The common bread mold, Neurospora crassa, is a model organism that encodes an amidinotransferase similar to those found in humans and in a variety of bacteria. Amidinotransferase enzymes catalyze the transfer of an amidino group from a donor molecule (usually arginine) to an acceptor molecule bearing a primary amine. They play a role in a variety of essential metabolic pathways, and they are also found in some fungi, including N. crassa, where their biochemical function is unknown. This project sought to investigate the biochemical function of this conserved amidinotransferase from N. crassa (NcAMT). The gene encoding NcAMT from strain ATCC 24698 was cloned into the prokaryotic expression vector pET28b using a gene synthesis strategy based on accession number Q1K5W2 from the UniProt database. Recombinant NcAMT was expressed in E. coli and purified using immobilized metal affinity chromatography. The oligomeric state of NcAMT was investigated using size exclusion chromatography and found to be a dimer. An HPLC-based amidinotransferase functional assay was carried out to investigate the amidino acceptor substrate specificity and the pH- and temperature-activity profiles of the enzyme and to measure the KM for several substrates. NcAMT used arginine as an amidino donor, with the KM determined to be 15 mM. Of the amidino acceptor substrates examined, lysine appeared to be the most efficient, with a KM of 1.5 mM; putrescine and other short polyamines also showed some activity. NcAMT displayed optimal enzymatic activity at pH 8.0 and 50 °C.

Neurospora crassa, a common bread mold, encodes an arginine-lysine amidinotransferase

Macromolecular Chemistry: The Second Century

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ACS Spring 2020 National Meeting & Expo

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.