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.
Highlights
SfmD is a hypothetical protein thought to be a hydroxylase in saframycin A biosynthesis
SacD, a counterpart of SfmD involved in safracin B biosynthesis, was characterized as a heme-containing peroxidase, suggesting that SfmD-like heme-containing peroxidases may be commonly involved in the biosynthesis of Saframycin A (SFM-A) and its analogs
In the in vitro assays, SacD used H2O2 as the oxidant, displayed an optimal activity at pH 9.0, and converted 3-Me-Tyr to the corresponding hydroxylation product, 3-OH-5-Me-Tyr (Fig. 5B). These results indicated that, like SfmD, SacD is a heme-containing peroxidase that catalyzes the hydroxylation of aromatic amino acids using H2O2 as the oxidant
Summary
SfmD is a hypothetical protein thought to be a hydroxylase in saframycin A biosynthesis. 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. The hypothetical proteins, SfmD and its homolog SacD, were deduced to be hydroxylases responsible for the 3-hydroxy group construction based on heterologous expression results [7, 8] These proteins have no sequence similarity to the known characterized proteins, and little is known about these enzymes or the reactions that they catalyze. From kinetic studies of SfmD and enzyme assays of SfmM2, the true substrate of SfmD was identified as 3-methyltyrosine (3-Me-Tyr), and the biosynthetic pathway of 3-OH-5Me-OMe-Tyr was elucidated This type of heme-containing peroxidase was further demonstrated to be a potentially common factor in the biosynthesis of SFM-A and its analogues, which possess the same core quinone structure as SFM-A, as determined by characterization of SacD in vitro. Comparative analyses of UV-Vis and Magnetic Circular Dichroism (MCD) spectra of SfmD wild-type and SfmD mutants revealed that the conserved motif HXXXC plays a crucial role in heme binding
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