Domain Organization and Active Site Architecture of a Polyketide Synthase <i>C</i>-methyltransferase

Skiba, Meredith A.; Sikkema, Andrew P.; Fiers, William D.; Gerwick, William H.; Sherman, David H.; Aldrich, Courtney C.; Smith, Janet L.

doi:10.1021/acschembio.6b00759

Cited by 46 publications

(87 citation statements)

References 49 publications

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“…In connection with these considerations, while this manuscript was under review, a structure of an excised CMeT from a modular PKS was determined and closely agrees with the present data on a comparable domain from an iterative PKS (Skiba et al, 2016). …”

Section: Discussionsupporting

confidence: 78%

Functional and Structural Analysis of Programmed C-Methylation in the Biosynthesis of the Fungal Polyketide Citrinin

Storm

Herbst

Maier

et al. 2017

Cell Chemical Biology

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Summary Fungal polyketide synthases (PKSs) are large, multi-domain enzymes that biosynthesize a wide range of natural products. A hallmark of these megasynthases is the iterative use of catalytic domains to extend and modify a series of enzyme-bound intermediates. A subset of these iterative PKSs (iPKSs) contains a C-methyltransferase (CMeT) domain that adds one or more S-adenosylmethionine (SAM)-derived methyl groups to the carbon framework. Neither the basis by which only specific positions on the growing intermediate are methylated (“programming”) nor the mechanism of methylation are well understood. Domain dissection and reconstitution of PksCT, the fungal non-reducing PKS (NR-PKS) responsible for the first isolable intermediate in citrinin biosynthesis, demonstrates the role of CMeT-catalyzed methylation in precursor elongation and pentaketide formation. The crystal structure of the S-adenosyl-homocysteine (SAH) coproduct-bound PksCT CMeT domain reveals a two-subdomain organization with a novel N-terminal subdomain characteristic of PKS C-methyltransferase domains and provides insights into cofactor and ligand recognition.

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Section: Discussionsupporting

confidence: 78%

Functional and Structural Analysis of Programmed C-Methylation in the Biosynthesis of the Fungal Polyketide Citrinin

Storm

Herbst

Maier

et al. 2017

Cell Chemical Biology

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“…Of the several hundred MT structures in the structure database, AprA MT1 most closely resembles the MTs from PKS extension steps 28, 29 . All class I SAM-dependent MTs are thought to have a common ancestor, but the superfamily has several highly diverged branches.…”

Section: Resultsmentioning

confidence: 99%

“…The lid domains are topologically similar at the N-termini, but the AprA MT1 lid (formerly known as AR) is much larger due to a C-terminal extension. We previously showed that conserved His and Glu amino acids are essential to methyl transfer by the CurJ extension-module MT, and proposed that the His imidazole is the catalytic base that accepts a proton from the α-carbon of the β-ketoacyl-ACP substrate 28 . By structure superposition, the critical His and Glu of extension-module MTs correspond to AprA MT1 His369 and Glu431, which are also conserved in GNAT loading modules (Supplementary Figure 4d, 4e).…”

Section: Resultsmentioning

confidence: 99%

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A Mononuclear Iron-Dependent Methyltransferase Catalyzes Initial Steps in Assembly of the Apratoxin A Polyketide Starter Unit

et al. 2017

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Natural product biosynthetic pathways contain a plethora of enzymatic tools to carry out difficult biosynthetic transformations. Here we discover an unusual mononuclear iron-dependent methyltransferase that acts in the initiation steps of apratoxin A biosynthesis (AprA MT1). Fe3+-replete AprA MT1 catalyzes one or two methyl transfer reactions on the substrate malonyl-ACP (acyl carrier protein), whereas Co2+, Fe2+, Mn2+ and Ni2+ support only a single methyl transfer. MT1 homologs exist within the “GNAT” (GCN5-related N-acetyltransferase) loading modules of several modular biosynthetic pathways with propionyl, isobutyryl or pivaloyl starter units. GNAT domains are thought to catalyze decarboxylation of malonyl-CoA and acetyl transfer to a carrier protein. In AprA the GNAT domain lacks both decarboxylation and acyl transfer activity. A crystal structure of the AprA MT1-GNAT didomain with bound Mn2+, malonate and the methyl donor S-adenosylmethionine (SAM) reveals that the malonyl substrate is a bidentate metal ligand, indicating that the metal acts as a Lewis acid to promote methylation of the malonyl α-carbon. The GNAT domain is truncated relative to functional homologs. These results afford an expanded understanding of MT1-GNAT structure and activity, and permit the functional annotation of homologous GNAT loading modules both with and without methyltransferases, additionally revealing their rapid evolutionary adaptation in different biosynthetic contexts.

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“…Although a number of excised PKS C -methyltranferases have been expressed as recombinant proteins and shown to catalyze the expected SAM-dependent methylation of 3-ketoacylthioesters, the stereochemistry of the resulting 2-methyl-3-ketoacyl thioester products has not been determined. 8,9 …”

mentioning

confidence: 99%

Elucidation of the Stereospecificity of C-Methyltransferases from trans-AT Polyketide Synthases

2017

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S-Adenosyl methionine (SAM)-dependent C-methyltransferases are responsible for the C2-methylation of 3-ketoacyl-acyl carrier protein (ACP) intermediates to give the corresponding 2-methy-3-ketoacyl-ACP products during bacterial polyketide biosynthesis mediated by trans-AT polyketide synthases that lack integrated acyl transferase (AT) domains. A coupled ketoreductase (KR) assay was used to assign the stereochemistry of the C-methyltransferase-catalyzed reaction. Samples of chemoenzymatically-generated 3-ketopentanoyl-ACP (9) were incubated with SAM and BonMT2 from module 2 of the bongkrekic acid polyketide synthase. The resulting 2-methyl-3-ketopentanoyl-ACP (10) was incubated separately with five (2R)- or (2S)-methyl specific KR domains. Analysis of the derived 2-methyl-3-hydroxypentanoate methyl esters (8) by chiral GC-MS established that the BonMT2-catalyzed methylation generated exclusively (2R)-2-methyl-3-ketopentanoyl-ACP ((2R)-10). Identical results were also obtained with three additional C-methyltransferases – BaeMT9, DifMT1, and MupMT1 – from the bacillaene, difficidin, and mupirocin trans-AT polyketide synthases

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Domain Organization and Active Site Architecture of a Polyketide Synthase C-methyltransferase

Cited by 46 publications

References 49 publications

Functional and Structural Analysis of Programmed C-Methylation in the Biosynthesis of the Fungal Polyketide Citrinin

Functional and Structural Analysis of Programmed C-Methylation in the Biosynthesis of the Fungal Polyketide Citrinin

A Mononuclear Iron-Dependent Methyltransferase Catalyzes Initial Steps in Assembly of the Apratoxin A Polyketide Starter Unit

Elucidation of the Stereospecificity of C-Methyltransferases from trans-AT Polyketide Synthases

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