Nontypeable Haemophilus influenzae Carbonic Anhydrase Is Important for Environmental and Intracellular Survival

Item does not contain fulltextNontypeable Haemophilus influenzae (NTHi) is one of the leading causes of noninvasive mucosal infections, such as otitis media, sinusitis, and conjunctivitis. During its life cycle, NTHi is exposed to different CO2 levels, which vary from approximately 0.04% in ambient air during transmission to a new host to over 5% in the respiratory tract and tissues of the human host during colonization and disease. We used the next-generation sequencing Tn-seq technology to identify genes essential for NTHi adaptation to changes in environmental CO2 levels. It appeared that H. influenzae carbonic anhydrase (HICA), which catalyzes the reversible hydration of CO2 to bicarbonate, is a molecular factor that is conditionally essential for NTHi survival in ambient air. Growth of NTHi Deltacan strains was restored under 5% CO2-enriched conditions, by supplementation of the growth medium with sodium bicarbonate, or by genetic complementation with the can gene. Finally, we showed that HICA not only is essential for environmental survival but also appeared to be important for intracellular survival in host cells. Hence, HICA is important for NTHi niche adaptation

Structural studies of β-Carbonic Anhydrase from the Green Alga Coccomyxa : Inhibitor complexes with Anions and Acetazolamide

The β-class carbonic anhydrases (β-CAs) are widely distributed among lower eukaryotes, prokaryotes, archaea, and plants. Like all CAs, the β-enzymes catalyze an important physiological reaction, namely the interconversion between carbon dioxide and bicarbonate. In plants the enzyme plays an important role in carbon fixation and metabolism. To further explore the structure-function relationship of β-CA, we have determined the crystal structures of the photoautotroph unicellular green alga Coccomyxa β-CA in complex with five different inhibitors: acetazolamide, thiocyanate, azide, iodide, and phosphate ions. The tetrameric Coccomyxa β-CA structure is similar to other β-CAs but it has a 15 amino acid extension in the C-terminal end, which stabilizes the tetramer by strengthening the interface. Four of the five inhibitors bind in a manner similar to what is found in complexes with α-type CAs. Iodide ions, however, make contact to the zinc ion via a zinc-bound water molecule or hydroxide ion - a type of binding mode not previously observed in any CA. Binding of inhibitors to Coccomyxa β-CA is mediated by side-chain movements of the conserved residue Tyr-88, extending the width of the active site cavity with 1.5-1.8 Å. Structural analysis and comparisons with other α- and β-class members suggest a catalytic mechanism in which the movements of Tyr-88 are important for the CO(2)-HCO(3) (-) interconversion, whereas a structurally conserved water molecule that bridges residues Tyr-88 and Gln-38, seems important for proton transfer, linking water molecules from the zinc-bound water to His-92 and buffer molecules