LAUSR

Currently, there are more than 216 million annotated protein sequences available in public databases, a number that doubles every 28 months, and just like the deep sea floor, only a minuscule portion of this territory has been explored. Each sequence encodes for a protein with a unique composition and order of amino acids that dictate its fold, and in the case of an enzyme, the reactions it can catalyze. However, predicting function based on sequence is not an easy feat. Typically, function has been experimentally determined through labor-intensive protein expression and isolation coupled with experimental characterization of enzymes from primary metabolism and natural product biosynthetic pathways. In this issue of ACS Central Science, Lewis and co-workers survey the activity across one family of enzymes in order to profile reactivity and selectivity across a range of substrates. Well-characterized enzymes have historically served as benchmarks for predicting function of uncharacterized enzymes. For example, flavin-dependent monooxygenases (FDMOs) can mediate various transformations depending on their fold. One known function for a subset of monooxygenases, class F flavin adenine dinucleotide (FAD)dependent monooxygenases, is halogenation. This class of enzymes shares a structurally similar nucleotide binding site to class A aromatic hydroxylases; however, a unique tryptophan cage provides class F FAD-dependent monooxygenases with a characteristic sequence fingerprint. To predict function and mechanism, experimental findings on class F FDMOs coupled with the amino acid fingerprint are often applied to related sequences. While this approach can lead to accurate function assignments in some cases, there other instances in which enzymes possess slightly altered motifs and can be overlooked in such a function assignment. By constructing a sequence similarity network (SSN) containing sequences with the highest similarity to wellstudied flavin-dependent halogenases (FDHs) involved in indole alkaloid biosynthesis, the authors define the sequence space hypothesized to have conserved halogenase activity. This SSN contained nearly 4000 sequences, of which 129 had been previously characterized. Lewis and co-workers canvassed the FDH family and identified 128 putative FDHs based on a sequence motif conserved across characterized halogenases. To profile how these “unknown” sequences fit within the family, the authors profiled the activity of these enzymes against a panel of substrates and halide sources. This allowed the researchers to identify trends in reactivity across this family of enzymes and ultimately identify wildtype enzymes capable of halogenating previously intractable substrates in a site-selective manner.