Pyridoxal-5'-phosphate-dependent alkyl transfer in nucleoside antibiotic biosynthesis.

Several nucleoside antibiotics are structurally characterized by a 5″-amino-5″-deoxyribose (ADR) appended via a glycosidic bond to a high-carbon sugar nucleoside (5'S,6'S)-5'-C-glycyluridine (GlyU). GlyU is further modified with an N-alkylamine linker, the biosynthetic origin of which has yet to be established. By using a combination of feeding experiments with isotopically labeled precursors and characterization of recombinant proteins from multiple pathways, the biosynthetic mechanism for N-alkylamine installation for ADR-GlyU-containing nucleoside antibiotics has been uncovered. The data reveal S-adenosyl-L-methionine (AdoMet) as the direct precursor of the N-alkylamine, but, unlike conventional AdoMet- or decarboxylated AdoMet-dependent alkyltransferases, the reaction is catalyzed by a pyridoxal-5'-phosphate-dependent aminobutyryltransferase (ABTase) using a stepwise γ-replacement mechanism that couples γ-elimination of AdoMet with aza-γ-addition onto the disaccharide alkyl acceptor. In addition to using a conceptually different strategy for AdoMet-dependent alkylation, the newly discovered ABTases require a phosphorylated disaccharide alkyl acceptor, revealing a cryptic intermediate in the biosynthetic pathway.

Pyridoxal-5'-phosphate as an oxygenase cofactor: Discovery of a carboxamide-forming, α-amino acid monooxygenase-decarboxylase.

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 ß-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.

Merging Natural Products: Muraymycin-Sansanmycin Hybrid Structures as Novel Scaffolds for Potential Antibacterial Agents.

To overcome bacterial resistances, the need for novel antimicrobial agents is urgent. The class of so-called nucleoside antibiotics furnishes promising candidates for the development of new antibiotics, as these compounds block a clinically unexploited bacterial target: the integral membrane protein MraY, a key enzyme in cell wall (peptidoglycan) biosynthesis. Nucleoside antibiotics exhibit remarkable structural diversity besides their uridine-derived core motifs. Some sub-classes also show specific selectivities towards different Gram-positive and Gram-negative bacteria, which are poorly understood so far. Herein, the synthesis of a novel hybrid structure is reported, derived from the 5'-defunctionalized uridine core moiety of muraymycins and the peptide chain of sansanmycin B, as a new scaffold for the development of antimicrobial agents. The reported muraymycin-sansanmycin hybrid scaffold showed nanomolar activity against the bacterial target enzyme MraY, but displayed no significant antibacterial activity against S. aureus, E. coli, and P. aeruginosa.

Muraymycin Nucleoside Antibiotics: Structure-Activity Relationship for Variations in the Nucleoside Unit.

Muraymycins are a subclass of naturally occurring nucleoside antibiotics with promising antibacterial activity. They inhibit the bacterial enzyme translocase I (MraY), a clinically yet unexploited target mediating an essential intracellular step of bacterial peptidoglycan biosynthesis. Several structurally simplified muraymycin analogues have already been synthesized for structure-activity relationship (SAR) studies. We now report on novel derivatives with unprecedented variations in the nucleoside unit. For the synthesis of these new muraymycin analogues, we employed a bipartite approach facilitating the introduction of different nucleosyl amino acid motifs. This also included thymidine- and 5-fluorouridine-derived nucleoside core structures. Using an in vitro assay for MraY activity, it was found that the introduction of substituents in the 5-position of the pyrimidine nucleobase led to a significant loss of inhibitory activity towards MraY. The loss of nucleobase aromaticity (by reduction of the uracil C5-C6 double bond) resulted in a ca. tenfold decrease in inhibitory potency. In contrast, removal of the 2'-hydroxy group furnished retained activity, thus demonstrating that modifications of the ribose moiety might be well-tolerated. Overall, these new SAR insights will guide the future design of novel muraymycin analogues for their potential development towards antibacterial drug candidates.

Enzymatic Synthesis of the Ribosylated Glycyl-Uridine Disaccharide Core of Peptidyl Nucleoside Antibiotics.

Muraymycins belong to a family of nucleoside antibiotics that have a distinctive disaccharide core consisting of 5-amino-5-deoxyribofuranose (ADR) attached to 6'- N-alkyl-5'- C-glycyluridine (GlyU). Here, we functionally assign and characterize six enzymes from the muraymycin biosynthetic pathway involved in the core assembly that starts from uridine monophosphate (UMP). The biosynthesis is initiated by Mur16, a nonheme Fe(II)- and α-ketoglutarate-dependent dioxygenase, followed by four transferase enzymes: Mur17, a pyridoxal-5'-phosphate (PLP)-dependent transaldolase; Mur20, an aminotransferase; Mur26, a pyrimidine phosphorylase; and Mur18, a nucleotidylyltransferase. The pathway culminates in glycosidic bond formation in a reaction catalyzed by an additional transferase enzyme, Mur19, a ribosyltransferase. Analysis of the biochemical properties revealed several noteworthy discoveries including that (i) Mur16 and downstream enzymes can also process 2'-deoxy-UMP to generate a 2-deoxy-ADR, which is consistent with the structure of some muraymycin congeners; (ii) Mur20 prefers l-Tyr as the amino donor source; (iii) Mur18 activity absolutely depends on the amine functionality of the ADR precursor consistent with the nucleotidyltransfer reaction occurring after the Mur20-catalyzed aminotransfer reaction; and (iv) the bona fide sugar acceptor for Mur19 is (5' S,6' S)-GlyU, suggesting that ribosyltransfer occurs prior to N-alkylation of GlyU. Finally, a one-pot, six-enzyme reaction was utilized to generate the ADR-GlyU disaccharide core starting from UMP.

Muraymycin nucleoside-peptide antibiotics: uridine-derived natural products as lead structures for the development of novel antibacterial agents.

Muraymycins are a promising class of antimicrobial natural products. These uridine-derived nucleoside-peptide antibiotics inhibit the bacterial membrane protein translocase I (MraY), a key enzyme in the intracellular part of peptidoglycan biosynthesis. This review describes the structures of naturally occurring muraymycins, their mode of action, synthetic access to muraymycins and their analogues, some structure-activity relationship (SAR) studies and first insights into muraymycin biosynthesis. It therefore provides an overview on the current state of research, as well as an outlook on possible future developments in this field.