Repurposing the GNAT Fold in the Initiation of Polyketide Biosynthesis

Skiba, Meredith A.; Tran, Collin L.; Dan, Qingyun; Sikkema, Andrew P.; Klaver, Zachary; Gerwick, William H.; Sherman, David H.; Smith, Janet L.

doi:10.1016/j.str.2019.11.004

Cited by 13 publications

(24 citation statements)

References 67 publications

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“…Based on many studies of the chemical and kinetic mechanisms of GNAT enzymes, it is clear this superfamily has evolved to utilize a diversity of chemistries and active site residues for more complex and targeted modifications of acceptor substrates. Identification of additional non-acyl transfer reactions for GNATs, including decarboxylation, methylcarbamoyl transfer, and condensation ( Gu et al, 2007 ; Izoré et al, 2019 ; Karambelkar et al, 2020 ; Skiba et al, 2020 ) highlight the shear multitude of chemistries that enzymes from this superfamily can accomplish. Thus, GNATs appear to have a highly tunable scaffold that has evolved to modify a diverse range of substrates.…”

Section: Discussionmentioning

confidence: 99%

“…The majority of GNATs that have been functionally characterized perform N- acetylation of primary amines ( Vetting et al, 2005b ; Favrot et al, 2016 ; Burckhardt and Escalante-Semerena, 2020 ), but a few examples of O- acetylation of hydroxyl groups exist ( Daigle et al, 1999 ; Hegde et al, 2001 ). Recent studies have also shown that the GNAT fold has been repurposed by some organisms to catalyze decarboxylation instead of acyl transfer, thus highlighting the sheer diversity of reaction capabilities of members of this superfamily of proteins ( Skiba et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

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Gcn5-Related N-Acetyltransferases (GNATs) With a Catalytic Serine Residue Can Play Ping-Pong Too

Baumgartner

Mohammad

Czub

et al. 2021

Front. Mol. Biosci.

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Enzymes in the Gcn5-related N-acetyltransferase (GNAT) superfamily are widespread and critically involved in multiple cellular processes ranging from antibiotic resistance to histone modification. While acetyl transfer is the most widely catalyzed reaction, recent studies have revealed that these enzymes are also capable of performing succinylation, condensation, decarboxylation, and methylcarbamoylation reactions. The canonical chemical mechanism attributed to GNATs is a general acid/base mechanism; however, mounting evidence has cast doubt on the applicability of this mechanism to all GNATs. This study shows that the Pseudomonas aeruginosa PA3944 enzyme uses a nucleophilic serine residue and a hybrid ping-pong mechanism for catalysis instead of a general acid/base mechanism. To simplify this enzyme’s kinetic characterization, we synthesized a polymyxin B substrate analog and performed molecular docking experiments. We performed site-directed mutagenesis of key active site residues (S148 and E102) and determined the structure of the E102A mutant. We found that the serine residue is essential for catalysis toward the synthetic substrate analog and polymyxin B, but the glutamate residue is more likely important for substrate recognition or stabilization. Our results challenge the current paradigm of GNAT mechanisms and show that this common enzyme scaffold utilizes different active site residues to accomplish a diversity of catalytic reactions.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Gcn5-Related N-Acetyltransferases (GNATs) With a Catalytic Serine Residue Can Play Ping-Pong Too

Baumgartner

Mohammad

Czub

et al. 2021

Front. Mol. Biosci.

