Identification of the dioxygenase-generated intermediate formed during biosynthesis of the dihydropyrrole moiety common to anthramycin and sibiromycin

Saha, Shalini; Li, Wei; Gerratana, Barbara; Rokita, Steven E.

doi:10.1016/j.bmc.2014.12.024

Cited by 18 publications

(24 citation statements)

References 49 publications

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“…Successful activation of SibD AT also provided a platform to test which biosynthetic intermediate derived from l ‐Tyr might be loaded onto the NRPS. l ‐DOPA, the first intermediate of this process, was available commercially and a later intermediate, 1 , was prepared enzymatically, as described previously (Scheme ). A final intermediate in dihydropyrrole biosynthesis, 2 b , was assembled by using a key allyl‐coupling step (Scheme S1).…”

Section: Methodsmentioning

confidence: 99%

An Activator of an Adenylation Domain Revealed by Activity but Not Sequence Homology

Saha

Rokita

2016

ChemBioChem

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Nonribosomal peptide synthetases (NRPSs), responsible for synthesizing many medicinally important natural products, frequently use adenylation domain activators (ADAs) to promote substrate loading. Although ADAs are usually MbtH-like proteins (MLPs), a new type of ADA appears to promote an NRPS-dependent incorporation of a dihydropyrrole unit into sibiromycin. The adenylation and thiolation didomain of the NRPS SibD catalyzes the adenylation of a limited number of amino acids including L-tyr, the precursor in dihydropyrrole biosynthesis, using a standard radioactivity exchange assay. LC-MS/MS analysis confirmed loading of L-tyr onto the thiolation domain. SibB, a small protein with no prior functional assignment nor sequence homology to MLPs, was found to promote the exchange activity. MLPs from bacteria expressing homologous biosynthetic pathways were unable to replace this function of SibB. Discovery of this new type of ADA demonstrates the importance of searching beyond the conventional MLP standard for proteins affecting NRPS activity. Graphical AbstractNonribosomal peptide synthetases (NRPSs) frequently use MbtH-like proteins to promote activity of their adenylation domains. Sibiromycin biosynthesis provides an example of a pathway that lacks such a protein and instead uses an atypical adenylation domain activator, SibB, to promote the activity of the NRPS SibD.Correspondence to: Shalini Saha; Steven E. Rokita. Experimental SectionAll experimental details including the construction of expression vectors, production and purification of proteins, synthesis and characterization of all compounds, and assays for radioisotope exchange and NRPS acylation are described in supporting information. Elongation modules include an additional condensation (C) domain for coupling two NRPSconjugates through a peptide bond. Termination modules contain yet another domain most typically a thioesterase (TE) domain or infrequently a reductase (R) domain that promotes hydrolysis of the nascent peptide from the NRPS. These modules often are complemented by a protein known as an A domain activator (ADA). [2] ADAs are small proteins that stimulate A domain activity and were first found necessary for successful biosynthesis of capreomycin and viomycin. [3] In a few cases, NRPSs were not even observed or isolated in a soluble form after heterologous expression without coexpression of an appropriate ADA. [4] The first ADAs reported were all MbtH-like proteins (MLPs) named after a homolog (MbtH) in Mycobacterium tuberculosis and identifiable by a Pfam domain (PF03621). [5] More recently, two new ADAs have been discovered that are devoid of the MLP signature sequences. One of these resembles an incomplete C domain and the other shares very low sequence similarity to known NRPSs (E > 0.002). [6] Both of these ADAs as well as one example of an MLP are covalently fused to their A domains. [6][7] In contrast, the vast majority of ADAs exist as independent proteins encoded by genes within their associated biosynthetic gene cluster...

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Section: Methodsmentioning

confidence: 99%

An Activator of an Adenylation Domain Revealed by Activity but Not Sequence Homology

Saha

Rokita

2016

ChemBioChem

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“…The addition of L-DOPA to the biocatalytic system promptly results in the appearing of a broad absorption band with a maximum at 414 nm and shoulders at around 380 nm and 450 nm, which have been attributed, respectively, to 2,3-and 4,5-seco-DOPAs (Saha et al, 2015) and to betaxanthins (Fig. 3a).…”

Section: Determination Of Catalytic Constantsmentioning

confidence: 99%

“…3a). The enzyme activity measured via absorption spectroscopy refers to the overall conversion of L-DOPA into several products because the band centered at 414 nm may refer to betalamic acid, muscaflavin, OBDC (Saha et al, 2015), and any other substance showing high molar absorption coefficient at this wavelength.…”

Section: Determination Of Catalytic Constantsmentioning

confidence: 99%

“…L-4-(2-oxo-3-butenoic-acid)-4,5-dihydropyrrole-2-carboxylic-acid (OBDC) (Barth et al, 1979;Saha et al, 2015), on the other hand, is the key intermediate for the biosynthesis of antitumor pyrrolo [1,4]benzodiazepines, the bacterial hormone hormaomycin, and the lincosamide antibiotic lincomycin (Jiraskova et al, 2016). Betalamic acid, muscaflavin and OBDC show absorption maxima at 414 nm at pH 8 (Saha et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

