Design of a chimeric glycosyltransferase OleD for the site‐specific O‐monoglycosylation of 3‐hydroxypyridine in nosiheptide

Abstract

AbstractTo identify the potential role of the 3‐hydroxyl group of the pyridine ring in nosiheptide (NOS) for its antibacterial activity against Gram‐positive pathogens, enzymatic glycosylation was utilized to regio‐selectively create a monoglycosyl NOS derivative, NOS‐G. For this purpose, we selected OleD, a UDP glycosyltransferase from Streptomyces antibioticus that has a low productivity for NOS‐G. Activity of the enzyme was increased by swapping domains derived from OleI, both single and in combination. Activity enhancement was best in mutant OleD‐10 that contained four OleI domains. This chimer was engineered by site‐directed mutagenesis (single and in combination) to increase its activity further, whereby variants were screened using a newly‐established colorimetric assay. OleD‐10 with I117F and T118G substitutions (FG) had an increased NOS‐G productivity of 56%, approximately 70 times higher than that of wild‐type OleD. The reason for improved activity of FG towards NOS was structurally attributed to a closer distance (<3 Å) between NOS/sugar donor and the catalytic amino acid H25. The engineered enzyme allowed sufficient activity to demonstrate that the produced NOS‐G had enhanced stability and aqueous solubility compared to NOS. Using a murine MRSA infection model, it was established that NOS‐G resulted in partial protection within 20 h of administration and delayed the death of infected mice. We conclude that 3‐hydroxypyridine is a promising site for structural modification of NOS, which may pave the way for producing nosiheptide derivatives as a potential antibiotic for application in clinical treatment.