Crystal structure of carbonic anhydrase from Neisseria gonorrhoeae and its complex with the inhibitor acetazolamide

Huang, Sijia; Xue, Yafeng; Sauer‐Eriksson, A. Elisabeth; Chirică, Laura C.; Lindskog, Sven; Jonsson, Bengt‐Harald

doi:10.1006/jmbi.1998.2077

Cited by 104 publications

(118 citation statements)

References 35 publications

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“…We have termed the M3 position the swivel position owing to this conformational variability as well as the open character of the active site cleft in this region. Additionally, the presence of a histidine (His-273) at a key position of the active site suggests that the communication proceeds via a proton shuttle (19)(20)(21)(22). Histidine residues are well suited to be both donors and acceptors of protons at physiological pH, and the presence of His-273 in coordinating the active site and making contact with Asp-270 may indicate such a role.…”

Section: Resultsmentioning

confidence: 99%

Golgi α-mannosidase II cleaves two sugars sequentially in the same catalytic site

Shah

Kuntz

Rose

2008

Proc. Natl. Acad. Sci. U.S.A.

101

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Golgi ␣-mannosidase II (GMII) is a key glycosyl hydrolase in the N-linked glycosylation pathway. It catalyzes the removal of two different mannosyl linkages of GlcNAcMan 5GlcNAc2, which is the committed step in complex N-glycan synthesis. Inhibition of this enzyme has shown promise in certain cancers in both laboratory and clinical settings. Here we present the high-resolution crystal structure of a nucleophile mutant of Drosophila melanogaster GMII (dGMII) bound to its natural oligosaccharide substrate and an oligosaccharide precursor as well as the structure of the unliganded mutant. These structures allow us to identify three sugar-binding subsites within the larger active site cleft. Our results allow for the formulation of the complete catalytic process of dGMII, which involves a specific order of bond cleavage, and a major substrate rearrangement in the active site. This process is likely conserved for all GMII enzymes-but not in the structurally related lysosomal mannosidase-and will form the basis for the design of specific inhibitors against GMII.cancer therapy ͉ enzyme mechanism ͉ N-glycosylation pathway ͉ x-ray crystallography ͉ glycoside hydrolase

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Section: Resultsmentioning

confidence: 99%

Golgi α-mannosidase II cleaves two sugars sequentially in the same catalytic site

Shah

Kuntz

Rose

2008

Proc. Natl. Acad. Sci. U.S.A.

101

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“…A sequence-similarity search against proteins with known structure in the RCSB Protein Data Bank revealed that Hp CA shares 39% identity with CA from Sulfurihydrogenibium yellowstonense YO3AOP1 (PDB entry 4g7a; Di Fiore et al, 2013) in a 228-residue overlap and 38% identity with carbonic anhydrase from Neisseria gonorrhoeae (PDB code 1kop; Huang et al, 1998) in a 231-residue overlap. Molecular replacement was performed with Phaser using data to 2.5 Å resolution (McCoy et al, 2005).…”

Section: Molecular Replacementmentioning

confidence: 99%

Cloning, purification and preliminary crystallographic analysis of the complex ofHelicobacter pyloriα-carbonic anhydrase with acetazolamide

2013

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Helicobacter pylori infection of the stomach can lead to severe gastroduodenal diseases such as gastritis, peptic ulcers and gastric cancers. Periplasmic H. pylori -carbonic anhydrase (Hp CA) is essential for the acclimatization of the bacterium to the acidity of the stomach. Through the action of urease and carbonic anhydrases, the H. pylori periplasmic pH is maintained at around 6 in an environment with a pH as low as 2, which in turn facilitates the maintenance of a cytoplasmic pH close to neutral, allowing growth in the gastric niche. Crystals of Hp CA in complex with the inhibitor acetazolamide have been grown by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitating agent. The crystals have the symmetry of space group P2 1 2 1 2 1 , with unit-cell parameters a = 37.0, b = 82.4, c = 150.8 Å . An X-ray diffraction data set was collected from a single crystal to 1.7 Å resolution. Calculation of the self-rotation function using this data and molecular replacement showed that the asymmetric unit contains an Hp CA dimer.

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“…To assign the bridge to suitable positions, we took advantage of the recently described structure of the enzyme designated NGCA, a related carbonic anhydrase found in the bacterium Neisseria gonorrhoeae (12). Although the homology between HCA II and NGCA is not very high (sequence identity 38.5%) (12), the three-dimensional structure of the two enzymes is highly similar ( Figure 1A). In addition, NGCA has a disulfide bridge that corresponds to the buried residues Ala23 and Leu203 in HCA II, which makes this protein extremely stable (13) and therefore a suitable candidate model.…”

mentioning

confidence: 99%

Dramatic Stabilization of the Native State of Human Carbonic Anhydrase II by an Engineered Disulfide Bond

2002

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To find a disulfide pair that could stabilize the enzyme human carbonic anhydrase II (HCA II), we grafted the disulfide bridge from the related and unusually stable carbonic anhydrase form from Neisseria gonorrhoeae (NGCA) into the human enzyme. Thus, the two Cys residues at positions 23 and 203 were engineered into a pseudo-wild-type form of HCA II (C206S), giving the mutant C206S/A23C/ L203C. The disulfide bond was not formed spontaneously. The native state of the reduced form of the mutant was markedly destabilized (2.9 kcal/mol) compared to that of HCA II. Formation of a disulfide bridge was achieved by treatment by oxidized glutathione. This led to a significant stabilization of the native conformation. Compared to HCA II the unfolding midpoint for the variant was increased from 0.9 to 1.7 M guanidine HCl, corresponding to a stabilization of 3.7 kcal/mol. This makes the human enzyme almost as stable as the model protein NGCA, for which the unfolding of the native state has a midpoint at 2.1 M guanidine HCl. The stabilized protein underwent, contrary to all other investigated variants of HCA II, an apparent two-state unfolding transition, as judged from intrinsic Trp fluorescence measurements. A molten-globule intermediate is nevertheless formed but is suppressed because of the high denaturant pressure it faces upon rupture of the native state.

show abstract

Crystal structure of carbonic anhydrase from Neisseria gonorrhoeae and its complex with the inhibitor acetazolamide

Cited by 104 publications

References 35 publications

Golgi α-mannosidase II cleaves two sugars sequentially in the same catalytic site

Golgi α-mannosidase II cleaves two sugars sequentially in the same catalytic site

Cloning, purification and preliminary crystallographic analysis of the complex ofHelicobacter pyloriα-carbonic anhydrase with acetazolamide

Dramatic Stabilization of the Native State of Human Carbonic Anhydrase II by an Engineered Disulfide Bond

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