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Alonso-Mori, R.

Publications and source records attributed to Alonso-Mori, R..

2 recordsLinked to original sources

Structure of active methyl-CoM reductase, Earth's main methane producer

Our work reveals the structure of the active state of Methyl-Coenzyme M Reductase (MCR), the key and rate-limiting enzyme in biological methane formation. We find large differences between the active Ni(I) and inactive Ni(II) proteins and provide insight into how nature makes and breaks the C-H bond of methane. The Ni(II)-F430 center in inactive MCR contains four planar nitrogen ligands, a lower axial glutamine oxo, and an upper axial thiolate. The Ni(I)-enzyme replaces the axial ligands with a single water. The one-electron redox change results in movement of the Ni ion and upward swing of the {beta}-lactam ring in the tetrapyrrole coupled to a domino-like protein quake through second sphere residues, inter-subunit interactions, a substrate tunnel, affecting even the dimensions of the unit cell. These structural changes lead Ni(I)-MCR to release a charge clamp that, in the Ni(II) state, locks down substrate Coenzyme B. Determining the Ni(I)-MCR structure required development of rigorous anaerobic crystallographic techniques. Validation of the MCR redox state was accomplished by in-line and parallel spectroscopic and unit cell analyses. This structure has large implications for developing technologies to limit methane emissions and efficiently produce biofuels. Methodology described here will enhance structural biology for other oxygen-sensitive enzymes.

biochemistry↗

Cysteine modification can gate non-equilibrium conformational dynamics during enzyme catalysis

Summary ParagraphProtein dynamics play an important role in enzyme catalysis1-4. Many enzymes form covalent catalytic intermediates that can alter enzyme structure and conformational dynamics5,6. How these changes in enzyme structure and dynamics facilitate passage along the reaction coordinate is a fundamental unanswered question in structural enzymology. Here, we use Mix-and-Inject Serial Femtosecond X-ray Crystallography (MISC) at an X-ray Free Electron Laser (XFEL)7-10, ambient temperature X-ray crystallography, computer simulations, and enzyme kinetics to characterize how covalent modification of the active site cysteine residue in isocyanide hydratase (ICH) alters the enzymes conformational ensemble throughout the catalytic cycle. With MISC, we directly observe formation of a thioimidate covalent intermediate during ICH catalysis. The intermediate exhibits changes in the active site electrostatic environment, disrupting a hydrogen bond and triggering a cascade of conformational changes in ICH. X-ray-induced formation of a cysteine-sulfenic acid at the catalytic nucleophile (Cys101-SOH) with conventional crystallography at ambient temperature induces similar conformational shifts, demonstrating that these enzyme motions result from cysteine modification. Computer simulations show how cysteine modification-gated structural changes allosterically propagate through the ICH dimer. Mutations at Gly150 that modulate helical mobility reduce ICH catalytic turnover and alter its pre-steady state kinetic behavior, establishing that helical mobility is important for ICH catalytic efficiency. Taken together, our results demonstrate the potential of mix-and-inject XFEL crystallography to capture otherwise elusive mechanistic details of enzyme catalysis and dynamics from microcrystalline samples7,11. This approach can connect conformational dynamics to function for the large class of systems that rely on covalently modified cysteine residues for catalysis or regulation, resolving long-standing questions about enzyme mechanism and functionally relevant non-equilibrium enzyme motions.

biochemistry↗