Betalamic acid is the structural unit of all the natural pigments betalains. These are nitrogen-containing water-soluble compounds with high colorant and bioactive properties, characteristic of plants of the order Caryophyllales. The formation of betalamic acid from the precursor amino acid 3,4-dihydroxy-l-phenylalanine (l-DOPA) by the enzyme 4,5-DOPA-extradiol-dioxygenase was supposed to be restricted to plants of this order and two fungal species. Here, the first case of betalamic acid formation by an enzyme other than eukaryotes is reported with a homolog enzyme from Escherichia coli. The protein YgiD has been cloned, expressed, and purified to carry out its molecular and functional characterization. The enzyme was obtained as a monomeric active protein with a molecular mass of 32 kDa characterized by chromatography, electrophoresis, and MALDI-TOF analysis. Enzyme kinetic properties are characterized in the transformation of the relevant substrate l-DOPA. Reaction was analyzed spectrophotometrically and by HPLC-DAD, electrospray ionization mass spectrometry, and time-of-flight mass spectrometry.