Assay of tyrosine hydroxylase based on high‐performance liquid chromatography separation and quantification of <scp>l</scp>‐dopa and <scp>l</scp>‐tyrosine

Olšovská, Jana; Novotná, Jitka; Flieger, Miroslav; Spížek, J.

doi:10.1002/bmc.880

Cited by 18 publications

(12 citation statements)

References 21 publications

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“…The HPLC method was derived from a published method using isocratic elution profile (3/97/0.1, ACN/H 2 O/FA). 61 All the HPLC profiles are shown with absorbance at 280 nm except assays for 2 were at both 257 and 280 nm. The peaks eluted from HPLC were then collected and concentrated by SpeedVac (ThermoFisher) for further MS and NMR analysis.…”

Section: Methodsmentioning

confidence: 99%

Biocatalytic Carbon–Hydrogen and Carbon–Fluorine Bond Cleavage through Hydroxylation Promoted by a Histidyl-Ligated Heme Enzyme

et al. 2019

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LmbB2 is a peroxygenase-like enzyme that hydroxylates L-tyrosine to L-3,4-dihydroxyphenylalanine (DOPA) in the presence of hydrogen peroxide. However, its heme cofactor is ligated by a proximal histidine, not cysteine. We show that LmbB2 can oxidize L-tyrosine analogs with ring-deactivated substituents such as 3-nitro-, fluoro-, chloro-, iodo-L-tyrosine. We also found that the 4-hydroxyl group of the substrate is essential for reacting with the heme-based oxidant and activating the aromatic C-H bond. The most interesting observation of this study was obtained with 3-fluoro-L-tyrosine as a substrate and mechanistic probe. The LmbB2-mediated catalytic reaction yielded two hydroxylated products with comparable populations, i.e., oxidative C-H bond cleavage at C5 to generate 3-fluoro-5-hydroxyl-L-tyrosine and oxygenation at C3 concomitant with a carbon-fluorine bond cleavage to yield DOPA and fluoride. An iron protein-mediated hydroxylation on both C-H and C-F bonds with multiple turnovers is unprecedented. Thus, this finding reveals a significant potential of biocatalysis in C-H/C-X bond (X = halogen) cleavage. Further 18O-labeling results suggest that the source of oxygen for hydroxylation is a peroxide, and that a commonly expected oxidation by a high-valent iron intermediate followed by hydrolysis is not supported for the C-F bond cleavage. Instead, the C-F bond cleavage is proposed to be initiated by a nucleophilic aromatic substitution mediated by the iron-hydroperoxo species. Based on the experimental results, two mechanisms are proposed to explain how LmbB2 hydroxylates the substrate and cleaves C-H/C-F bond. This study broadens the understanding of heme enzyme catalysis and sheds light on enzymatic applications in medicinal and environmental fields.

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Section: Methodsmentioning

confidence: 99%

Biocatalytic Carbon–Hydrogen and Carbon–Fluorine Bond Cleavage through Hydroxylation Promoted by a Histidyl-Ligated Heme Enzyme

et al. 2019

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“…The amount of DOPA produced was determined as described previously [46]. The assay was performed in triplicates with a control sample containing the respective buffer instead of the enzyme.…”

Section: Methodsmentioning

confidence: 99%

“…The LmbB2 reaction product with the retention time corresponding to the DOPA standard was purified by RP-HPLC as described previously [46] and fractions containing the LmbB2 reaction product were pooled. Mass spectrometry was performed on a commercial APEX-Qe FTMS instrument equipped with a 9.4 T superconducting magnet (Bruker Daltonics, Billerica, MA).…”

