Allosteric reversion of Haemophilus influenzae β-carbonic anhydrase via a proline shift.

Haemophilus influenzae β-carbonic anhydrase (HICA) has been reverse-engineered in the allosteric site region to resemble the nonallosteric Pisum sativum enzyme in order to identify critical features of allostery and intersusbunit communication. Three variants (W39V/G41A, P48S/A49P, and W39V/G41A/P48S/A49P) were identified, through a comparison with a crystal structure of nonallosteric P. sativum β-carbonic anhydrase (PSCA, PDB 1EKJ ), to potentially revert HICA to a nonallosteric enzyme.

Identification and characterization of a carboxysomal γ-carbonic anhydrase from the cyanobacterium Nostoc sp. PCC 7120.

Carboxysomes are proteinaceous microcompartments that encapsulate carbonic anhydrase (CA) and ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco); carboxysomes, therefore, catalyze reversible HCO3 (-) dehydration and the subsequent fixation of CO2. The N- and C-terminal domains of the β-carboxysome scaffold protein CcmM participate in a network of protein-protein interactions that are essential for carboxysome biogenesis, organization, and function. The N-terminal domain of CcmM in the thermophile Thermosynechococcus elongatus BP-1 is also a catalytically active, redox regulated γ-CA.

Structure and catalytic mechanism of β-carbonic anhydrases.

The β-carbonic anhydrases (β-CAs) are a structurally distinct family of carbonic anhydrase that is widely distributed in microorganisms, algae, plants, and invertebrates. Like all carbonic anhydrases, β-CAs catalyze the reaction CO2 + H2O ⇆ HCO3 (-) + H(+), and is typically associated with other enzymes that produce or utilize CO2 or HCO3 (-). β-CA is required for normal growth for many organisms.

Structure and catalytic mechanism of nicotinate (vitamin B3) degradative enzyme maleamate amidohydrolase from Bordetella bronchiseptica RB50.

The penultimate reaction in the oxidative degradation of nicotinate (vitamin B(3)) to fumarate in several species of aerobic bacteria is the hydrolytic deamination of maleamate to maleate, catalyzed by maleamate amidohydrolase (NicF). Although it has been considered a model system for bacterial degradation of N-heterocyclic compounds, only recently have gene clusters that encode the enzymes of this catabolic pathway been identified to allow detailed investigations concerning the structural basis of their mechanisms. Here, the Bb1774 gene from Bordetella bronchiseptica RB50, putatively annotated as nicF, has been cloned, and the recombinant enzyme, overexpressed and purified from Escherichia coli, is shown to catalyze efficiently the hydrolysis of maleamate to maleate and ammonium ion.

Re(CO)(3)(H(2)O)(3)(+) binding to lysozyme: structure and reactivity.

The reaction of Re(CO)(3)(H(2)O)(3)(+) with hen egg white lysozyme in aqueous solution results in a single covalent adduct. Both NMR spectroscopy and single crystal X-ray diffraction show that the rhenium tricarbonyl cation binds to His15 via replacement of one of the coordinated water molecules. The formation of this adduct does not greatly affect the structure of the protein.

Co(II)-substituted Haemophilus influenzae β-carbonic anhydrase: spectral evidence for allosteric regulation by pH and bicarbonate ion.

Cobalt(II)-substituted Haemophilus influenzae β-carbonic anhydrase (HICA) has been produced by overexpression in minimal media supplemented with CoCl(2), enabling kinetic, structural, and spectroscopic characterization. Co(II)-substituted HICA (Co-HICA) has comparable catalytic activity to that of wild-type enzyme with k(cat)=82±19 ms(-1) (120% of wild-type). The X-ray crystal structure of Co-HICA was determined to 2.

Specific derivatization of lysozyme in aqueous solution with Re(CO)3(H2O)3(+).

The reaction of Re(CO)(3)(H(2)O)(3)(+) with hen egg lysozyme in aqueous solution results in a single covalent adduct; single crystal X-ray diffraction shows that the rhenium tricarbonyl cation binds to His15 in two significantly populated rotamer conformations.

Evidence for a bicarbonate "escort" site in Haemophilus influenzae beta-carbonic anhydrase .

The Haemophilus influenzae beta-carbonic anhydrase (HICA) allosteric site variants V47A and G41A were overexpressed and purified to homogeneity. These variants have k(cat)/K(m) values similar to that of the wild-type enzyme and exhibit a similar dramatic decrease in catalytic activity at pH <8.0.

