The Cyanobactin Heterocyclase Enzyme: A Processive Adenylase That Operates with a Defined Order of Reaction

Koehnke, Jesko; Bent, Andrew F.; Zollman, David; Smith, Kieran; Houssen, Wael E.; Zhu, Xiaofeng; Mann, Greg; Lébl, Tomáš; Scharff, Richard; Shirran, Sally L.; Botting, Catherine H.; Jaspars, Marcel; Schwarz‐Linek, Ulrich; Naismith, James H.

doi:10.1002/anie.201306302

Cited by 96 publications

(169 citation statements)

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“…Although the TOMM YcaO domain was first implicated in cyclodehydration reactions in the mid-1990s 6 , its exact role remains unclear 7,8 . The function of the TOMM YcaO (D protein) is intimately linked to members of the E1 ubiquitin-activating enzyme family (C protein) found in canonical TOMM biosynthetic clusters 6,9,10 .…”

mentioning

confidence: 99%

Discovery of a new ATP-binding motif involved in peptidic azoline biosynthesis

et al. 2014

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Despite intensive research, the cyclodehydratase responsible for azoline biogenesis in thiazole/oxazole-modified microcin (TOMM) natural products remains enigmatic. The collaboration of two proteins, C and D, is required for cyclodehydration. The C protein is homologous to E1 ubiquitin-activating enzymes, while the D protein is within the YcaO superfamily. Recent studies have demonstrated that TOMM YcaOs phosphorylate amide carbonyl oxygens to facilitate azoline formation. Here we report the X-ray crystal structure of an uncharacterized YcaO from Escherichia coli (Ec-YcaO). Ec-YcaO harbors an unprecedented fold and ATP-binding motif. This motif is conserved among TOMM YcaOs and is required for cyclodehydration. Furthermore, we demonstrate that the C protein regulates substrate binding and catalysis and that the proline-rich C-terminus of the D protein is involved in C protein recognition and catalysis. This study identifies the YcaO active site and paves the way for the characterization of the numerous YcaO domains not associated with TOMM biosynthesis.

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confidence: 99%

Discovery of a new ATP-binding motif involved in peptidic azoline biosynthesis

et al. 2014

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“…YcaO domain proteins activate backbone amide bonds by phosphorylation [26,27] or adenylation [28] of the carbonyl oxygen, and all YcaO domain proteins with ac haracterized activity have ap artner cyclodehydratase that aids catalysis of cyclization to oxazolines or thiazolines. [29] Theb ottromycin gene cluster encodes two YcaO domain proteins,B tmE and BtmF,b ut no cyclodehydratases.T herefore,w eh ypothesized that one participates in macrocyclization and the other is involved in the formation of the terminal thiazole.…”

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confidence: 99%

Dissecting Bottromycin Biosynthesis Using Comparative Untargeted Metabolomics

et al. 2016

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Bottromycin A 2 is astructurally unique ribosomally synthesized and post-translationally modified peptide (RiPP) that possesses potent antibacterial activity towards multidrugresistant bacteria. The structural noveltyofbottromycin stems from its unprecedented macrocyclic amidine and rare bmethylated amino acid residues.The N-terminus of aprecursor peptide (BtmD) is converted into bottromycin A 2 by tailoring enzymes encoded in the btm gene cluster.H owever,l ittle was knowna bout key transformations in this pathway,i ncluding the unprecedented macrocyclization. To understand the pathway in detail, an untargeted metabolomic approacht hat harnesses mass spectral networking was used to assess the metabolomes of aseries of pathway mutants.This analysis has yielded key information on the function of av ariety of previously uncharacterized biosynthetic enzymes,i ncluding aY caO domain protein and ap artner protein that together catalyze the macrocyclization.Ribosomally synthesized and post-translationally modified peptides (RiPPs) are natural products that are prevalent throughout nature, [1] and their biosynthetic pathways are capable of transforming simple proteinogenic amino acids into structurally complex compounds that have potent bioactivities. [2][3][4] However,e lucidating the biosynthesis of RiPPs can be hindered by the difficulty of isolating intermediates,asthe biosynthesis takes place on alarger precursor peptide,a nd intermediates may be rapidly proteolyzed. Therefore,i mproved methods for the identification of RiPP intermediates are desirable.Bottromycin A 2 (1,Scheme 1) [5][6][7][8] possesses potent antibacterial activity towards multidrugresistant bacteria, [9] and is structurally unique owing its unprecedented macrocyclic amidine,r are b-methylated amino acids residues,and aterminal thiazole.Nature employs av ariety of strategies for peptide macrocyclization, [10][11][12] but amidine formation has only been observed for bottromycin. Initial studies on bottromycin biosynthesis showed that its amino acids were b-methylated by radical SAM methyltransferases [5,7] (RSMTs), but the rest of the bottromycin pathway represented abiosynthetic black box, where little was known about key steps in the pathway,including the unprecedented macrocyclization. In this study,weemploy untargeted metabolomics and mass spectral networking to deduce the biosynthetic route to bottromycins in Streptomyces scabies.T his analysis identifies the enzymes responsible for macrocyclization, thiazole formation, and aspartate epimerization, thereby demonstrating the utility of an untargeted metabolomic approach for elucidating atargeted biosynthetic pathway.To assess the role of the putative tailoring genes in the bottromycin pathway (Supporting Information, Figure S1), we had previously generated S. scabies DbtmC, DbtmE, DbtmF, DbtmI,a nd DbtmJ,b ut were unable to identify bottromycin-like compounds in these mutants. [5] We therefore established that these deletions did not lead to deleterious polar effects on the pathway b...

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“…Since discovery of the first cyanobactin gene cluster (Schmidt et al, 2005), a series of biochemical studies characterized representatives of the most ubiquitous cyanobactin enzymes, including heterocyclase (TruD and homologs) (McIntosh et al, 2010a; McIntosh and Schmidt, 2010; Koehnke et al, 2013; Sardar et al, 2015b), the N-terminal and the C-terminal protease/macrocyclase (TruA and TruG homologs) (Lee et al, 2009; McIntosh et al, 2010b; Houssen et al, 2012; Koehnke et al, 2012; Agarwal et al, 2012) and the prenyltransferase (TruF1 and homologs) (McIntosh et al, 2011; Sardar et al, 2015a). An elegant alternative in vitro approach to probing enzyme activity was recently reported by Goto et al, using a cell-free translation system.…”

Section: Synthesis Of Cyanobactins In Vitromentioning

confidence: 99%

Directing Biosynthesis

Sardar¹,

Tianero²,

Schmidt³

2016

Methods in Enzymology

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The increasingly rapid accumulation of genomic information is revolutionizing natural products discovery. However, the translation of sequence data to chemical products remains a challenge. Here, we detail methods used to circumvent the supply problem of cyanobactin natural products, both by engineered synthesis in E. coli and by using purified enzymes in vitro. Such methodologies exploit nature’s strategies of combinatorial chemistry in the cyanobactin class of RiPP natural products. As a result, it is possible to synthesize a wide variety of natural and unnatural compounds.

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The Cyanobactin Heterocyclase Enzyme: A Processive Adenylase That Operates with a Defined Order of Reaction

Cited by 96 publications

References 17 publications

Discovery of a new ATP-binding motif involved in peptidic azoline biosynthesis

Discovery of a new ATP-binding motif involved in peptidic azoline biosynthesis

Dissecting Bottromycin Biosynthesis Using Comparative Untargeted Metabolomics

Directing Biosynthesis

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