The Structural Model of Salmonella typhimurium Ethanolamine Ammonia-Lyase Directs a Rational Approach to the Assembly of the Functional [(EutB–EutC)₂]₃ Oligomer from Isolated Subunits

Abstract

Ethanolamine ammonia-lyase (EAL) is a 5’-deoxyadenosylcobalamin (AdoCbl; coenzyme B12) –dependent bacterial enzyme that catalyzes the deamination of the short-chain vicinal amino alcohols, aminoethanol and [S]- and [R]-2-aminopropanol. The coding sequence for EAL is located within the 17-gene eut operon, which codes for the broad spectrum of proteins that comprise the eut metabolosome sub-organelle structure. A high-resolution structure of the ~500 kDa EAL [(EutB-EutC)2]3 oligomer from Escherichia coli has been determined by X-ray crystallography, but high-resolution spectroscopic determinations of reactant intermediate state structures, and detailed kinetic and thermodynamic studies of EAL, have been conducted for the Salmonella typhimurium enzyme. Therefore, a statistically robust homology model for the S. typhimurium EAL is constructed from the E. coli structure. The model structure is used to describe the hierarchy of EutB and EutC subunit interactions that construct the native EAL oligomer, and specifically, to address the long-standing challenge of reconstitution of the functional oligomer from isolated, purified subunits. Model prediction that the (EutB2)3 oligomer assembly will occur from isolated EutB, and that this hexameric structure will template the formation of the complete, native [(EutB-EutC)2]3 oligomer, is verified by biochemical methods. Prediction that cysteine residues on the exposed subunit-subunit contact surfaces of isolated EutB and EutC will interfere with assembly by cystine formation is verified by activating effects of disulfide reducing agents. Ångstrom-scale congruence of the reconstituted and native EAL in the active site region is shown by electron paramagnetic resonance spectroscopy. Overall, the hierarchy of subunit interactions and microscopic features of the contact surfaces, that are revealed by the homology model, guide and provide a rationale for a refined genetic and biochemical approach to reconstitution of the functional [(EutB-EutC)2]3 EAL oligomer. The results establish a platform for further advances toward understanding the molecular mechanism of EAL catalysis, and for insights into therapy-targeted manipulation of the bacterial ethanolamine utilization (eut) metabolosome.

Resolution and Characterization of Chemical Steps in Enzyme Catalytic Sequences by Using Low-Temperature and Time-Resolved, Full-Spectrum EPR Spectroscopy in Fluid Cryosolvent and Frozen Solution Systems

“…C. Ke, 2003). The simulation-derived R ee values and radical-cobalamin geometries are consistent with high-resolution X-ray crystallographic and modeled structures of EAL (Bovell & Warncke, 2013; Shibata et al, 2010). The substrate radical component of the radical pair EPR spectrum has been monitored in the ensemble kinetics studies, and step 1 (Section 2) and step 3 (Section 3) of the reaction cycle (Figure 1) are the foci in this chapter.…”

Two Dynamical Regimes of the Substrate Radical Rearrangement Reaction in B12-Dependent Ethanolamine Ammonia-Lyase Resolve Contributions of Native Protein Configurations and Collective Configurational Fluctuations to Catalysis

“…27 The specific activity of purified EAL with aminoethanol as substrate was 20 μ mol/min/mg ( T = 298 K, P = 1 atm), as determined by using the coupled assay with alcohol dehydrogenase and NADH. 45 …”

“…23 Thus, to reveal the effects of native protein interconfigurational motions on reaction chemistry, the configurational transition rates, displacement amplitudes (extents), or both, of these motions must be perturbed selectively, so that the reaction rate is impacted. In EAL, this condition is realized by studying an isolated, single reaction step in the catalytic sequence, the substrate radical rearrangement, over the cryogenic T range of 190–230 K. 24,25 The substrate radical intermediate state in EAL, S • (Figure 1), 26,27 is first accumulated in the steady-state at room temperature and then cryotrapped at 140 K. 28 Subsequently, T -step to ≥190 K triggers first-order decay of the substrate radical to the diamagnetic products state, P , (Figure 1) which is monitored by using time-resolved, full-spectrum electron paramagnetic resonance (EPR) spectroscopy of the decay of S • 28 . The P • state is not detected by EPR, 24 consistent with the calculated 5–9 kcal/mol higher energy of P •29–31 and rapid depletion by the subsequent P • → P step.…”

Cobinamide production of hydrogen in a homogeneous aqueous photochemical system, and assembly and photoreduction in a (βα)8 protein

“…Our long-term aim is to create protein-bound, site-specific Co macrocycle catalysts for fuels formation, that display photo- and chemical stability, and operate under biological conditions of pure water solution (at pH~7), and ambient temperature ( T ) and pressure ( p ). Here, we report H 2 production in water by the corrin-based macrocyclic Co complex, cobinamide (Cbi; Scheme 1), in a visible-light photosensitizer/sacrificial electron donor system [22, 23], and characterize the assembly and photoreduction of Cbi in a selected protein scaffold, the EutB protein of adenosylcobalamin-dependent ethanolamine ammonia-lyase [24, 25]. …”

“…The high-resolution X-ray crystallographic structure of EAL from Escherichia coli has been reported [24], and a homology model and subunit preparation procedure for S. typhimurium EutB have been described [25]. The EutB subunit dimerizes [24], and the dimers form a hexameric, “trimer of dimers” oligomer [24, 25], which is depicted in Figure 1. Each EutB monomer contains one cobalamin binding site.…”