NanC Crystal Structure, a Model for Outer-Membrane Channels of the Acidic Sugar-Specific KdgM Porin Family

Sialic acids are acidic sugars present mostly on vertebrate cell surfaces, which can be metabolized by bacteria and act as an inflammation signal. N-Acetylneuraminic acid, the most abundant sialic acid, can enter into Escherichia coli K12 through NanC, an N-acetylneuraminic acid-inducible outer-membrane channel. With its 215 residues, NanC belongs to the family of small monomeric KdgM-related porins. KdgM homologues are found in gammaproteobacteria, including major plant and human pathogens, and together they define a large family of putative acidic sugar/oligosaccharide transporters, which are as yet poorly characterized. Here, we present the first high-resolution structure of a KdgM family member. NanC folds into a 28-Å-high, 12-stranded β-barrel, resembling the β-domain of autotransporter NalP and defining an open pore with an average radius of 3.3 Å. The channel is lined by two strings of basic residues facing each other across the pore, a feature that appears largely conserved within the KdgM family and is likely to facilitate the diffusion of acidic oligosaccharides.     

The crystal structure of the cytosolic exopolyphosphatase from Saccharomyces cerevisiae reveals the basis for substrate specificity

Inorganic long-chain polyphosphate is a ubiquitous linear polymer in biology, consisting of many phosphate moieties linked by phosphoanhydride bonds. It is synthesized by polyphosphate kinase, and metabolised by a number of enzymes, including exo- and endopolyphosphatases. The Saccharomyces cerevisiae gene PPX1 encodes for a 45 kDa, metal-dependent, cytosolic exopolyphosphatase that processively cleaves the terminal phosphate group from the polyphosphate chain, until inorganic pyrophosphate is all that remains. PPX1 belongs to the DHH family of phosphoesterases, which includes: family-2 inorganic pyrophosphatases, found in Gram-positive bacteria; prune, a cyclic AMPase; and RecJ, a single-stranded DNA exonuclease. We describe the high-resolution X-ray structures of yeast PPX1, solved using the multiple isomorphous replacement with anomalous scattering (MIRAS) technique, and its complexes with phosphate (1.6 A), sulphate (1.8 A) and ATP (1.9 A). Yeast PPX1 folds into two domains, and the structures reveal a strong similarity to the family-2 inorganic pyrophosphatases, particularly in the active-site region. A large, extended channel formed at the interface of the N and C-terminal domains is lined with positively charged amino acids and represents a conduit for polyphosphate and the site of phosphate hydrolysis. Structural comparisons with the inorganic pyrophosphatases and analysis of the ligand-bound complexes lead us to propose a hydrolysis mechanism. Finally, we discuss a structural basis for substrate selectivity and processivity.     

Crystal structure of 3-hydroxybenzoate hydroxylase from Comamonas testosteroni has a large tunnel for substrate and oxygen access to the active site

The 3-hydroxybenzoate hydroxylase (MHBH) from Comamonas testosteroni KH122-3s is a single-component flavoprotein monooxygenase, a member of the glutathione reductase (GR) family. It catalyzes the conversion of 3-hydroxybenzoate to 3,4-dihydroxybenzoate with concomitant requirements for equimolar amounts of NADPH and molecular oxygen. The production of dihydroxy-benzenoid derivative by hydroxylation is the first step in the aerobic degradation of various phenolic compounds in soil microorganisms. To establish the structural basis for substrate recognition, the crystal structure of MHBH in complex with its substrate was determined at 1.8 A resolution. The enzyme is shown to form a physiologically active homodimer with crystallographic 2-fold symmetry, in which each subunit consists of the first two domains comprising an active site and the C-terminal domain involved in oligomerization. The protein fold of the catalytic domains and the active-site architecture, including the FAD and substrate-binding sites, are similar to those of 4-hydroxybenzoate hydroxylase (PHBH) and phenol hydroxylase (PHHY), which are members of the GR family, providing evidence that the flavoprotein aromatic hydroxylases share similar catalytic actions for hydroxylation of the respective substrates. Structural comparison of MHBH with the homologous enzymes suggested that a large tunnel connecting the substrate-binding pocket to the protein surface serves for substrate transport in this enzyme. The internal space of the large tunnel is distinctly divided into hydrophilic and hydrophobic regions. The characteristically stratified environment in the tunnel interior and the size of the entrance would allow the enzyme to select its substrate by amphiphilic nature and molecular size. In addition, the structure of the Xe-derivative at 2.5 A resolution led to the identification of a putative oxygen-binding site adjacent to the substrate-binding pocket. The hydrophobic nature of the xenon-binding site extends to the solvent through the tunnel, suggesting that the tunnel could be involved in oxygen transport.