Alkaline phosphatases play a crucial role in phosphate acquisition by microorganisms. To expand our understanding of catalysis by this class of enzymes we have determined the structure of the widely-occurring microbial alkaline phosphatase PhoX. The enzyme contains a complex active site cofactor comprising two antiferromagnetically-coupled Fe 3+ ions, three Ca 2+ ions, and a μ 3 -bridging oxo group. Notably, the main part of the cofactor resembles synthetic oxide-centered triangular metal complexes. Structures of PhoX-ligand complexes reveal how the active site metal ions bind substrate and implicate the cofactor oxo group in the catalytic mechanism. The presence of iron in PhoX raises the possibility that iron bioavailability limits microbial phosphate acquisition.Phosphate-containing macromolecules and metabolites are essential components of living cells. Under conditions of phosphate deficiency microorganisms obtain phosphate from biologically-derived organic compounds by producing extra-cytoplasmic alkaline phosphatases (1, 2). Prominent amongst these enzymes are phosphate monoesterases of the PhoA and PhoX families which are found in all three domains of life. The archetypal PhoA enzyme of Escherichia coli has been extensively studied (2) but PhoX alkaline phosphatases are minimally characterized and do not exhibit sequence similarity to other phosphotransfer enzymes. Genes encoding PhoX are abundant in ocean bacteria (3)(4)(5) and are also present in bloom-forming cyanobacteria (6), human pathogens (7,8), and eukaryotic green algae including the model organism Chlamydomonas reinhardtii (9).

The twin arginine translocation (Tat) system moves folded proteins across the cytoplasmic membrane of bacteria and the thylakoid membrane of plant chloroplasts. Signal peptide-bearing substrates of the Tat pathway (precursor proteins) are recognized at the membrane by the TatBC receptor complex. The only established preparation of the TatBC complex uses the detergent digitonin, rendering it unsuitable for biophysical analysis. Here we show that the detergent glyco-diosgenin (GDN) can be used in place of digitonin to isolate homogeneous TatBC complexes that bind precursor proteins with physiological specificity. We use this new preparation to quantitatively characterize TatBC–precursor interactions in a fully defined system. Additionally, we show that the GDN-solubilized TatBC complex co-purifies with substantial quantities of phospholipids.