Discovery, Characterization and Mechanistic Study of a Novel L-Tyrosine Hydroxylase in the Biosynthesis of Anthramycin
| dc.contributor.advisor | Gerratana, Barbara | en_US |
| dc.contributor.advisor | Rokita, Steven E | en_US |
| dc.contributor.author | Connor, Katherine Lindsey | en_US |
| dc.contributor.department | Biochemistry | en_US |
| dc.contributor.publisher | Digital Repository at the University of Maryland | en_US |
| dc.contributor.publisher | University of Maryland (College Park, Md.) | en_US |
| dc.date.accessioned | 2012-10-11T06:01:45Z | |
| dc.date.available | 2012-10-11T06:01:45Z | |
| dc.date.issued | 2012 | en_US |
| dc.description.abstract | A tyrosine hydroxylase coded by <italic>orf13</italic> of the anthramycin biosynthesis gene cluster is proposed to catalyze the <italic>ortho</italic>-hydroxylation of L-tyrosine to L-DOPA as the initial step of a unique transformation to the hydropyrrole moiety found in anthramycin. The sequence of Orf13 is not similar to any known characterized proteins, nor does it contain conserved domains or motifs characteristic of enzymes performing aromatic hydroxylation. The lack of information for this common enzymatic reaction suggests the identification of a new class of tyrosine hydroxylases which may have novel cofactor requirements, novel folds and/or chemical mechanisms. Heme B has been identified in purified Orf13 and full heme B occupancy is achieved during expression with iron (III) citrate in <italic>E. coli</italic>. Maximal L-tyrosine to L-DOPA conversion is observed in the presence of hydrogen peroxide (H<sub>2</sub>O<sub>2</sub>). This confirmed heme B as the required catalytic cofactor and the putative function of Orf13 as a tyrosine hydroxylase. This information also classified Orf13 as a heme peroxidase. Spectroscopic data from a reduced-CO (g) spectrum of Orf13 and electron paramagnetic resonance of ferric-heme Orf13 are consistent with histidyl-ligated heme peroxidases. The steady-state kinetics of L-tyrosine hydroxylation show similar catalytic efficiency for L-tyrosine and H<sub>2</sub>O<sub>2</sub>. Orf13 has a secondary tyrosine hydroxylation activity in the presence of molecular oxygen (O<sub>2</sub>) and dihydroxyfumaric acid (DHFA), which is also found with histidyl-heme peroxidases. Orf13 is substrate specific and stereoselective for L-tyrosine. Turnover is only observed with <italic>para</italic>-substituted phenols but not with D-tyrosine, implicating the <italic>para</italic>-phenol substituent is required for hydroxylation. Although the catalytic requirements of heme B and H<sub>2</sub>O<sub>2</sub> are in agreement with heme peroxidases, the resulting hydroxylated product (L-DOPA) by a H<sub>2</sub>O<sub>2</sub> dependent pathway is unprecedented. Heme dependent aromatic hydroxylation is typically catalyzed by cytochrome P450s through an O<sub>2</sub> dependent pathway. Mechanistic investigation of Orf13 revealed H<sub>2</sub>O<sub>2</sub> as the oxygen source in a labeling study using H<sub>2</sub><super>18</super>O<sub>2</sub>. A proposed mechanism of L-tyrosine hydroxylation is suggested to proceed through an oxygen rebound mechanism similar to cytochrome P450 aromatic hydroxylation. Therefore, Orf13 represents a new class of heme-histidyl ligated H<sub>2</sub>O<sub>2</sub> dependent hydroxylases and is the first identified bacterial tyrosine hydroxylase. | en_US |
| dc.identifier.uri | http://hdl.handle.net/1903/13207 | |
| dc.subject.pqcontrolled | Biochemistry | en_US |
| dc.subject.pquncontrolled | anthramycin | en_US |
| dc.subject.pquncontrolled | aromatic hydroxylation | en_US |
| dc.subject.pquncontrolled | heme peroxidase | en_US |
| dc.subject.pquncontrolled | pyrrolo[1,4]benzodiazepine | en_US |
| dc.subject.pquncontrolled | tyrosine hydroxylase | en_US |
| dc.title | Discovery, Characterization and Mechanistic Study of a Novel L-Tyrosine Hydroxylase in the Biosynthesis of Anthramycin | en_US |
| dc.type | Dissertation | en_US |
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