Amide bond-containing (ABC) biomolecules are some of the most intriguing and functionally significant natural products with unmatched utility in medicine, agriculture and biotechnology. The enzymatic formation of an amide bond is therefore a particularly interesting platform for engineering the synthesis of structurally diverse natural and unnatural ABC molecules for applications in drug discovery and molecular design. As such, efforts to unravel the mechanisms involved in carboxylate activation and substrate selection has led to the characterization of a number of structurally and functionally distinct protein families involved in amide bond synthesis. Unlike ribosomal synthesis and thio-templated synthesis using nonribosomal peptide synthetases, which couple the hydrolysis of phosphoanhydride bond(s) of ATP and proceed via an acyl-adenylate intermediate, here we discuss two mechanistically alternative strategies: ATP-dependent enzymes that generate acylphosphate intermediates and ATP-independent transacylation strategies. Several examples highlighting the function and synthetic utility of these amide bond-forming strategies are provided.

The lipopeptidyl nucleoside antibiotics reperesented by A-90289, caprazamycin, and muraymycin, are structurally highlighted by a nucleoside core that contains a nonproteinogenic β-hydroxy-α-amino acid named 5′-C-glycyluridine (GlyU). Bioinformatic analysis of the biosynthetic gene clusters revealed a shared open reading frame encoding a protein with sequence similarity to serine hydroxymethyltransferases, resulting in the proposal that this shared enzyme catalyzes an aldol-type condensation with glycine and uridine-5′-aldehyde to furnish GlyU. Using LipK involved in A-90289 biosynthesis as a model, we now functionally assign and characterize the enzyme responsible for the C-C bond-forming event during GlyU biosynthesis as an l-threonine:uridine-5′-aldehyde transaldolase. Biochemical analysis revealed this transformation is dependent upon pyridoxal-5′-phosphate, the enzyme has no activity with alternative amino acids such as glycine or serine as aldol donors, and acetaldehyde is a co-product. Structural characterization of the enzyme product is consistent with stereochemical assignment as the threo diastereomer (5′S,6′S)-GlyU. Thus this enzyme orchestrates C-C bond breaking and formation with concomitant installation of two stereocenters to make a new l-α-amino acid with a nucleoside side chain.

Background: Several nucleoside antibiotics contain a uridine-5Ј-carboxamide core of unclear origin. Results: The A-102395 biosynthetic gene cluster was cloned, a genetic system was developed, and three enzymes were characterized in vivo and in vitro. Conclusion: Uridine-5Ј-carboxamide originates from UMP and L-Thr by sequential reactions catalyzed by a dioxygenase and transaldolase. Significance: The results provide the first opportunity to methodically interrogate the biosynthesis of these unusual antibiotics.

LipL and Cpr19 are non-heme, mononuclear Fe(II)-dependent, α-ketoglutarate (αKG):UMP oxygenases that catalyze the formation of CO2, succinate, phosphate, and uridine-5′-aldehyde, the last of which is a biosynthetic precursor for several nucleoside antibiotics that inhibit bacterial translocase I (MraY). To better understand the chemistry underlying this unusual oxidative dephosphorylation and establish a mechanistic framework for LipL and Cpr19, we report herein the synthesis of two biochemical probes – [1′,3′,4′,5′,5′-2H]UMP and the phosphonate derivative of UMP – and their activity with both enzymes. The results are consistent with a reaction coordinate that proceeds through the loss of one 2H atom of [1′,3′,4′,5′,5′-2H]UMP and stereospecific hydroxylation geminal to the phosphoester to form a cryptic intermediate, (5′R)-5′-hydroxy-UMP. Thus, these enzyme catalysts can additionally be assigned as UMP hydroxylase-phospholyases.

Using the ATP-independent transacylase CapW required for the biosynthesis of capuramycin-type antibiotics, we developed a biocatalytic approach for the synthesis of 43 analogues via a one-step aminolysis reaction from the methyl ester precursor as the acyl donor and various nonnative amines as acyl acceptors. Further examination of the donor substrate scope for CapW revealed this enzyme can also catalyze a direct transamidation reaction using the major capuramycin congener as a semisynthetic precursor. Biological activity tests revealed that a few of the new capuramycin analogues have significantly improved antibiotic activity against Mycobacterium smegmatis MC2 155 and Mycobacterium tuberculosis H37Rv. Furthermore, most of the analogues are able to be covalently modified by the phosphotransferase CapP/Cpr17 involved in self resistance, providing critical insight for future studies regarding clinical development of the capuramycin antimycobacterial antibiotics.