Biophysical Society Thematic Meeting | Trieste 2024

Emerging Theoretical Approaches to Complement Single-Particle Cryo-EM

Poster Abstracts

27-POS Board 27 PARAMETERIZING THE COVALENT INTERMEDIATES OF THE NITRILASE SUPERFAMILY Dewald P van Heerden ; Trevor Sewell 1 ; 1 University of Cape Town, Integrative Biomedical Sciences, Cape Town, South Africa Proteins of the nitrilases superfamily (which include amidases) have a structurally conserved active site grouping comprising two glutamates, a lysine, and a cysteine. All postulated mechanisms involve covalent modification of the cysteine. Such covalently modified amino acids cannot be modeled by the best available modeling tools used in the field. To address this shortcoming, we developed a procedure that enables the optimization of these amino acids using the Interactive Structure Optimization by Local Direct Exploration (ISOLDE) package incorporated in UCSF ChimeraX. In addition to coordinates that conventional docking programs rely on, ISOLDE employs electron density to minimize protein structures. The AmberTools23 suite of programs was used to generate force field parameters and semi-empirical (AM1-BCC) charges for the covalently modified cysteine residues of the thioester and thioimidate intermediates, enabling their minimization in ISOLDE.We previously characterized the amidase from Nesterenkonia sp. (NitN) that forms a dimer and displays activity against a range of amide substrates that are hydrolyzed to form ammonia and the corresponding carboxylic acid. To investigate the energy barrier involved in the deprotonation of the catalytically active cysteine by the conserved glutamate (i.e., Cys-S-H + Glu-COO(-) --> Cys-S(-) + Glu-COOH), truncated models of the active site were extracted from the molecular mechanics-minimized protein structures and further investigated using density functional theory.The ω B97X-D/def2-TZVP energy profiles indicate that Cys-S-H + Glu-COO(-) is approximately 5 kJ/mol lower in energy than Cys-S(-) + Glu-COOH). In addition, there is an ~20 kJ/mol energy barrier that must be overcome for the conventional base-catalyzed nucleophilic attack of the cysteine on an amide substrate to hold true. Furthermore, we have generated the natural bond orbitals (NBOs) of various docked amide and nitrile substrates and their thioester and thioimidate intermediates. Preliminary results indicate the correct stereoelectronic orientation for the progression of the enzymatic reactions.

51