Characterization of CamH from Methanosarcina thermophila, founding member of a subclass of the {gamma} class of carbonic anhydrases

Abstract

The homotrimeric enzyme Mt-Cam from Methanosarcina thermophila is the archetype of the gamma class of carbonic anhydrases. A search of databases queried with Mt-Cam revealed that a majority of the homologs comprise a putative subclass (CamH) in which there is major conservation of all of the residues essential for the archetype Mt-Cam except Glu62 and an acidic loop containing the essential proton shuttle residue Glu84. The CamH homolog from M. thermophila (Mt-CamH) was overproduced in Escherichia coli and characterized to validate its activity and initiate an investigation of the CamH subclass. The Mt-CamH homotrimer purified from E. coli cultured with supplemental zinc (Zn-Mt-CamH) contained 0.71 zinc and 0.15 iron per monomer and had k(cat) and k(cat)/K(m) values that were substantially lower than those for the zinc form of Mt-Cam (Zn-Mt-Cam). Mt-CamH purified from E. coli cultured with supplemental iron (Fe-Mt-CamH) was also a trimer containing 0.15 iron per monomer and only a trace amount of zinc and had an effective k(cat) (k(cat)(eff)) value normalized for iron that was 6-fold less than that for the iron form of Mt-Cam, whereas the k(cat)/K(m)(eff) was similar to that for Fe-Mt-Cam. Addition of 50 mM imidazole to the assay buffer increased the k(cat)(eff) of Fe-Mt-CamH more than 4-fold. Fe-Mt-CamH lost activity when it was exposed to air or 3% H(2)O(2), which supports the hypothesis that Fe(2+) has a role in the active site. The k(cat) for Fe-Mt-CamH was dependent on the concentration of buffer in a way that indicates that it acts as a second substrate in a "ping-pong" mechanism accepting a proton. The k(cat)/K(m) was not dependent on the buffer, consistent with the mechanism for all carbonic anhydrases in which the interconversion of CO(2) and HCO(3)(-) is separate from intermolecular proton transfer.

FIG. 1.

Phylogenetic tree of selected Cam and CamH homologs. Cam homologs are indicated by bold type. The CamH homolog from M. thermophila is indicated by an asterisk. The members of the Eukarya included are Vitis vinifera (accession no. gi|157335308), Zea mays (gi|219362885), Entamoeba dispar (gi|167389375), Arabidopsis thaliana (gi|10177532), Chlamydomonas reinhardtii (gi|159490549), and Yersinia pestis (gi|22127899). The members of the Bacteria included are Syntrophus aciditrophicus (gi|85859775), Pelobacter carbinolicus (gi|77919438), Vibrio cholerae (gi|153217568), Mycobacterium tuberculosis (gi|215432497), Dictyoglomus thermophilum (gi|206900416), and Escherichia coli (gi|563866). The members of the Archaea included are Sulfolobus solfataricus (gi|15897307), Methanospirillum hungatei (gi|88601918), Pyrococcus horikoshii (gi|14591369), Methanothermobacter thermoautotrophicus (gi|15679583), three Methanosarcina acetivorans strains (indicated by 2 [gi|20090000], 1 [gi|20091364], and 3 [gi|20089968]), three Methanosarcina mazei strains (indicated by 2 [gi|21228263], 1 [gi|21229190], and 3 [gi|21228253]), two Methanosarcina barkeri strains (indicated by 2 [gi|73669360] and 1 [gi|73670479]), Methanosarcina thermophila (gi|1827571) (Cam), and Methanosaeta thermophila (gi|116754979). The tree was constructed as described in reference .

FIG. 2.

Sequence alignment of Mt-Cam and Mt-CamH. +, catalytically relevant residues in Mt-Cam; %, metal ligands in Mt-Cam; #, structurally relevant residues in Mt-Cam. Acidic loop residues, including the proton shuttle residue Glu84 in Mt-Cam, are underlined. The sequences were aligned using the CLUSTAL 2.0.10 multiple-sequence alignment program.

FIG. 3.

Effect of oxidation on Fe-Mt-CamH activity. The enzyme (subunit concentration, 400 nM) was exposed to 1 atm air and assayed at the times indicated using the stopped-flow method at 25°C in 50 mM HEPES (pH 7.5). The CO₂ concentration used was 24.7 mM. A control incubated anaerobically exhibited 100% activity over the time period shown (data not shown).

FIG. 4.

pH dependence of CO₂ hydration catalyzed by Fe-Mt-CamH. Activities were measured in 50 mM buffer at an ionic strength of 150 mM and 25°C. The subunit concentration used was 1 μM. The CO₂ concentration was varied from 7.9 to 24.7 mM. Data were weighted based on the standard errors determined by fitting the observed initial rates to the Michaelis-Menten equation.

FIG. 5.

Buffer dependence of CO₂ hydration catalyzed by Fe-Mt-Cam. The CO₂ hydration activity (subunit concentration, 1.0 μM) was measured at pH 8.2, an ionic strength of 150 mM, and 25°C. The CO₂ concentration was varied from 7.9 to 24.7 mM, and the TAPS concentration was varied from 10 to 100 mM. The K_m for TAPS was 10.3 ± 0.2 mM, and the maximum k_cat was 1.9 × 10³ ± 0.01 × 10³ s⁻¹. Both kinetic parameters were determined by fitting the observed initial rates to the Michaelis-Menten equation.

FIG. 6.

Relative expression of the genes encoding Mt-Cam and Mt-CamH in acetate-, trimethylamine-, and methanol-grown M. acetivorans determined by quantitative RT-PCR. (A) Mt-Cam gene. (B) Mt-CamH gene. Ac, acetate; TMA, trimethylamine; MeOH, methanol.