Eukaryotes usually have two separate enzymes, while most prokaryotes have a single bifunctional protein that can carry out both catalyses, although exceptions occur in both cases.

While eukaryotic monofunctional riboflavin kinase is orthologous to the bifunctional prokaryotic enzyme,[2] the monofunctional FAD synthetase differs from its prokaryotic counterpart, and is instead related to the PAPS-reductase family.

However, archaeal riboflavin kinases (EC 2.7.1.161) in general utilize CTP rather than ATP as the donor nucleotide, catalyzing the reaction Riboflavin kinase can also be isolated from other types of bacteria, all with similar function but a different number of amino acids.

The riboflavin kinase enzyme isolated from Thermoplasma acidophilum contains 220 amino acids.

As of late 2007, 14 structures have been solved for this class of enzymes, with PDB accession codes 1N05, 1N06, 1N07, 1N08, 1NB0, 1NB9, 1P4M, 1Q9S, 2P3M, 2VBS, 2VBT, 3CTA, 2VBU, and 2VBV.