The ubiquinone (UQ) is a lipophilic molecule which is essential to respiratory chains, as it is involved in shuttling electrons between respiratory enzymes. The UQ biosynthetic pathway includes three hydroxylation reactions on three positions of the UQ precursor: C5, C1 and C6. In Escherichia coli, these three hydroxylation steps are performed by three different enzymes whereas some bacteria use two enzymes (e.g. UbiL and Coq7 for Rhodospirillum rubrum), or even a single enzyme (e.g. UbiM for Nesseiria meningitidis) to perform the three reactions [1]. This denotes a variation in “regio-selectivity” across UQ hydroxylases. Interestingly, the six known UQ hydroxylases belong to only two distinct protein families. Coq7 is a member of the di-iron carboxylate family, whereas all other hydroxylases identified so far (UbiF, -H, -I, -L, -M, thereafter called “Ubi-FMO”) belong to the large Flavin Mono-oxygenase family. The Ubi-FMO family offers the unique opportunity to study the evolution of enzymatic regio-selectivity among homologous enzymes. In this study, we combined experimental and computational approaches to investigate the evolution of regio-selectivity across UQ hydroxylases. Based on genomic distribution, we could predict expected regio-selectivity for the different UQ hydroxylases. We then performed phylogenetic analyses to (i) sample taxonomically representative sequences for experimental characterization, and (ii) investigate potential evolutionary scenarios for Ubi-FMO and the overall genomic repertoire in UQ hydroxylases. In order to test the enzymes’ predicted activity, experimental characterizations of UQ hydroxylases were performed on an unprecedented scale for >100 hydroxylases across Proteobacteria using heterologous expressions in relevant KO mutants of E. coli. We could systematically confirm the anticipated regio-selectivity, showing that regio-selectivity is evolutionarily conserved for each different protein sub-family. We propose an evolutionary scenario of the evolution of UQ hydroxylases’ regio-selectivity. Overall, this study provides an interesting case study for the evolution of enzymatic function, and paves the way for future research on the precise molecular mechanisms dictating enzymes’ regio-selectivity. [1] Pelosi L., Ducluzeau A. L., Loiseau L., Barras F., Schneider D., Junier I., Pierrel F. (2016) Evolution of Ubiquinone Biosynthesis: Multiple Proteobacterial Enzymes with Various Regioselectivities To Catalyze Three Contiguous Aromatic Hydroxylation Reactions. mSystems 1: e00091-16 DOI: 10.1128/mSystems.00091-16