Characterization of two distinct families of ornithine cyclodeaminases in Clostridium sporogenes: Biochemistry and genetics of microorganisms

Details

Supervisors

Desguin, Benoît

Faculty

Faculté des sciences

Degree label

Master [120] en biochimie et biologie moléculaire et cellulaire, à finalité approfondie

Abstract

enThe nickel-pincer nucleotide (NPN) is a novel organometallic cofactor that was initially identified in lactate racemase (LarA). It coordinates a nickel ion within a nucleotide scaffold, forming an unique stable metal–carbon bond. More recently, its functional scope has since been proposed to extend to the COG1915 family of ornithine cyclodeaminases (OCDs), which are thought to require a dinucleotidic variant, the nickel-pincer adenine dinucleotide (NPAD). The genome of Clostridium sporogenes encodes two distinct OCD families: an NAD-dependent enzyme (COG2423) and an NPAD-dependent enzyme (COG1915). Both are predicted to catalyse the ornithine-to-proline cyclodeamination reaction, raising the question of whether they function redundantly or under different metabolic conditions. This work aimed (i) to characterize and compare the biochemical properties of both OCDs in vitro, and (ii) to attempt their inactivation in vivo through CRISPR base editing. The NAD-dependent enzyme was expressed and purified from Escherichia coli, while the NPAD-dependent OCD from Clostridium sporogenes could not be cloned; instead, the ortholog from Aerococcus urinae was used. Both enzymes displayed true OCD activity but with distinct features: NAD-OCD showed higher thermal stability and broader activity, while NPAD-OCD was highly temperature-sensitive and rapidly lost activity outside optimal conditions. In parallel, CRISPR base editing using pCBEclos plasmids was applied in C. sporogenes. Although transconjugants were obtained, no stop codons were introduced at target sites. However, consistent bystander mutations confirmed that base editing is functional, although with limited efficiency and heterogeneity within colonies. Altogether, this thesis provides a first direct comparison between NAD- and NPAD-dependent OCDs, demonstrating distinct features despite catalysing the same reaction in the bacterium.