dependence on fossil fuels, microbial hydrocarbon biosynthesis pathways are being studied for renewable, tailored production of specialty chemicals and biofuels. One candidate is long-chain olefin biosynthesis, a widespread bacterial pathway that produces waxy hydrocarbons. Found in three- and four-gene clusters, oleABCD encode the enzymes necessary to produce cis-olefins that differ by alkyl chain length, degree of unsaturation, and alkyl chain branching. The first enzyme in the pathway, OleA, catalyzes the Claisen condensation of two fatty acyl-coenzyme A molecules to form a β-keto acid. In this report, the mechanistic role of Xanthomonas campestris OleA Glu117 is investigated through mutant enzymes. Crystal structures were determined for each mutant as well as their complex with the inhibitor cerulenin. Complemented by substrate modelling, these structures suggest that Glu117 aids in substrate positioning for productive carbon-carbon bond formation. Analysis of acyl-coenzyme A substrate hydrolysis shows diminished activity in all mutants. When the active site lacks an acidic residue in the 117 position, OleA cannot form condensed product, demonstrating Glu117 has a critical role upstream of the essential condensation reaction. Profiling of pH dependence shows that the apparent pKa for Glu117 is impacted by mutagenesis. Taken together, we propose that Glu117 is the general base needed to prime condensation via deprotonation of the second, non-covalently bound substrate during turnover. This is the first example of a member of the thiolase superfamily of condensing enzymes to contain an active site base originating from the second monomer of the dimer. Summary Statement OleA is the first reported thiolase enzyme whose active site general base, a glutamic acid (Glu117), is derived from a homodimeric interface. Structural and functional characterization of glutamatic acid mutants establishes its mechanistic role in initiating olefin biosynthesis.

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In vitro reconstitution of α-pyrone ring formation in myxopyronin biosynthesis

Abstract: α-Pyrone rings exist in many polyketide synthase (PKS) derived natural products. We report the first in vitro reconstitution of α-pyrone ring formation by a type I PKS using chemically synthesized substrates.

Cited by 47 publications

References 35 publications

Substrate Trapping in Crystals of the Thiolase OleA Identifies Three Channels That Enable Long Chain Olefin Biosynthesis

Substrate Trapping in Crystals of the Thiolase OleA Identifies Three Channels That Enable Long Chain Olefin Biosynthesis

Biomimetic Thioesters as Probes for Enzymatic Assembly Lines: Synthesis, Applications, and Challenges

OleA Glu117 is key to condensation of two fatty-acyl coenzyme A substrates in long-chain olefin biosynthesis

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