The amino acid L-arginine is an essential component of all living organisms. Its importance resides in the variety of functions that arginine itself, along with some intermediary metabolites involved in its de novo synthesis in the cell. In many prokaryotes, fungi, and plants the de novo biosynthesis of arginine proceeds from glutamate in eight enzymatic steps (Figure 2). The first committed step of this pathway is the N-acetylation of glutamate. Acetylation of the early precursors of arginine distinguishes them from the analogous intermediates in the biosynthesis of proline. Although each and every step of the pathway is essential for its completion, transcarbamylation of ornithine to produce citrulline is one of the crucial steps in the pathway. This reaction is catalyzed by the enzyme ornithine transcarbamylase (OTC). The transcarbamylases family of enzymes belongs to the carboxyl- and carbamoyltransferases group, EC 2.1.3, a family that comprises enzymes that catalyze the transfer of a carbamoyl group from carbamylphosphate (CP) to an amino or oxygen group of a second substrate. Members of the transcarbamylase family can be identified based on sequence identities in the N-terminal or CP-binding domain as all the members of this family share common residues involved in the binding of CP to the enzyme. All the evolutionary conserved motifs present in the transcarbamylase family have led to the erroneous annotation of a large, yet increasing, number of genes as OTCases without experimental confirmation of such activity. The case of the transcarbamylase-like gene found in B. fragilis illustrates this statement. The present thesis work is focused in the case of two families of misannotated microbial transcarbamylases that have been identified as essential for arginine biosynthesis, but which lacked the ability to catalyze the conversion of ornithine into citrulline. The objectives of the present thesis work are the biochemical and molecular characterization of the N-acetylornithine transcarbamylase (AOTC) activity of XcArgF’ as well as the elucidation of the enzymatic activity of BfArgF’ and its biochemical and molecular characterization. We show that Xanthomonads and Bacteroidetes use novel pathways for the de novo arginine biosynthesis mediated by acetyl- and succinyl-ornithine transcarbamylases, we identify a recognition motif for these enzymes and we provide evidence supporting the view that the existence of succinyl-ornithine transcarbamylase requires that the first five steps of the arginine biosynthesis pathway use succinylated rather than acetylated intermediates.