Increased catalytic efficiency following gene fusion of bifunctional methionine sulfoxide reductase enzymes from Shewanella oneidensis

Abstract

Methionine sulfoxide reductase enzymes MsrA and MsrB have complementary stereospecificities that reduce the S and R stereoisomers of methionine sulfoxide (MetSO), respectively, and together function as critical antioxidant enzymes. In some pathogenic and metal-reducing bacteria, these genes are fused to form a bifunctional methionine sulfoxide reductase (i.e., MsrBA) enzyme. To investigate how gene fusion affects the substrate specificity and catalytic activities of Msr, we have cloned and expressed the MsrBA enzyme from Shewanella oneidensis, a metal-reducing bacterium and fish pathogen. For comparison, we also cloned and expressed the wild-type MsrA enzyme from S. oneidensis and a genetically engineered MsrB protein. MsrBA is able to completely reduce (i.e., repair) MetSO in the calcium regulatory protein calmodulin (CaM), while only partial repair is observed using both MsrA and MsrB enzymes together at 25 degrees C. A restoration of the normal protein fold is observed co-incident with the repair of MetSO in oxidized CaM (CaMox by MsrBA, as monitored by time-dependent increases in the anisotropy associated with the rigidly bound multiuse affinity probe 4',5'-bis(1,3,2-dithioarsolan-2-yl)fluorescein (FlAsH). Underlying the efficient repair of MetSO in CaMox is the coordinate activity of the two catalytic domains in the MsrBA fusion protein, which results in a 1 order of magnitude rate enhancement in comparison to those of the individual MsrA or MsrB enzyme alone. The coordinate binding of both domains of MsrBA permits the full repair of all MetSO in CaMox. The common expression of Msr fusion proteins in bacterial pathogens is consistent with an important role for this enzyme activity in the maintenance of protein function necessary for bacterial survival under highly oxidizing conditions associated with pathogenesis or bioremediation.

Figure 1. Conserved Active Site Sequences of Msr Proteins

Tertiary structures (top) and sequences (bottom) of Msr proteins highlighting identical (yellow) and conserved (green) amino acids. Side chains implicated in catalysis or substrate recognition are highlighted (12, 49, 50). Structures correspond to MsrB (left) or MsrA (right). Sequences compare amino acids between MsrB from Neisserium gonorrhoeae and MsrA from E. coli with Shewanella oneidensis MR-1 proteins MsrA (SO2337) and MsrBA (SO2588), as determined using the BLAST algorithm (http://www.ncbi.nlm.nih.gov/sutils/genom_table.cgi). Structures correspond to 2gt3.pdb for MsrA from E. coli or 1l1d.pdb for MsrB from Neisseria gonorrhoeae (12, 51, 52), and were drawn using the program RASMOL (53).

Figure 2. Complete Repair of CaM_ox by MsrBA

(Top) SDS-PAGE (14% Tris-glycine) gel showing time-dependent mobility changes of CaM_ox in the presence of either MsrA or MsrBA. (Bottom) Intact protein ESI-MS spectra of unoxidized CaM (A) and oxidized CaM prior to (B) and following incubation for 12 hours with MsrA (C), MsrB (D), an equimolar mixture of MsrA + MsrB (E), and MsrBA (F). Experimental conditions involved CaM (10 μM) incubated with indicated Msr isoforms (1.0 μM) of either MsrBA, MsrA, or MsrB in 10 mM MOPS (pH 7.5), 50 mM KCl, and 15 mM DTT at 25°C. No MetSO reduction is observed in the absence of added DTT, which acts to reduce the active site of Msr enzymes to permit reduction of MetSO.

Figure 3. Substrate-Dependence of MsrBA Mediated Repair of CaM_ox

MsrBA-dependent rates of repair of CaM_ox as a function of NADPH (A), thioredoxin (B), or thioredoxin reductase (C). Experimental conditions included MsrBA (0.5 μM) and CaM_ox (30 μM) in 10 mM Tris-HCl (pH 7.5) containing indicated substrate concentrations and saturating concentrations of NADPH (400 μM) (B and C), thioredoxin (50 μM) (A and C) or thioredoxin reductase (2 μM) (A and B) at 24 °C. Line in panel B represents nonlinear least-squares fit to the Michaelis-Menten rate equation, where K_M = 3.3 ± 0.8 μM.

Figure 4. Restoration of CaM_ox Protein Fold Upon Repair of Met(SO) by MsrBA

Steady-state anisotropies for FlAsH-labeled CaM_ox (1.0 μM) in the presence of MsrBA (◆), MsrA (○) and MsrB (●). Reaction mixture consists of NADPH (400 μM), thioredoxin (50 μM), thioredoxin reductase (2 μM), and indicated isoform of Msr (1.0 μM) in 50 mM HEPES (pH 7.5), 140 mM KCl, and 0.2 mM CaCl₂ in a total volume of 2 mL. Initial rates associated with MsrBA-dependent increases in anisotropy are (5.9 ± 1.1) × 10⁻³/min, (0.53 ± 0.12) × 10⁻³/min, and (0.19 ± 0.02) × 10⁻³/min for MsrBA, MsrA and MsrB, respectively. Excitation was at 500 nm and emitted light was measured at 530 nm; slit widths were set at 5 nm. Inset: Electrophoretic mobility on SDS-PAGE of FlAsH-labeled CaM (10 μg) prior to (lane 1) and following oxidation of all nine methionines (lane 2) using a 14% Tris-Glycine gel visualized by fluorescence detection (right) or following Coomassie blue staining (left).

Figure 5. Preferential Recognition and Repair of CaM_ox

Catalytic activities of Msr isoforms (0.5 μM) against L-MetSO (A) and CaM_ox (B) for MsrBA (◆; solid lines), MsrA (○; dashed lines) and MsrB (●; dotted lines). Experimental conditions involved 10 mM Tris-HCl (pH 7.5), NADPH (400 μM), thioredoxin (50 μM), and thioredoxin reductase (2 μM) in a total volume of 0.2 mL at 24 °C. Lines represent fits to the Michaelis-Menten rate equation (see Table 1).

Figure 6. Coordinated Binding at Multiple MetSO in CaM_ox by MsrBA Facilitates Repair

Depiction of proposed role of MsrBA fusion protein (gray connected circles denoted A and B) in binding and stabilizing unfolded state of oxidized CaM (white cylinders)(step 1), permitting reduction of all nine MetSO (yellow) to their native Met structure through the coordinate binding of both active sites A and B in MsrBA (top) at multiple MetSO sites (step 2), where diffusional steps are minimized due to the role of the complemetary protein domain in anchoring MsrBA to the oxidized protein. Underlying thecapacity of MsrBA to fully repair CaM_ox is an ability to stabilize the oxidized and partially unfolded CaM to promote recognition of exposed MetSO by the Msr enzymes (Figure 4). The ability to maintain the MetSO within protein substrates in an accessible partially unfolded state results in an increased catalytic rates of repair (Figure 5). Following MetSO reduction, MsrBA dissociates, releasing the fully repaired protein (step 3). In contrast, while binding of MsrA (bottom) or MsrB (not depicted) to fully oxidized CaM (step 4) readily occurs, neither MsrA or MsrB alone can fully repair all S- or R-MetSO in CaM_ox under these experimental conditions, where there is substantial protein refolding at 25 °C (step 5) that competes with the ability of the individual Msr enzymes to recognize and bind MetSO in CaM_ox (step 5) (5, 6, 25, 32). Following protein refolding, there is no additional reduction of MetSO (step 6 is blocked), resulting in the retention of MetSO as occurs during biological aging (2).