Section: Methodsmentioning

confidence: 99%

Lincomycin Biosynthesis Involves a Tyrosine Hydroxylating Heme Protein of an Unusual Enzyme Family

et al. 2013

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The gene lmbB2 of the lincomycin biosynthetic gene cluster of Streptomyces lincolnensis ATCC 25466 was shown to code for an unusual tyrosine hydroxylating enzyme involved in the biosynthetic pathway of this clinically important antibiotic. LmbB2 was expressed in Escherichia coli, purified near to homogeneity and shown to convert tyrosine to 3,4-dihydroxyphenylalanine (DOPA). In contrast to the well-known tyrosine hydroxylases (EC 1.14.16.2) and tyrosinases (EC 1.14.18.1), LmbB2 was identified as a heme protein. Mass spectrometry and Soret band-excited Raman spectroscopy of LmbB2 showed that LmbB2 contains heme b as prosthetic group. The CO-reduced differential absorption spectra of LmbB2 showed that the coordination of Fe was different from that of cytochrome P450 enzymes. LmbB2 exhibits sequence similarity to Orf13 of the anthramycin biosynthetic gene cluster, which has recently been classified as a heme peroxidase. Tyrosine hydroxylating activity of LmbB2 yielding DOPA in the presence of (6R)-5,6,7,8-tetrahydro-L-biopterin (BH4) was also observed. Reaction mechanism of this unique heme peroxidases family is discussed. Also, tyrosine hydroxylation was confirmed as the first step of the amino acid branch of the lincomycin biosynthesis.

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“…The aqueous layer (65 μL) was transferred to new tube containing 2.5 M NaOH (10 μL). 20 μL from each sample were analyzed by HPLC-FLD as described for the detection of l -tyrosine and l -DOPA (21). l -Tyrosine and l -DOPA stocks were quantified spectrophotometrically at ε 275 = 1.4 mM -1 cm -1 (22) and ε 280 = 2.63 mM -1 cm -1 (23) respectively.…”

Section: Methodsmentioning

confidence: 99%

A Heme Peroxidase with a Functional Role as an l-Tyrosine Hydroxylase in the Biosynthesis of Anthramycin

2011

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We report the first characterization and classification of Orf13 (S. refuineus) as a heme dependent peroxidase catalyzing the ortho-hydroxylation of l-tyrosine to l-DOPA. The putative tyrosine hydroxylase coded by orf13 of the anthramycin biosynthesis gene cluster has been expressed and purified. Heme b has been identified as the required cofactor for catalysis and maximal l-tyrosine conversion to l-DOPA is observed in the presence of hydrogen peroxide. Pre-incubation of l-tyrosine with Orf13 prior to the addition of hydrogen peroxide is required for l-DOPA production. However, the enzyme becomes inactivated by hydrogen peroxide during catalysis. Steady state kinetic analysis of l-tyrosine hydroxylation revealed similar catalytic efficiency for both l-tyrosine and hydrogen peroxide. Spectroscopic data from a reduced-CO (g) UV-visible spectrum of Orf13 and electron paramagnetic resonance of ferric-heme Orf13 are consistent with heme peroxidases that have a histidyl-ligated heme-iron. Contrary to the classical heme peroxidase oxidation reaction with hydrogen peroxide that produces coupled aromatic products such as o,o'-dityrosine, Orf13 is novel in its ability to catalyze aromatic amino acid hydroxylation with hydrogen peroxide, in the substrate addition order and for its substrate specificity for l-tyrosine. Peroxygenase activity of Orf13 for the ortho-hydroxylation of l-tyrosine to l-DOPA by a molecular oxygen dependent pathway in the presence of dihydroxyfumaric acid is also observed. This reaction behavior is consistent with peroxygenase activity reported with horseradish peroxidase for the hydroxylation of phenol. Overall, the putative function of Orf13 as a tyrosine hydroxylase has been confirmed and establishes the first bacterial class of tyrosine hydroxylases.

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Assay of tyrosine hydroxylase based on high‐performance liquid chromatography separation and quantification of l‐dopa and l‐tyrosine

Cited by 18 publications

References 21 publications

Biocatalytic Carbon–Hydrogen and Carbon–Fluorine Bond Cleavage through Hydroxylation Promoted by a Histidyl-Ligated Heme Enzyme

Biocatalytic Carbon–Hydrogen and Carbon–Fluorine Bond Cleavage through Hydroxylation Promoted by a Histidyl-Ligated Heme Enzyme

Lincomycin Biosynthesis Involves a Tyrosine Hydroxylating Heme Protein of an Unusual Enzyme Family

A Heme Peroxidase with a Functional Role as an l-Tyrosine Hydroxylase in the Biosynthesis of Anthramycin

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