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

Abstract

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.

Characterization of SfmD as a Heme Peroxidase That Catalyzes the Regioselective Hydroxylation of 3-Methyltyrosine to 3-Hydroxy-5-methyltyrosine in Saframycin A Biosynthesis

“…SfmD shows no homology to these hemecontaining peroxidases listed in the PeroxiBase, and is a novel heme-containing peroxidase reported to catalyze the aromatic ring hydroxylation of Tyr or its derivatives using H 2 O 2 as the oxidant. Very recently, Orf13 involved in the biosynthesis of anthramycin was also reported to be a heme-containing perox-idase catalyzing the hydroxylation of Tyr to Dopa in the presence of H 2 O 2 around the time we submitted this article (40). And Orf13 also had the ability to catalyze the hydroxylation of Tyr to Dopa by a molecular oxygen-dependent pathway in the presence of dihydroxyfumaric acid or ascorbic acid (40).…”

“…Very recently, Orf13 involved in the biosynthesis of anthramycin was also reported to be a heme-containing perox-idase catalyzing the hydroxylation of Tyr to Dopa in the presence of H 2 O 2 around the time we submitted this article (40). And Orf13 also had the ability to catalyze the hydroxylation of Tyr to Dopa by a molecular oxygen-dependent pathway in the presence of dihydroxyfumaric acid or ascorbic acid (40). But SfmD showed no hydroxylation activity under the same conditions as reported for Orf13 using oxygen as the oxidant (data not shown).…”

Enhancing the Enzymatic Activity of a Heme-Dependent Peroxidase through Genetic Modification

“…However, hydrogen peroxide has been found to inactivate the enzyme during catalysis [17] and has a deleterious effect on enzymes [21].It is possible that hydrogen peroxide may interfere the secondary structures of the enzymes during catalysis. Herein the change of secondary structures of the enzymes in the presence of H 2 O 2 with different concentrations was monitored by measuring CD spectra.…”

“…The heme-dependent peroxidase (HDP) catalyzed the ortho-hydroxylation of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA)in the presence of hydrogen peroxide [17]. Here both HDP and HDP-ELP have been used to catalyze the ortho-hydroxylation of L-tyrosine with various hydrogen peroxide concentrations from 0.5 to 3 mM.…”

“…The hydroxylation of L-tyrosine to L-3,4-dihydroxyphenylalanine (L-DOPA) can be catalyzed by a heme-dependent peroxidase in the presence of hydrogen peroxide [17]. L-DOPA is the precursor to synthesize neurotransmitters dopamine, norepinephrine and epinephrine.…”