Bioinformatic analysis of alpha/beta-hydrolase fold enzymes reveals subfamily-specific positions responsible for discrimination of amidase and lipase activities

Abstract

Superfamily of alpha-beta hydrolases is one of the largest groups of structurally related enzymes with diverse catalytic functions. Bioinformatic analysis was used to study how lipase and amidase catalytic activities are implemented into the same structural framework. Subfamily-specific positions--conserved within lipases and peptidases but different between them--that were supposed to be responsible for functional discrimination have been identified. Mutations at subfamily-specific positions were used to introduce amidase activity into Candida antarctica lipase B (CALB). Molecular modeling was implemented to evaluate influence of selected residues on binding and catalytic conversion of amide substrate by corresponding library of mutants. In silico screening was applied to select reactive enzyme-substrate complexes that satisfy knowledge-based criteria of amidase catalytic activity. Selected CALB variants with substitutions at subfamily-specific positions Gly39, Thr103, Trp104, and Leu278 were produced and showed significant improvement of experimentally measured amidase activity. Based on these results, we suggest that value of subfamily-specific positions should be further explored in order to develop a systematic tool to study structure-function relationship in enzymes and to use this information for rational enzyme engineering.

Targeting Lipid Esterases in Mycobacteria Grown Under Different Physiological Conditions Using Activity-based Profiling with Tetrahydrolipstatin (THL)

“…We illustrate how substratedocking studies (computational) in combination with the (experimental) THL target list can be extended to learn more about orientation of the natural product inhibitor in enzymatic cavities. The basis for this analysis is (1) the assumption that the amino acids comprising the catalytic triad are oriented sterically comparable; (2) that the binding energies are minimal between known substrates (phthiocerol and vinyl ester for TesA and LipH, respectively) and the inhibitor (THL for both TesA and LipH); and (3) with a minimal distance to active site (serine OH group) which describes a near-to-attack conformation of the ligand (substrate/inhibitor) in the active site (60). The minimum distance from the serine OH group to the carbonyl carbon of docked ligands ("active site distance" in Fig.…”

Hybrid Computing Clusters To Study Protein Structure, Function And Regulation

“…Таким образом, установлена противотуберкулез-ная активность предложенных соединений. Подходы, аналогичные использованным в этой работе, были ранее применены нами для изучения структуры и функции фермен-тов из других суперсемейств, поиска и характеристики новых центров связывания, а также получения препаратов ферментов с улучшенными свойствами [16][17][18][19][20].…”

Probing the Substrate Specificity and Intersubunit Interactions of <i>Brevundimonas Diminuta</i> Glutaryl Acylase with Site-Directed Mutagenesis

“…All single mutants, as well as all double and triple combinations of the selected mutations were considered. Protocols for molecular docking with the Auto dock software (Huey et al, 2007) and preparation of in silico libraries were discussed previously (Suplatov et al, 2012). Structural filtration of the resulting enzymesubstrate complexes was used as a post-docking tool to select mutants that were able to stabilize the productive binding state in the active site.…”