Defining a kinetic mechanism for l-DOPA 2,3 dioxygenase, a single-domain type I extradiol dioxygenase from Streptomyces lincolnensis

Colabroy, Keri L.; Smith, Ian R.; Vlahos, Alexander H.S.; Markham, Androo J.; Jakubik, Matthew E.

doi:10.1016/j.bbapap.2013.12.005

Cited by 18 publications

(20 citation statements)

References 46 publications

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“…Integration of these signals indicate the presence of approximately 5% 3b . The measured extinction coefficient for 3a at pH 8.0 (ε 414nm = 50 ± 3 mM −1 cm −1 ) closely matches that reported for the LmbB1 product (ε 413nm = 48 ± 2 and ε 414nm = 45 ± 2 mM −1 cm −1 ) 36,43 and provides confirmation that Orf12 generates the same product as LmbB1. However, LmbB1 was reported earlier to form 3b .…”

Section: Resultssupporting

confidence: 83%

“…This cyclization occurs spontaneously since its rate is independent of enzyme concentration in the LmbB1-dependent production of 2 . 43 Whether downstream biosynthetic enzyme(s) prefer the acyclic compound 2 or cyclic compound 3a as substrates is not yet known.…”

Section: Resultsmentioning

confidence: 99%

“…Exposure to a basic environment slows the cyclization of 2 43 suggesting that dioxygenation reactions performed at pH > 8 may shift the equilibrium from 3a to 2 . To assess the effects of increasing basicity on the equilibrium between 2 and 3a , the dioxygenation reaction was performed over a range of pH conditions (Figures 1, S3 and S8).…”

Section: Resultsmentioning

confidence: 99%

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

Saha

Gerratana

et al. 2015

Bioorganic & Medicinal Chemistry

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A description of pyrrolo[1,4]benzodiazepine (PBD) biosynthesis is a prerequisite for engineering production of analogs with enhanced antitumor activity. Predicted dioxygenases Orf12 and SibV associated with dihydropyrrole biosynthesis in PBDs anthramycin and sibiromycin, respectively, were expressed and purified for activity studies. UV-visible spectroscopy revealed that these enzymes catalyze the regiospecific 2,3-extradiol dioxygenation of L-3,4-dihydroxyphenylalanine (L-DOPA) to form L-2,3-secodopa (λmax = 368 nm). 1H NMR spectroscopy indicates that L-2,3-secodopa cyclizes into the α-keto acid tautomer of L-4-(2-oxo-3-butenoic-acid)-4,5-dihydropyrrole-2-carboxylic acid (λmax = 414 nm). Thus, the dioxygenases are key for establishing the scaffold of the dihydropyrrole moiety. Kinetic studies suggest the dioxygenase product is relatively labile and is likely consumed rapidly by subsequent biosynthetic steps. The enzymatic product and dimeric state of these dioxygenases are conserved in dioxygenases involved in dihydropyrrole or pyrrolidine biosynthesis within both PBD and non-PBD pathways.

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Section: Resultssupporting

confidence: 83%

Section: Resultsmentioning

confidence: 99%

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

Saha

Gerratana

et al. 2015

Bioorganic & Medicinal Chemistry

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“…KinTek Explorer simulates binding reactions by direct numerical integration of coupled rate equations and fits with experimental data to yield estimates for rate constants of microscopic kinetic steps, which has been widely used to determine the kinetic mechanism of enzymatic reactions (54–60). The model described in Scheme V was used to simulate peptide association kinetics and fit with experimental data.…”

Section: Methodsmentioning

confidence: 99%

Evaluating the Role of HLA-DM in MHC Class II–Peptide Association Reactions

Yin

Maben

Becerra

et al. 2015

The Journal of Immunology

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Antigen presentation by major histocompatibility complex class II molecules (MHC II) to CD4+ T cells plays a key role in the regulation of the adaptive immune response. Loading of antigenic peptides onto MHC II is catalyzed by HLA-DM (DM), a non-classical MHC II molecule. The mechanism of DM-facilitated peptide loading is an outstanding problem in the field of antigen presentation. In this study we systemically explored possible kinetic mechanisms for DM-catalyzed peptide association, by measuring real time peptide association kinetics using fluorescence polarization assays and comparing the experimental data with numerically modeled peptide association reactions. We found that DM does not facilitate peptide association by stabilizing peptide-free MHC II against aggregation. Moreover, DM does not promote transition of an inactive peptide-averse conformation of MHC II to an active peptide-receptive conformation. Instead, DM forms an intermediate with MHC II that binds peptide with faster kinetics than MHC II in the absence of DM. In the absence of peptides, interaction of MHC II with DM leads to inactivation and formation of a peptide-averse form. This study provides novel insights into how DM efficiently catalyzes peptide loading during antigen presentation.

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“…Mechanistically similar enzymes that utilize substrates in which one of ortho -hydroxyl groups is replaced by an amine (Scheme 1D) [2] or where the hydroxyl groups are para to one another (Scheme 1E&F) have also been studied [3, 4]. Other extradiol cleaving catechol dioxygenases are involved in natural product biosynthesis pathways [5]. In humans, related dioxygenases are involved in the metabolism of aromatic amino acids, where mutations are associated with several severe diseases including the neurodegenerative disorder Huntington’s chorea and degenerative arthritis [6, 7].…”

Section: Introductionmentioning

confidence: 99%

A two-electron-shell game: intermediates of the extradiol-cleaving catechol dioxygenases

2014

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Extradiol catechol ring-cleaving dioxygenases function by binding both the organic substrate and O2 at a divalent metal center in the active site. They have proven to be a particularly versatile group of enzymes with which to study the O2 activation process. Here, recent studies of homoprotocatechuate 2,3-dioxygenase (HPCD) are summarized with the objective of showing how Nature can utilize the enzyme structure and the properties of the metal and the substrate to select among many possible chemical paths to achieve both specificity and efficiency. Possible intermediates in the mechanism have been trapped by swapping active site metals, introducing active site amino acid substituted variants, and using substrates with different electron donating capacities. While each of these intermediates could form part of a viable reaction pathway, kinetic measurements significantly limit the likely candidates. Structural, kinetic, spectroscopic and computational analysis of the various intermediates shed light on how catalytic efficiency can be achieved.

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Defining a kinetic mechanism for l-DOPA 2,3 dioxygenase, a single-domain type I extradiol dioxygenase from Streptomyces lincolnensis

Cited by 18 publications

References 46 publications

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

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

Evaluating the Role of HLA-DM in MHC Class II–Peptide Association Reactions

A two-electron-shell game: intermediates of the extradiol-cleaving catechol dioxygenases

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