Document Type

Thesis

Publication Date

4-2015

Disciplines

Chemistry | Physical Sciences and Mathematics

Advisor

Rachel Hutcheson, Chemistry

Abstract

Radical S-adenosylmethionine (SAM) enzymes are a superfamily of enzymes that require the cofactor SAM for catalysis. These enzymes have diverse functions across all 3 domains of life, and are known to play roles in many biological pathways, including vitamin biosynthesis, synthesis of complex metal clusters, posttranslational RNA modification,DNA repair, and development of antibiotic resistance, among others. Radical SAM enzymes are characterized by the CX3CX2C motif, which is conserved throughout most members of the family. A number of genes have been identified which contain the CX3CX2C motif and other amino acid sequences characteristic to radical SAM enzymes, however the majority of these suspected members of the radical SAM superfamily remain poorly understood. To date, only 14 enzymes out of the suspected 50,000 have been fully characterized, including a crystal structure. In order to gain a more complete understanding of the superfamily and their biological significance, it is therefore essential that more of these suspected enzymes are characterized.

For this project, two of these uncharacterized genes were chosen from the structure-function linkage database (SFLD) for further study. The first, referred to as yfgB, showed sequence similarity to the class A radical SAM methyltransferases, and was picked based on the presence of a homolog in the BL21(DE3) E. Coli genomic DNA available to the laboratory. This homolog was successfully amplified via polymerase chain reaction (PCR) and sequentially digested with restriction enzymes Blp1 and BamH1, and is now ready to be ligated into the bacterial expression vector pet14b. The second gene, referred to as RS11, codes for an uncharacterized radical SAM enzyme in subgroup 11 (from Thermotoga neapolitana DSM 4359, NCBI taxonomy ID: 309803). This subgroup is pyridoxal phosphate (PLP)-dependent, and members show sequence homology to aminomutases. A pEX-N-His bacterial expression vector was purchased with the RS11 gene inserted from Blue Heron Gene Company and transformed into BL21(DE3) E. Coli. Attempts to express the RS11 gene and purify the corresponding protein have been unsuccessful thus far.

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