Observation of a calcium-binding site in the γ-class carbonic anhydrase fromPyrococcus horikoshii

Abstract

Carbonic anhydrases are zinc-containing metalloenzymes that catalyze the interconversion of carbon dioxide and bicarbonate. Three crystal structures of gamma-class carbonic anhydrase (one of which is bound to a bicarbonate molecule) from the aerobic OT3 strain of the hyperthermophilic archeon Pyrococcus horikoshii have been solved by molecular replacement in space group F4(1)32. The asymmetric unit contains a monomer of 173 amino acids and a catalytic Zn2+ ion. The protein fold is a regular prism formed by a left-handed beta-helix, similar to previously reported structures. The active-site Zn2+ ion located at the interface between the two monomers is bound to three histidyl residues and a water molecule in a tetrahedral fashion. In addition to the 20 beta-strands comprising the beta-helix, there is also a long C-terminal alpha-helix. For the first time, Ca2+ ions have been observed in addition to the catalytic Zn2+ ion. It is hypothesized that Tyr159 (which corresponds to the catalytically important Asn202 in previously reported structures) utilizes C-H...pi interactions to fulfill its functions. This study may shed light on the catalytic mechanism of the enzyme and throw open new questions on the mechanism of product removal in carbonic anhydrases.

Characterization of CamH from Methanosarcina thermophila , Founding Member of a Subclass of the γ Class of Carbonic Anhydrases

“…Thus, in the absence of accessory proteins to incorporate iron, zinc would be expected to occupy the active site of Mt-CamH when it is overproduced in E. coli cultured with supplemental zinc. Despite the presence of zinc in the crystal structure of the CamH homolog from the anaerobe P. horikoshii (14), the results presented here suggest that a role for iron in the active site needs to be examined considering that the enzyme was overproduced in E. coli.…”

“…The results of sequence analyses and the lack of CA activity of purified CamH homologs (14,27) have called into question the catalytic potential and other properties of the CamH subclass. Thus, Mt-CamH was overproduced in E. coli and characterized to begin to investigate the CamH subclass and increase our overall understanding of the ␥ class and the physiological role of CAs in Methanosarcina species.…”

“…In particular, the sequence analyses indicated that many homologs lack the acidic loop and the essential PSR Glu84 of Mt-Cam, suggesting that these homologs belong to a subclass of the ␥ class. However, the absence of CA activity reported for the two purified proteins belonging to the putative subclass (14,27) calls into question the catalytic activity, metal content, and other properties. Here we describe a more recent sequence analysis of the ␥ class and biochemical characterization of a representative of the putative subclass, Mt-CamH from M. thermophila, that was overproduced in E. coli and had substantial CA activity, although it has properties that distinguish it from Mt-Cam.…”

“…With only one exception, all of the sequences showed conservation with the Mt-Cam residues essential for metal binding and catalysis except Glu62 and acidic loop residues that include Glu84. The crystal structure of an Mt-Cam homolog from the anaerobic archaeon Pyrococcus horikoshii that was overproduced in E. coli (14) has left-handed parallel ␤-helix fold and active site architecture similar to that of MtCam (13) with bicarbonate bound to the active site zinc. However, the acidic loop and the PSR Glu84 of Mt-Cam are not conserved in the P. horikoshii enzyme, and CA activity was not examined.…”

Carbonic Anhydrases of Environmentally and Medically Relevant Anaerobic Prokaryotes

“…The CA from Thermosynechococcus elongatus (strain BP-1) of the domain Bacteria is the only other catalytically active CamH subclass homolog characterized (51). Crystal structures of two other CamH subclass homologs from E. coli (52) and Pyrococcus horikoshii (53) are available, although CA activity was not reported for either. However, both enzymes were purified in the presence of air and contain zinc in the active site, which presents the possibility that an active site iron is essential for activity.…”