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).
Character count:Current: 171 words in first paragraph 1497 words in rest of text (excluding methods, references, legends).Permitted: 200 words (300 words max) for first paragraph 1500 words max for rest (excluding methods, references, legends). The crystal structure of the fimbrial usher protein FimD revealed an intricate, five-domain architecture: a β-barrel domain, incorporated into which is a β-sandwich plug domain, an Nterminal periplasmic domain (NTD) and two C-terminal periplasmic domains (CTD1 and CTD2) 6,7 ( Supplementary Fig. S1). The mechanism for folding and assembling such a large, multi-domains, protein into the outer membrane is unclear, and currently no assay system is available to study the process in vivo. A candidate to catalyze this assembly reaction was the β-barrel assembly machinery (BAM complex): composed of the essential proteins BamA and BamD, assisted by the proteins BamB, BamC and BamE; Fig. 1a. In addition, bacteria haveTamA and TamB (Fig. 1a) that constitute a translocation and assembly module (TAM) [10][11][12] . The
SummaryAdhesive chaperone-usher pili are long, supramolecular protein fibers displayed on the surface of many bacterial pathogens. The type 1 and P pili of uropathogenic Escherichia coli (UPEC) play important roles during urinary tract colonization, mediating attachment to the bladder and kidney, respectively. The biomechanical properties of the helical pilus rods allow them to reversibly uncoil in response to flow-induced forces, allowing UPEC to retain a foothold in the unique and hostile environment of the urinary tract. Here we provide the 4.2-Å resolution cryo-EM structure of the type 1 pilus rod, which together with the previous P pilus rod structure rationalizes the remarkable “spring-like” properties of chaperone-usher pili. The cryo-EM structure of the type 1 pilus rod differs in its helical parameters from the structure determined previously by a hybrid approach. We provide evidence that these structural differences originate from different quaternary structures of pili assembled in vivo and in vitro.
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