Structure and catalytic mechanism of the beta-carbonic anhydrases.

The beta-carbonic anhydrases (beta-CAs) are a diverse but structurally related group of zinc-metalloenzymes found in eubacteria, plant chloroplasts, red and green algae, and in the Archaea. The enzyme catalyzes the rapid interconversion of CO(2) and H(2)O to HCO(3)(-) and H(+), and is believed to be associated with metabolic enzymes that consume or produce CO(2) or HCO(3)(-). For many organisms, beta-CA is essential for growth at atmospheric concentrations of CO(2).

Allosteric site variants of Haemophilus influenzae beta-carbonic anhydrase.

Haemophilus influenzae beta-carbonic anhydrase (HICA) is hypothesized to be an allosteric protein that is regulated by the binding of bicarbonate ion to a non-catalytic (inhibitory) site that controls the ligation of Asp44 to the catalytically essential zinc ion. We report here the X-ray crystallographic structures of two variants (W39F and Y181F) involved in the binding of bicarbonate ion in the non-catalytic site and an active-site variant (D44N) that is incapable of forming a strong zinc ligand. The alteration of Trp39 to Phe increases the apparent K(i) for bicarbonate inhibition by 4.

Identification of a novel noncatalytic bicarbonate binding site in eubacterial beta-carbonic anhydrase.

The structures of beta class carbonic anhydrases (beta-CAs) determined so far fall into two distinct subclasses based on the observed coordination of the catalytic zinc (Zn2+) ion. The subclass of beta-CAs that coordinate Zn2+ tetrahedrally with four protein-derived ligands is represented by the structures of orthologues from Porphyridium purpureum, Escherichia coli, and Mycobacterium tuberculosis. Here we present the structure of an additional member of that subclass, that from Haemophilus influenzae, as well as detailed kinetic analysis, revealing the correspondence between structural classification and kinetic profile for this subclass.

Complementation of the yeast deletion mutant DeltaNCE103 by members of the beta class of carbonic anhydrases is dependent on carbonic anhydrase activity rather than on antioxidant activity.

In recent years, members of the beta class of CAs (carbonic anhydrases) have been shown to complement Delta NCE103, a yeast strain unable to grow under aerobic conditions. The activity required for complementation of Delta NCE103 by tobacco chloroplast CA was studied by site-directed mutagenesis. E196A (Glu196-->Ala), a mutated tobacco CA with low levels of CA activity, complemented Delta NCE103.

Examination of the role of Gln-158 in the mechanism of CO(2) hydration catalyzed by beta-carbonic anhydrase from Arabidopsis thaliana.

We have cloned and overexpressed a variant of Arabidopsis thaliana beta-carbonic anhydrase (Q158A) that deletes the functional equivalent of the backbone amide NH of Thr-199 in human alpha-carbonic anhydrase II. The latter residue is hypothesized to be important in catalyzing the rate of CO(2)(-) HCO (3)(-) interconversion in alpha-carbonic anhydrase but this hypothesis is not directly testable in that enzyme. Kinetic studies of a variant of the functionally equivalent residue in A.

Chemical rescue of proton transfer in catalysis by carbonic anhydrases in the beta- and gamma-class.

Catalysis of the dehydration of HCO(3)(-) by carbonic anhydrase requires proton transfer from solution to the zinc-bound hydroxide. Carbonic anhydrases in each of the alpha, beta, and gamma classes, examples of convergent evolution, appear to have a side chain extending into the active site cavity that acts as a proton shuttle to facilitate this proton transfer, with His 64 being the most prominent example in the alpha class. We have investigated chemical rescue of mutants in two of these classes in which a proton shuttle has been replaced with a residue that does not transfer protons: H216N carbonic anhydrase from Arabidopsis thaliana (beta class) and E84A carbonic anhydrase from the archeon Methanosarcina thermophila (gamma class).

Kinetic characterization of wild-type and proton transfer-impaired variants of beta-carbonic anhydrase from Arabidopsis thaliana.

We have cloned and overexpressed a truncated, recombinant form of beta-carbonic anhydrase from Arabidopsis thaliana. The wild-type enzyme and two site-directed variants, H216N and Y212F, have been kinetically characterized both at steady state by stopped-flow spectrophotometry and at chemical equilibrium by (18)O isotope exchange methods. The wild-type enzyme has a maximal k(cat) for CO2 hydration of 320 ms(-1) and is rate limited by proton transfer involving two residues with apparent pK(a) values of 6.