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L-DOPA dioxygenase of the fly agaric toadstool: revision of thedodAgene sequence and mechanism of enzymatic pigment production

Dmm

Gonçalves²,

Co³

et al. 2020

Preprint

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L-DOPA extradiol dioxygenases (DODAs) catalyze the production of betalains and hygroaurins pigments. The sequence of the DODAs found in Caryophyllales and Basidiomycetes are not conserved, although betalains are produced both by plants and fungi. Here we revise the coding region of the dodA gene of fly agaric [Amanita muscaria (L.) Lam.] and describe an alternative start codon downstream that enables the heterologous expression of AmDODA, a promiscuous L-DOPA dioxygenase. AmDODA is 43-amino acid residues shorter than the recombinant DODA previously reported but catalyzes the formation of two isomeric seco-DOPAs that are the biosynthetic precursors of betalains and hygroaurins. The putative active site of AmDODA contains two distinct His-His-Glu motifs that can explain the dual cleavage of L-DOPA according to the mechanism proposed for non-heme iron-dependent dioxygenases. Upon addition of excess L-DOPA, both the betaxanthin and hygroaurin adducts of L-DOPA are produced. The kinetic parameters of enzymatic catalysis at pH 8.5 are similar to those reported for other L-DOPA dioxygenases. The rate constants for the conversion of L-DOPA into the betalamic acid and muscaflavin were estimated by kinetic modelling allowing the proposal of a mechanism of pigment formation. These results contribute to understanding the biosynthesis of bacterial, fungal and plant pigments, for the biotechnological production of hygroaurins, and for the development of more promiscuous dioxygenases for environmental remediation.

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“…L-DOPA subsequently underwent an extradiol cleavage by Apd2 L-DOPA-2,3-dioxygenase, resulting in intermediate 1, which was immediately subjected to a spontaneous intramolecular cyclization to form the yellow-coloured heterocyclic compound 3 (LmbB1, 30,96,98 Orf12, 97,99 SibV, 99 ). Apd1 belongs to a single-domain type I extradiol dioxygenase of the vicinal oxygen chelate superfamily of enzymes, which use non-heme Fe(II) to bind and activate molecular oxygen for the subsequent insertion of two oxygen atoms via cleavage of the aromatic ring of L-DOPA.…”

Section: Current Knowledge On Apd Biosynthesismentioning

confidence: 99%

Biosynthesis and incorporation of an alkylproline-derivative (APD) precursor into complex natural products

et al. 2018

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Covering: up to 2017This review covers the biosynthetic and evolutionary aspects of lincosamide antibiotics, antitumour pyrrolobenzodiazepines (PBDs) and the quorum-sensing molecule hormaomycin. These structurally and functionally diverse groups of complex natural products all incorporate rarely occurring 4-alkyl-l-proline derivatives (APDs) biosynthesized from l-tyrosine through an unusual specialized pathway catalysed by a common set of six proteins named Apd1-Apd6. We give an overview of APD formation, which involves unusual enzyme activities, and its incorporation, which is based either on nonribosomal peptide synthetase (PBDs, hormaomycin) or a unique hybrid ergothioneine-dependent condensation system followed by mycothiol-dependent sulphur atom incorporation (lincosamides). Furthermore, within the public databases, we identified 36 novel unannotated biosynthetic gene clusters that putatively encode the biosynthesis of APD compounds. Their products presumably include novel PBDs, but also novel classes of APD compounds, indicating an unprecedented potential for the diversity enhancement of these functionally versatile complex metabolites. In addition, phylogenetic analysis of known and novel gene clusters for the biosynthesis of APD compounds allowed us to infer novel evolutionary hypotheses: Apd3 methyltransferase originates from a duplication event in a hormaomycin biosynthetic gene cluster ancestor, while putative Apd5 isomerase is evolutionarily linked to PhzF protein from the biosynthesis of phenazines. Lastly, we summarize the achievements in preparing hybrid APD compounds by directing their biosynthesis, and we propose that the number of nature-like APD compounds could by multiplied by replacing l-proline residues in various groups of complex metabolites with APD, i.e. by imitating the natural process that occurs with lincosamides and PBDs, in which the replacement of l-proline for APD has proved to be an evolutionary successful concept.

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Identification of the dioxygenase-generated intermediate formed during biosynthesis of the dihydropyrrole moiety common to anthramycin and sibiromycin

Cited by 18 publications

References 49 publications

An Activator of an Adenylation Domain Revealed by Activity but Not Sequence Homology

An Activator of an Adenylation Domain Revealed by Activity but Not Sequence Homology

L-DOPA dioxygenase of the fly agaric toadstool: revision of thedodAgene sequence and mechanism of enzymatic pigment production

Biosynthesis and incorporation of an alkylproline-derivative (APD) precursor into complex natural products

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