title: Characterization of SfmD as a Heme peroxidase that catalyzes the regioselective hydroxylation of 3-methyltyrosine to 3-hydroxy-5-methyltyrosine in saframycin A biosynthesis.
abstract: Saframycin A (SFM-A) is a potent antitumor antibiotic that belongs to the tetrahydroisoquinoline family. Biosynthetic studies have revealed that its unique pentacyclic core structure is derived from alanine, glycine, and non-proteinogenic amino acid 3-hydroxy-5-methyl-O-methyltyrosine (3-OH-5-Me-OMe-Tyr). SfmD, a hypothetical protein in the biosynthetic pathway of SFM-A, was hypothesized to be responsible for the generation of the 3-hydroxy group of 3-OH-5-Me-OMe-Tyr based on previously heterologous expression results. We now report the in vitro characterization of SfmD as a novel heme-containing peroxidase that catalyzes the hydroxylation of 3-methyltyrosine to 3-hydroxy-5-methyltyrosine using hydrogen peroxide as the oxidant. In addition, we elucidated the biosynthetic pathway of 3-OH-5-Me-OMe-Tyr by kinetic studies of SfmD in combination with biochemical assays of SfmM2, a methyltransferase within the same pathway. Furthermore, SacD, a counterpart of SfmD involved in safracin B biosynthesis, was also characterized as a heme-containing peroxidase, suggesting that SfmD-like heme-containing peroxidases may be commonly involved in the biosynthesis of SFM-A and its analogs. Finally, we found that the conserved motif HXXXC is crucial for heme binding using comparative UV-Vis and Magnetic Circular Dichroism (MCD) spectra studies of SfmD wild-type and mutants. Together, these findings expand the category of heme-containing peroxidases and set the stage for further mechanistic studies. In addition, this study has critical implications for delineating the biosynthetic pathway of other related tetrahydroisoquinoline family members.
authors.affiliation: State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China.
authors.forename: Man-Cheng
authors.initials: MC
authors.lastname: Tang
authors.forename: Cheng-Yu
authors.initials: CY
authors.lastname: Fu
authors.forename: Gong-Li
authors.initials: GL
authors.lastname: Tang
mesh_terms: D020816:Amino Acid Motifs; D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006900:Hydroxylation / Q000502:physiology; D007546:Isoquinolines / Q000378:metabolism; D008781:Methyltyrosines / Q000235:genetics / Q000378:metabolism*; D009195:Peroxidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
chemical_list: C544271:3-hydroxy-5-methyl-O-methyltyrosine; D001425:Bacterial Outer Membrane Proteins; D029968:Escherichia coli Proteins; D007546:Isoquinolines; D008781:Methyltyrosines; C505399:sfmD protein, E coli; C032745:methyl-3-tyrosine; D009195:Peroxidase; C026925:saframycin A