First Advisor

Dirk Iwata-Reuyl

Date of Publication

Summer 8-10-2016

Document Type

Dissertation

Degree Name

Doctor of Philosophy (Ph.D.) in Chemistry

Department

Chemistry

Language

English

Subjects

Enzymes, Transfer RNA, Biosynthesis

DOI

10.15760/etd.3098

Physical Description

1 online resource (viii, 114 pages)

Abstract

The tunneling fold (T-Fold) superfamily is a small superfamily of enzymes found in organisms encompassing all kingdoms of life. Seven members have been identified thus far. Despite sharing a common three-dimensional structure these enzymes perform very diverse chemistries.

QueF is a bacterial NADPH-dependent oxidoreductase that catalyzes the reduction of the nitrile group of 7-cyano-7-deazaguanine (preQ0) to a primary amine (preQ1) in the queuosine biosynthetic pathway. Previous work on this enzyme has revealed the mechanism of reaction but the cofactor binding residues remain unknown. The experiments discussed herein aim to elucidate the role of residues lysine 80, lysine 83, and arginine 125 (B. subtilis numbering) in NADPH binding. The biological role of the disulfide bond between the conserved residues cysteine 55 and cysteine 99 observed in several crystal structures is also examined.

Characterization of QueF mutants K80A, K83, R125A and R125K revealed lysine 80, lysine 83 and arginine 125 are required for turnover. Further analysis of turnover rates for R125K are consistent with this residue and both lysines being involved in cofactor binding presumably by interacting with the negatively charged phosphate tail of NADPH and are therefore involved in cofactor binding. Based on bond angles and energies, the disulfide bond between Cys55 and Cys99 was characterized as non-structural. Enzyme oxidation assays were consistent with the bond serving to protect QueF against irreversible oxidation of Cys55, which would render the enzyme inactive. This is the only known example of a stress protective mechanism in the Tunneling-fold superfamily.

QueF-like is an amidinotransferase found in some species of Crenarchaeota and involved in the biosynthesis of archaeosine-tRNA. The work presented here is focused on the preliminary characterization of this enzyme, including the elucidation of the natural substrate as well as the source of ammonia. The structure of the enzyme was solved and is also discussed.

Substrate analysis for QueF-like indicated this enzyme is capable of binding both preQ0 and preQ0-tRNA and reacting to form a thioimide intermediate analogous to QueF but only the latter serves as a substrate for the reaction. This makes QueF-like the first example of a nucleic acid binding enzyme in the Tunneling-fold superfamily. Ammonia, glutamine and asparagine were tested as nitrogen sources and unlike most known amidotransferases, QueF-like can only use free ammonia to produce the archaeosine-tRNA product. The crystal structure of P. calidifontis QueF-like indicates the functional enzyme is a dimer of pentamers pinned together by a large number of salt bridges. The structure presents a high degree of similarity to that of QueF albeit the higher twist of the QueF-like pentamers with respect to QueF results in a more compact structure.

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Persistent Identifier

http://archives.pdx.edu/ds/psu/18077

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