Structural basis of the oxidative activation of the carboxysomal gamma-carbonic anhydrase, CcmM

Abstract

Cyanobacterial RuBisCO is sequestered in large, icosahedral, protein-bounded microcompartments called carboxysomes. Bicarbonate is pumped into the cytosol, diffuses into the carboxysome through small pores in its shell, and is then converted to CO(2) by carbonic anhydrase (CA) prior to fixation. Paradoxically, many beta-cyanobacteria, including Thermosynechococcus elongatus BP-1, lack the conventional carboxysomal beta-CA, ccaA. The N-terminal domain of the carboxysomal protein CcmM is homologous to gamma-CA from Methanosarcina thermophila (Cam) but recombinant CcmM derived from ccaA-containing cyanobacteria show no CA activity. We demonstrate here that either full length CcmM from T. elongatus, or a construct truncated after 209 residues (CcmM209), is active as a CA-the first catalytically active bacterial gamma-CA reported. The 2.0 A structure of CcmM209 reveals a trimeric, left-handed beta-helix structure that closely resembles Cam, except that residues 198-207 form a third alpha-helix stabilized by an essential Cys194-Cys200 disulfide bond. Deleting residues 194-209 (CcmM193) results in an inactive protein whose 1.1 A structure shows disordering of the N- and C-termini, and reorganization of the trimeric interface and active site. Under reducing conditions, CcmM209 is similarly partially disordered and inactive as a CA. CcmM protein in fresh E. coli cell extracts is inactive, implying that the cellular reducing machinery can reduce and inactivate CcmM, while diamide, a thiol oxidizing agent, activates the enzyme. Thus, like membrane-bound eukaryotic cellular compartments, the beta-carboxysome appears to be able to maintain an oxidizing interior by precluding the entry of thioredoxin and other endogenous reducing agents.

Fig. 1.

CcmM from T. elongatus is a carboxysomally associated γ-carbonic anhydrase. (A) CA activity assays illustrating the catalytic competency of the constructs. Assay condition: 100 mM N-(2-hyroxyethyl)piperazine-N ^′-(3-propanesulfonic acid) (EPPS) /NaOH, pH 8.0, 20 mM MgSO₄, 600 μM at 30 °C; CcmM193, 0.34 mg·mL^-1; CcmM652 (0.22 mg.mL^-1); CcmM209 (0.11 mg·mL^-1); bovine CAII (Sigma) (0.03 mg·mL^-1). EZA inhibits CcmM652 activity, but not completely. The uncatalyzed rate of ¹⁸O exchange was nearly identical to the CcmM193 trace. (B) CA activity catalyzed by partially purified T. elongatus carboxysomes (cbx) at 4.2 mg·mL^-1; this activity is inhibited by EZA and suppressed by boiling. (C) Negatively stained electron micrograph, showing a T. elongatus carboxysome.

Fig. 2.