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“…In cis -AT modular PKSs, it is catalyzed by the N -terminal KS Q domain of PKSs 34 – 37 (Fig. 5a ), while in trans -AT modular PKSs, it is performed by a GCN5-related N -acetyltransferase (GNAT)-like domain 38 , 39 (Fig. 5b ).…”

Section: Discussionmentioning

confidence: 99%

Initiating polyketide biosynthesis by on-line methyl esterification

Chen

Tang

et al. 2021

Nat Commun

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Aurantinins (ARTs) are antibacterial polyketides featuring a unique 6/7/8/5-fused tetracyclic ring system and a triene side chain with a carboxyl terminus. Here we identify the art gene cluster and dissect ART’s C-methyl incorporation patterns to study its biosynthesis. During this process, an apparently redundant methyltransferase Art28 was characterized as a malonyl-acyl carrier protein O-methyltransferase, which represents an unusual on-line methyl esterification initiation strategy for polyketide biosynthesis. The methyl ester bond introduced by Art28 is kept until the last step of ART biosynthesis, in which it is hydrolyzed by Art9 to convert inactive ART 9B to active ART B. The cryptic reactions catalyzed by Art28 and Art9 represent a protecting group biosynthetic logic to render the ART carboxyl terminus inert to unwanted side reactions and to protect producing organisms from toxic ART intermediates. Further analyses revealed a wide distribution of this initiation strategy for polyketide biosynthesis in various bacteria.

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“…Type C LM has a CoA-ligase domain rather than AT domain to incorporate carboxylic acid substrate in an ATP-dependent manner. Lastly, type D LM has a GCN5 N-acetyltransferaselike domain rather than AT domain that recently repurposed to catalyze decarboxylation (Skiba et al, 2020). A common characteristic of LMs is the absence of the condensation domain, which results a more flexible substrate specificity of the AT domain than those of downstream modules.…”

Section: Modular Pks and Nrps Architecture And Mechanismmentioning

confidence: 99%

Repurposing Modular Polyketide Synthases and Non-ribosomal Peptide Synthetases for Novel Chemical Biosynthesis

Hwang

Lee

Cho

et al. 2020

Front. Mol. Biosci.

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In nature, various enzymes govern diverse biochemical reactions through their specific three-dimensional structures, which have been harnessed to produce many useful bioactive compounds including clinical agents and commodity chemicals. Polyketide synthases (PKSs) and non-ribosomal peptide synthetases (NRPSs) are particularly unique multifunctional enzymes that display modular organization. Individual modules incorporate their own specific substrates and collaborate to assemble complex polyketides or non-ribosomal polypeptides in a linear fashion. Due to the modular properties of PKSs and NRPSs, they have been attractive rational engineering targets for novel chemical production through the predictable modification of each moiety of the complex chemical through engineering of the cognate module. Thus, individual reactions of each module could be separated as a retro-biosynthetic biopart and repurposed to new biosynthetic pathways for the production of biofuels or commodity chemicals. Despite these potentials, repurposing attempts have often failed owing to impaired catalytic activity or the production of unintended products due to incompatible protein-protein interactions between the modules and structural perturbation of the enzyme. Recent advances in the structural, computational, and synthetic tools provide more opportunities for successful repurposing. In this review, we focused on the representative strategies and examples for the repurposing of modular PKSs and NRPSs, along with their advantages and current limitations. Thereafter, synthetic biology tools and perspectives were suggested for potential further advancement, including the rational and large-scale high-throughput approaches. Ultimately, the potential diverse reactions from modular PKSs and NRPSs would be leveraged to expand the reservoir of useful chemicals.

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Repurposing the GNAT Fold in the Initiation of Polyketide Biosynthesis

Abstract: Highlights d GNAT superfamily catalyzes decarboxylation in addition to acyltransfer d A GNAT-like enzyme initiates biosynthesis of some polyketide natural products d Polyketide synthase GNAT-like enzymes catalyze only decarboxylation

Cited by 13 publications

References 67 publications

Gcn5-Related N-Acetyltransferases (GNATs) With a Catalytic Serine Residue Can Play Ping-Pong Too

Gcn5-Related N-Acetyltransferases (GNATs) With a Catalytic Serine Residue Can Play Ping-Pong Too

Initiating polyketide biosynthesis by on-line methyl esterification

Repurposing Modular Polyketide Synthases and Non-ribosomal Peptide Synthetases for Novel Chemical Biosynthesis

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