Converting the E. coli Isochorismatase Nicotinamidase into γ-Lactamase

Guo, Xiaoyan; Chang, Licao; Jin, Huixia; Zhu, Jingxian; Tao, Yong; Wu, Sheng; Wang, Jianjun

doi:10.1128/spectrum.00985-21

Cited by 2 publications

(3 citation statements)

References 26 publications

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“…For 4H2B, the binding pocket of LnRCR was capacious. As proposed previously, compact binding pockets provided a conducive environment for small substrate catalysis (Guo et al, 2022). From this point of view, the binding pocket of LnRCRS154K seemed tight and energetically stable.…”

Section: Mechanism Underlying the Enhanced Catalytic Efficiency Of Ln...supporting

confidence: 67%

A short‐chain carbonyl reductase mutant is an efficient catalyst in the production of (R)‐1,3‐butanediol

Guo

Gao

Liu

et al. 2023

Microbial Biotechnology

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R-1,3-butanediol (R-1,3-BDO) is an important chiral intermediate of penem and carbapenem synthesis. Among the different synthesis methods to obtain pure enantiomer R-1,3-BDO, oxidation-reduction cascades catalysed by enzymes are promising strategies for its production. Dehydrogenases have been used for the reduction step, but the enantio-selectivity is not high enough for further organic synthesis efforts. Here, a short-chain carbonyl reductase (LnRCR) was evaluated for the reduction step and developed via protein engineering. After docking result analysis with the substrate 4-hydroxy-2-butanone (4H2B), residues were selected for virtual mutagenesis, their substrate-binding energies were compared, and four sites were selected for saturation mutagenesis. High-throughput screening helped identify a Ser154Lys mutant which increased the catalytic efficiency by 115% compared to the parent enzyme. Computer-aided simulations indicated that after single residue replacement, movements in two flexible areas (VTDPAF and SVGFANK) facilitated the volumetric compression of the 4H2B-binding pocket. The number of hydrogen bonds between the stabilized 4H2B-binding pocket of the mutant enzyme and substrate was higher (from four to six) than the wild-type enzyme, while the substrate-binding energy was decreased (from −17.0 kJ/mol to −29.1 kJ/mol). Consequently, the catalytic efficiency increased by approximately 115% and enantio-selectivity increased from 95% to 99%. Our findings indicate that compact and stable substrate-binding pockets are critical for enzyme catalysis. Lastly, the utilization of a microbe expressing the Ser154Lys mutant enzyme was proven to be a robust process to conduct the oxidation-reduction cascade at larger scales.

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Section: Mechanism Underlying the Enhanced Catalytic Efficiency Of Ln...supporting

confidence: 67%

A short‐chain carbonyl reductase mutant is an efficient catalyst in the production of (R)‐1,3‐butanediol

Guo

Gao

Liu

et al. 2023

Microbial Biotechnology

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“…To confirm the right composition of rhIL‐31 or rhIL‐31RA‐hFc, the recombinant proteins were subjected to the sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) gel, and then the target bands were digested with trypsin in‐gel. The peptide samples were subjected to an EASY‐nLC 1000 interfaced via a Nanospray Flex ion source to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific, MA, USA) for analysis 21,22 . Mass spectra/mass spectra (MS/MS) data were processed using Mascot search engine (v.2.5.1, 2014, http://www.matrixscience.com; Matrix Science, London, UK).…”

Section: Methodsmentioning

confidence: 99%

“…The peptide samples were subjected to an EASY-nLC 1000 interfaced via a Nanospray Flex ion source to an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific, MA, USA) for analysis. 21,22 Mass spectra/mass spectra (MS/MS) data were processed using Mascot search engine (v.2.5.1, 2014, http://www. matrixscience.com; Matrix Science, London, UK).…”

Section: Mass Spectra Analysis Of Rhil-31 and Rhil-31ra-hfcmentioning

confidence: 99%

Expression, purification, and biological characterization of recombinant human interleukin‐31 protein

Chen

Zheng

Wang

et al. 2023

Biotech and App Biochem

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Interleukin‐31 (IL‐31), belonging to the IL‐6 cytokine family, is involved in skin inflammation and pruritus, as well as some tumors’ progression. Here, we reported the expression and purification of recombinant human IL‐31 (rhIL‐31) using a prokaryotic system. This recombinant protein was expressed in the form of inclusion bodies, refolded and purified by size‐exclusion chromatography. Circular dichroism analysis revealed that the secondary structure of rhIL‐31 was mainly composed of alpha‐helix, which is in consistence with the 3D model structure built by AlphaFold server. In vitro studies showed that rhIL‐31 exhibited a good binding ability to the recombinant hIL‐31 receptor alpha fused with human Fc fragment (rhIL‐31RA‐hFc) with EC50 value of 16.36 µg/mL in ELISA assay. Meanwhile, flow cytometry demonstrated that rhIL‐31 was able to bind to hIL‐31RA or hOSMRβ expressed on the cell surface, independently. Furthermore, rhIL‐31 could induce the phosphorylation of STAT3 in A549 cells. In conclusion, the prepared rhIL‐31 in this study possesses the binding ability to its receptors, and can activate the signal pathway of JAK/STAT. Thus, it can be applied in further studies, including investigation of hIL‐31‐related diseases, structural analysis, and development of therapeutic drugs, and monoclonal antibodies targeting hIL‐31.

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Converting the E. coli Isochorismatase Nicotinamidase into γ-Lactamase

Abstract: γ-Lactamases are important enzymatic catalysts in preparing optically pure γ-lactam enantiomers, which are high-value chiral intermediates. Different studies have presumed that the isochorismatase superfamily is a candidate to obtain novel (+) γ-lactamases.

Cited by 2 publications

References 26 publications

A short‐chain carbonyl reductase mutant is an efficient catalyst in the production of (R)‐1,3‐butanediol

A short‐chain carbonyl reductase mutant is an efficient catalyst in the production of (R)‐1,3‐butanediol

Expression, purification, and biological characterization of recombinant human interleukin‐31 protein

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