The structure of CcmM (A) Structure of the CcmM209 monomer, with secondary structure labeled. (B) CcmM209 trimer viewed down the 3-fold axis. The zinc binding histidine residues, as well as R121 (which binds a structural chloride ion), are shown in sticks. The zinc ion is shown as a pink sphere. (C) The CcmM209 trimer viewed orthogonal to the 3-fold axis. Individual monomers are arranged with the β-helical axis parallel to one another, while αC interacts with the adjacent protomer. (D) Details of the inset area, with σA weighted 2mF_o-DF_c electron density contoured at 1.0σ (blue) shown for residues E171–L208 of chain A (orange sticks), and for residues P9– L17 of chain B (yellow sticks). Electron density in this region is well defined, with temperature factors comparable to elsewhere in the structure. Density is also contoured at 3.0σ (green surface) for C194 and C200, showing the density associated with the sulfur atoms participating in the disulfide bond. For comparison, V182 is the last residue ordered in the CcmM193 structure. (E) Superposition of apo zinc Cam (1qrg; white and yellow) on CcmM209 (blue, αC in orange). Aside from the highlighted localized differences, the two structures are overall very similar. (F) Ribbon diagram of CcmM193 (white) superimposed on CcmM209 (blue) demonstrating that structural differences are localized, but substantial. Residues 4–16 (orange in CcmM209) are disordered in the CcmM193 structure. Of residues 172–208, which in CcmM209 comprise αB and αC (brown), only residues 172–182 are partially ordered in the CcmM193 structure (red), and these residues are displaced from the position seen in CcmM209. (G) Inset showing electron density for the CcmM193 structure. Density is contoured at 1σ (light blue) and 4σ (dark blue). The electron density for αB is considerably less defined than for the rest of the structure. (H) The oligomeric organization differs between CcmM209 (blue) and CcmM193 (white) structures. The structures were superimposed with reference to the protomer on the right only. The motion the CcmM193 and CcmM209 structures can be described as the rotation of each protomer approximately 6° away from the 3-fold axis, with the fulcrum located near R121. (I) Details of the CcmM209 catalytic site. The αB helix, which covers the catalytic site, is shown in cyan in transparent cartoon representation. (J) Details of the CcmM193 putative catalytic site. Residues labeled “b” are contributed by a symmetry related molecule. (K) Superposition of the CcmM209 active site (blue) on CcmM193 (white). Note that many of the residues contributed by the “b” side of the pocket are misplaced in CcmM193 and fail to form hydrogen bonds important for catalytic activity. (L) Overlay of the CcmM209 (blue) and Cam (yellow) catalytic sites. Note that, despite the low overall sequence identity (∼35%), all catalytic site residues are conserved and adopt identical conformations. See Fig. S3 for details of the metal ion geometry, and Fig. S4 for further details of the catalytic site.

Fig. 3.

Reduction of the Cys194–Cys200 disulfide bond inactivates and partially disorders CcmM209. (A) Effect of 10 mM TCEP on the catalytic activity of CcmM209 (0.02 mg·mL^-1) and CcmMχ173 (0.01 mg·mL^-1). CcmMχ173 is constitutively active under both oxidizing and reducing (10 mM TCEP) conditions, whereas CcmM209 is active under oxidizing conditions, but activity is reduced to uncatalyzed rates in the presence of 10 mM TCEP. The CcmM209C200S variant is inactive under both oxidizing and reducing conditions. Assay condition: 200 mM EPPS/NaOH, pH 7.8, 1 mM NaCl, 600 μM at 30 °C. (B) Tryptophan fluorescence of CcmM193, CcmM209, and CcmMχ173 under oxidizing and reducing (10 mM DTT) conditions. The constitutively active CcmMχ173 chimera is strongly fluorescent under both conditions, the constitutively inactive construct CcmM193 is more weakly fluorescent under both conditions, while the CcmM209 switches from strongly fluorescent under oxidizing, to weakly fluorescent under reducing conditions. This is consistent with the quenching of W13 caused by the unstructuring of the β1–β2 loop and the α2 and α3 helices upon reduction of the C194–C200 disulfide bond. (C) E. coli’s cytosolic reducing machinery keeps CcmM209 in an inactive state. Activity traces for fresh cell lysate containing over expressed CcmM209 in the absence and presence (25 mM) of the thiol oxidizing agent diamide. Activation of CcmM209 also occurs during the purification process due to oxidation of the protein by molecular O₂. Cell lysate containing the CcmM209C200S variant remains inactive in the presence of diamide, indicating that the activation mechanism is dependent upon the potential to form a C194–C200 disulfide bond. (D) Details showing the regions of the β1–β2 loop and α2–α3 helices that are necessary for stabilizing the protein in an active conformation. (E) Excerpt of an alignment of CcmM sequences. Species are abbreviated as Te,Thermosynechococcus elongatus-BP1; Gv, Gloeobacter violaceus PCC 7421; Tric, Trichodesmium erythraeum IMS101; Np, Nostoc punctiforme PCC 73102; Cw, Crocosphaera watsonii WH 8501; 6803, Synechocystis sp. PCC 6803; 7942, Synechococcus elongatus PCC 7942; 7335, Synechococcus sp. PCC 7335. Among the shown species, Te, Gv, and Tric contain no ccaA homolog; the other species shown do. The sequences for 6803, 7942, and 7335 do not conserve C194, C200 (green ellipses), the β1–β2 loop (cyan box with W13 marked with a cyan ellipse), or critical elements in the α2 and α3 helices (orange box, N184 magenta ellipse). Consequently, they (along with six other species not depicted) are unlikely to show CA activity.