Elucidation of the Biosynthetic Pathway for 7-Deazapurines

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.

Name thesis:degree_name

Ph.D.

Level thesis:degree_level

doctoral

Discipline thesis:degree_discipline

Graduate College

Grantor dc:publisher

The University of Arizona.

Year dc:date.issued

2011

Author dc:creator

• McCarty, Reid Michael

Advisor dc:contributor.advisor

• Bandarian, Vahe

Committee members dc:contributor.committeemember

• Cordes, Matthew H.
• Molnar, Istvan
• McEvoy, Megan M.

• antibiotics
• enzymology
• natural products
• tRNA

Statement dc:rights

• Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.

Language dc:language.iso

en

Handle dc:identifier.uri

http://hdl.handle.net/10150/204334

OAI identifier oai:identifier

oai:repository.arizona.edu:10150/204334