Mechanism and Structural Insights Into a Novel Esterase, E53, Isolated From Erythrobacter longus

Abstract

Esterases are a class of enzymes that split esters into an acid and an alcohol in a chemical reaction with water, having high potential in pharmaceutical, food and biofuel industrial applications. To advance the understanding of esterases, we have identified and characterized E53, an alkalophilic esterase from a marine bacterium Erythrobacter longus. The crystal structures of wild type E53 and three variants were solved successfully using the X-ray diffraction method. Phylogenetic analysis classified E53 as a member of the family IV esterase. The enzyme showed highest activity against p-nitrophenyl butyrate substrate at pH 8.5–9.5 and 40°C. Based on the structural feature, the catalytic pocket was defined as R1 (catalytic center), R2 (pocket entrance), and R3 (end area of pocket) regions. Nine variants were generated spanning R1–R3 and thorough functional studies were performed. Detailed structural analysis and the results obtained from the mutagenesis study revealed that mutations in the R1 region could regulate the catalytic reaction in both positive and negative directions; expanding the bottleneck in R2 region has improved the enzymatic activity; and R3 region was associated with the determination of the pH pattern of E53. N166A in R3 region showed reduced activity only under alkaline conditions, and structural analysis indicated the role of N166 in stabilizing the loop by forming a hydrogen bond with L193 and G233. In summary, the systematic studies on E53 performed in this work provide structural and functional insights into alkaliphilic esterases and further our knowledge of these enzymes.

Discovery, characterization and mechanism of a Microbacterium esterase for key d-biotin chiral intermediate synthesis

“…2 b, where the residual activity dropped to just 10% after a 1-hour incubation period. The optimal temperature for EstSIT01 closely aligns with that of esterase E53 (40 °C) (Ding et al 2021 ). Both of these enzymes belong to the mesophilic enzyme category, and temperatures exceeding 45 °C may potentially disrupt their tertiary structure.…”

The Mobility of the Cap Domain Is Essential for the Substrate Promiscuity of a Family IV Esterase from Sorghum Rhizosphere Microbiome

“…Furthermore, as seen below, it should be noted that the two proline residues are located at the entrance of the narrow tunnel giving access to the active site, an issue that ascribes a prominent role to both residues in binding activity and specificity. Structural details of the EH 0 active site and assignment of the acyl and alcohol moieties were explored by comparison with its homologs E53 complexed with 4-nitrophenyl hexanoate (PDB code 6KEU) (28) and EH 1AB1 in complex with a derivative of methyl-4-nitrophenylhexylphosphonate (PDB code 6RB0) (31). However, the acyl and alcohol moieties of these complexes are located in opposite sites (Fig.…”

“…Analysis of EH 0 folding using the DALI server ( 27 ) was employed to search for homologous proteins. The closest homologs are E53 isolated from Erythrobacter longus , with 46% identity and a root mean square deviation (RMSD) of 2.3 Å on 296 Cα atoms (PDB code 7W8N ) ( 28 ), Est8 isolated from Parvibaculum , with 38% identity and an RMSD of 2.5 Å on 291 Cα atoms (PDB code 4YPV ) ( 26 ), PestE isolated from Pyrobaculum calidifontis , with 33% identity and an RMSD of 2.6 Å on 294 Cα atoms (PDB code 3ZWQ ) ( 29 ), and EstA isolated from Sulfolobus islandicus REY15A, with 31% identity and an RMSD of 2.6 Å on 281 Cα atoms (PDB code 5LK6 ). The structural superimposition of these proteins reveals a high conservation of the corresponding catalytic domains and also a common spatial arrangement of the helices at the cap domains in all the proteins except EH 0 , where α2 and the long α7 are visibly shifted very close to its EH 0 active site and are apparently impeding the entrance of substrates ( Fig.…”

“…Structural details of the EH 0 active site and assignment of the acyl and alcohol moieties were explored by comparison with its homologs E53 complexed with 4-nitrophenyl hexanoate (PDB code 6KEU ) ( 28 ) and EH 1AB1 in complex with a derivative of methyl-4-nitrophenylhexylphosphonate (PDB code 6RB0 ) ( 31 ). However, the acyl and alcohol moieties of these complexes are located in opposite sites ( Fig.…”

The mobility of the cap domain is essential for the substrate promiscuity of a family IV esterase from sorghum rhizosphere microbiome

“…Analysis of EH 0 folding using the DALI server (27) was employed to search for homologous proteins. The closest homologs are E53 isolated from Erythrobacter longus , with 46% identity and RMSD of 2.3 Å on 296 Cα atoms (PDB code 7W8N) (28); Est8 isolated from Parvibaculum , with 38% identity and RMSD of 2.5Å on 291 Cα atoms (PDB code 4YPV) (26); PestE isolated from Pyrobaculum calidifontis , with 33% identity and RMSD of 2.6Å on 294 Cα atoms (PDB code 3ZWQ) (29); and EstA isolated from Sulfolobus islandicus REY15A , with 31% identity and RMSD of 2.6Å on 281 Cα atoms (PDB code 5LK6). The structural superimposition of these proteins reveals a high conservation of the corresponding catalytic domains, and also a common spatial arrangement of the helices at the cap domains in all the proteins except EH 0 , where α2 and the long α7 are visibly shifted very close to its EH 0 active site and apparently impeding the entrance of substrates (Fig.…”