Elucidation of the Biosynthetic Pathway for 7-Deazapurines

Abstract

Small molecules containing a 7-deazapurine moiety are ubiquitous in nature. They comprise a broad range of structurally diverse antibiotics produced by terrestrial and marine microorganisms that possess demonstrated antibiotic and antineoplastic activity. In addition, queuosine, a hypermodified nucleoside located in the wobble position of select tRNAs that is almost universally conserved throughout biology, contains a 7-deazapurine functional group. The since their initial identification over 50 years ago, the chemical transformations underlying the biosynthesis of 7-deazapurines have remained elusive. This work describes the identification of a cluster of co-localized genes in the Streptomyces rimosus chromosome that are responsible for the biosynthesis of the 7-deazapurine containing antibiotics toyocamycin and sangivamycin. Further, the in vitro conversion of GTP to the previously identified queuosine biosynthetic intermediate 7-cyano-7-deazaguanine (preQ₀) is demonstrated using purified, recombinant enzymes. Also included herein is a kinetic, spectroscopic, and mechanistic characterization of QueE, an enzyme that catalyzes the third step in the biosynthesis of 7-deazapurines using a radical-mediated rearrangement. A possible mechanism for the reaction catalyzed by QueD, the second step in the deazapurine biosynthetic pathway, is explored based on X-ray crystallographic data of site directed QueD mutants containing bound substrate. Finally, hitherto unrecognized gene clusters that are likely devoted to the biosynthesis of 7-deazapurines other than queuosine are described.