Pseudouridine-Modifying Enzymes SapB and SapH Control Entry into the Pseudouridimycin Biosynthetic Pathway

Abstract

Pseudouridimycin is a microbial C-nucleoside natural product that specifically inhibits bacterial RNA polymerases by binding to the active site and competing with uridine triphosphate for the nucleoside triphosphate (NTP) addition site. Pseudouridimycin consists of 5′-aminopseudouridine and formamidinylated, N-hydroxylated Gly−Gln dipeptide moieties to allow Watson−Crick base pairing and to mimic protein−ligand interactions of the triphosphates of NTP, respectively. The metabolic pathway of pseudouridimycin has been studied in Streptomyces species, but no biosynthetic steps have been characterized biochemically. Here, we show that the flavin-dependent oxidase SapB functions as a gate-keeper enzyme selecting pseudouridine (K M = 34 μM) over uridine (K M = 901 μM) in the formation of pseudouridine aldehyde. The pyridoxal phosphate (PLP)-dependent SapH catalyzes transamination, resulting in 5′-aminopseudouridine with a preference for arginine, methionine, or phenylalanine as cosubstrates as amino group donors. The binary structure of SapH in complex with pyridoxamine-5′-phosphate and site-directed mutagenesis identified Lys289 and Trp32 as key residues for catalysis and substrate binding, respectively. The related C-nucleoside oxazinomycin was accepted as a substrate by SapB with moderate affinity (K M = 181 μM) and was further converted by SapH, which opens possibilities for metabolic engineering to generate hybrid C-nucleoside pseudouridimycin analogues in Streptomyces.

C-Ribosylating Enzymes in the (Bio)Synthesis of C-Nucleosides and C-Glycosylated Natural Products

“…Malayamycin ( 6 ) has antifungal activity by inhibiting sporulation . Pseudouridimycin ( 7 ) exhibits antimicrobial activity by inhibiting the RNA polymerase of bacteria. ,,,− Malayamycin biosynthesis in Streptomyce s sp. , starts from ΨMP ( 2c ), as shown in Figure S2. MalD, a PUS-like enzyme belonging to the TruD subfamily, is involved in providing free ΨMP ( 2c ).…”

Biosynthesis and Genome Mining Potentials of Nucleoside Natural Products

“…The nucleosides biosynthesized through the 5'-aldehyde intermediate are represented by the MraY-targeting antibacterial antibiotics. The C5' oxidation is catalyzed by either α-KG and Fe-dependent oxygenase (e. g. liposidomycin [13] and caprazamycin, [14] Figure 1b), or flavin-dependent oxidase (pacidamycin [16] and pseudouridimycin [27] ). Of the two mecha- nisms of C5' oxidation, more pathways employ the α-KG and Fe-dependent oxygenase.…”