Functional assignment of enzymes encoded by the Mycobacterium tuberculosis genome is largely incomplete despite recent advances in genomics and bioinformatics. Here, we applied an activity-based metabolomic profiling method to assign function to a unique phosphatase, Rv1692. In contrast to its annotation as a nucleotide phosphatase, metabolomic profiling and kinetic characterization indicate that Rv1692 is a D,L- glycerol 3-phosphate phosphatase. Crystal structures of Rv1692 reveal a unique architecture, a fusion of a predicted haloacid dehalogenase fold with a previously unidentified GCN5-related N -acetyltransferase region. Although not directly involved in acetyl transfer, or regulation of enzymatic activity in vitro, this GCN5-related N-acetyltransferase region is critical for the solubility of the phosphatase. Structural and biochemical analysis shows that the active site features are adapted for recognition of small polyol phosphates, and not nucleotide substrates. Functional assignment and metabolomic studies of M. tuberculosis lacking rv1692 demonstrate that Rv1692 is the final enzyme involved in glycerophospholipid recycling/catabolism, a pathway not previously described in M. tuberculosis .

Controlled halogenation of chemically versatile substrates is difficult to achieve. Here we describe a unique flavin-dependent halogenase, PltM, which is capable of utilizing a wide range of halides for installation on a diverse array of phenolic compounds, including FDA-approved drugs and natural products, such as terbutaline, fenoterol, resveratrol, and catechin. Crystal structures of PltM in complex with phloroglucinol and FAD in different states yield insight into substrate recognition and the FAD recycling mechanism of this halogenase.