期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：2018
卷号：115
期号：5
页码：974-979
DOI：10.1073/pnas.1718667115
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Capuramycins are antimycobacterial antibiotics that consist of a modified nucleoside named uridine-5′-carboxamide (CarU). Previous biochemical studies have revealed that CarU is derived from UMP, which is first converted to uridine-5′-aldehyde in a reaction catalyzed by the dioxygenase CapA and subsequently to 5′-C-glycyluridine (GlyU), an unusual β–hydroxy-α-amino acid, in a reaction catalyzed by the pyridoxal-5′-phosphate (PLP)-dependent transaldolase CapH. The remaining steps that are necessary to furnish CarU include decarboxylation, O atom insertion, and oxidation. We demonstrate that Cap15, which has sequence similarity to proteins annotated as bacterial, PLP-dependent l -seryl-tRNA(Sec) selenium transferases, is the sole catalyst responsible for complete conversion of GlyU to CarU. Using a complementary panel of in vitro assays, Cap15 is shown to be dependent upon substrates O2 and (5′ S ,6′ R )-GlyU, the latter of which was unexpected given that (5′ S ,6′ S )-GlyU is the isomeric product of the transaldolase CapH. The two products of Cap15 are identified as the carboxamide-containing CarU and CO2. While known enzymes that catalyze this type of chemistry, namely α-amino acid 2-monooxygenase, utilize flavin adenine dinucleotide as the redox cofactor, Cap15 remarkably requires only PLP. Furthermore, Cap15 does not produce hydrogen peroxide and is shown to directly incorporate a single O atom from O2 into the product CarU and thus is an authentic PLP-dependent monooxygenase. In addition to these unusual discoveries, Cap15 activity is revealed to be dependent upon the inclusion of phosphate. The biochemical characteristics along with initiatory mechanistic studies of Cap15 are reported, which has allowed us to assign Cap15 as a PLP-dependent (5′ S ,6′ R )-GlyU:O2 monooxygenase-decarboxylase.
关键词：natural products ; biosynthesis ; antibiotic ; enzyme function
