Re-designing the substrate binding pocket of laccase for enhanced oxidation of sinapic acid

Iterative saturation mutagenesis was performed over six residues delimiting the substrate binding pocket of a high redox potential chimeric laccase with the aim of enhancing its activity over sinapic acid, a lignin-related phenol of industrial interest.

Download Full-text

Substantially improving the enantioconvergence of PvEH1, a Phaseolus vulgaris epoxide hydrolase, towards m-chlorostyrene oxide by laboratory evolution

Microbial Cell Factories ◽

10.1186/s12934-019-1252-4 ◽

2019 ◽

Vol 18 (1) ◽

Cited By ~ 2

Author(s):

Xun-Cheng Zong ◽

Chuang Li ◽

Yao-Hui Xu ◽

Die Hu ◽

Bo-Chun Hu ◽

...

Keyword(s):

Phaseolus Vulgaris ◽

Epoxide Hydrolase ◽

Substrate Binding ◽

Binding Pocket ◽

Saturation Mutagenesis ◽

Substrate Binding Pocket ◽

Laboratory Evolution ◽

E Coli ◽

Enantioconvergent Hydrolysis ◽

Hydrolysis Of

Abstract Background Epoxide hydrolase can regioselectively catalyze the oxirane ring-opening hydrolysis of rac-epoxides producing the corresponding chiral diols. In our laboratory, a gene named pveh1 encoding an EH from Phaseolus vulgaris was cloned. Although the directed modification of PvEH1 was carried out, the mutant PvEH1Y3 showed a limited degree of enantioconvergence towards racemic (rac-) m-chlorostyrene oxide (mCSO). Results PvEH1 and PvEH1Y3 were combinatively subjected to laboratory evolution to further enhance the enantioconvergence of PvEH1Y3 towards rac-mCSO. Firstly, the substrate-binding pocket of PvEH1 was identified using a CAVER 3.0 software, and divided into three zones. After all residues in zones 1 and 3 were subjected to leucine scanning, two E. coli transformants, E. coli/pveh1Y149L and /pveh1P184L, were selected, by which rac-mCSO was transformed into (R)-m-chlorophenyl-1,2-ethanediol (mCPED) having 55.1% and 27.2% eep. Secondly, two saturation mutagenesis libraries, E. coli/pveh1Y149X and /pveh1P184X (X: any one of 20 residues) were created at sites Y149 and P184 of PvEH1. Among all transformants, both E. coli/pveh1Y149L (65.8% αS and 55.1% eep) and /pveh1P184W (66.6% αS and 59.8% eep) possessed the highest enantioconvergences. Finally, the combinatorial mutagenesis was conducted by replacements of both Y149L and P184W in PvEH1Y3, constructing E. coli/pveh1Y3Z2, whose αS reached 97.5%, higher than that (75.3%) of E. coli/pveh1Y3. In addition, the enantioconvergent hydrolysis of 20 mM rac-mCSO was performed by E. coli/pveh1Y3Z2, giving (R)-mCPED with 95.2% eep and 97.2% yield. Conclusions In summary, the enantioconvergence of PvEH1Y3Z2 was successfully improved by laboratory evolution, which was based on the study of substrate-binding pocket by leucine scanning. Our present work introduced an effective strategy for the directed modification of enantioconvergence of PvEH1.

Download Full-text

Catalytic Efficiency of Basidiomycete Laccases: Redox Potential versus Substrate-Binding Pocket Structure

Catalysts ◽

10.3390/catal8040152 ◽

2018 ◽

Vol 8 (4) ◽

pp. 152 ◽

Cited By ~ 15

Author(s):

Olga Glazunova ◽

Nikita Trushkin ◽

Konstantin Moiseenko ◽

Ivan Filimonov ◽

Tatyana Fedorova

Keyword(s):

Redox Potential ◽

Substrate Binding ◽

Catalytic Efficiency ◽

Binding Pocket ◽

Structural Features ◽

Redox Potentials ◽

Substrate Binding Pocket ◽

Trametes Hirsuta ◽

Special Cases ◽

Almost All

Laccases are copper-containing oxidases that catalyze a one-electron abstraction from various phenolic and non-phenolic compounds with concomitant reduction of molecular oxygen to water. It is well-known that laccases from various sources have different substrate specificities, but it is not completely clear what exactly provides these differences. The purpose of this work was to study the features of the substrate specificity of four laccases from basidiomycete fungi Trametes hirsuta, Coriolopsis caperata, Antrodiella faginea, and Steccherinum murashkinskyi, which have different redox potentials of the T1 copper center and a different structure of substrate-binding pockets. Enzyme activity toward 20 monophenolic substances and 4 phenolic dyes was measured spectrophotometrically. The kinetic parameters of oxidation of four lignans and lignan-like substrates were determined by monitoring of the oxygen consumption. For the oxidation of the high redox potential (>700 mV) monophenolic substrates and almost all large substrates, such as phenolic dyes and lignans, the redox potential difference between the enzyme and the substrate (ΔE) played the defining role. For the low redox potential monophenolic substrates, ΔE did not directly influence the laccase activity. Also, in the special cases, the structure of the large substrates, such as dyes and lignans, as well as some structural features of the laccases (flexibility of the substrate-binding pocket loops and some amino acid residues in the key positions) affected the resulting catalytic efficiency.

Download Full-text

Crystal structure of steroid reductase SRD5A reveals conserved steroid reduction mechanism

Nature Communications ◽

10.1038/s41467-020-20675-2 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yufei Han ◽

Qian Zhuang ◽

Bo Sun ◽

Wenping Lv ◽

Sheng Wang ◽

...

Keyword(s):

Crystal Structure ◽

Biochemical Characterization ◽

Substrate Binding ◽

Binding Pocket ◽

Substrate Binding Pocket ◽

Transmembrane Segments ◽

Therapeutic Molecules ◽

Binding Cavity ◽

Immune System Regulation ◽

Mediated Reduction

AbstractSteroid hormones are essential in stress response, immune system regulation, and reproduction in mammals. Steroids with 3-oxo-Δ4 structure, such as testosterone or progesterone, are catalyzed by steroid 5α-reductases (SRD5As) to generate their corresponding 3-oxo-5α steroids, which are essential for multiple physiological and pathological processes. SRD5A2 is already a target of clinically relevant drugs. However, the detailed mechanism of SRD5A-mediated reduction remains elusive. Here we report the crystal structure of PbSRD5A from Proteobacteria bacterium, a homolog of both SRD5A1 and SRD5A2, in complex with the cofactor NADPH at 2.0 Å resolution. PbSRD5A exists as a monomer comprised of seven transmembrane segments (TMs). The TM1-4 enclose a hydrophobic substrate binding cavity, whereas TM5-7 coordinate cofactor NADPH through extensive hydrogen bonds network. Homology-based structural models of HsSRD5A1 and -2, together with biochemical characterization, define the substrate binding pocket of SRD5As, explain the properties of disease-related mutants and provide an important framework for further understanding of the mechanism of NADPH mediated steroids 3-oxo-Δ4 reduction. Based on these analyses, the design of therapeutic molecules targeting SRD5As with improved specificity and therapeutic efficacy would be possible.

Download Full-text