Site-directed Saturation Mutagenesis to Improve the Catalytic Performance of 11α-hydroxylase from Aspergillus ochraceus

doi:10.13560/j.cnki.biotech.bull.1985.2023-0751

Abstract

Abstract:

【Objective】11α,17α-dihydroxyprogesterone is an important steroid hormone drug intermediate with high application value. The 11α hydroxylase CYP68J5 from Aspergillus ochraceus CICC 41473 is a key enzyme for the bioconversion from 17α-hydroxyprogesterone to 11α,17α-dihydroxyprogesterone, but its catalytic performance needs to be further improved. 【Methods】Based on the key amino acid sites D118, F216 and M488 identified in our previous works, the excellent mutants were screened by site-directed saturation mutagenesis and substrate transformation experiments. Molecular docking between enzymes and substrates was performed using AutoDock, and the intermolecular interaction forces and molecular dynamic simulations were studied by Discovery Studio and Gromacs, respectively. 【Results】Mutants D118V, F216W, M488L, and M488W possessed catalytic properties, among which, D118V showed the highest activity. At a substrate concentration of 0.5 g/L, the production intensity of D118V was 431.66 mg/（L·d）, which was 2.12-fold higher than that of the wild type CYP68J5. This might be attributed to the fact that a new hydrophobic interaction was generated between the site and substrate when the aspartic acid at position 118 was changed to valine, consequently enhancing the hydrophobicity of the substrate-binding pocket of the enzyme. Molecular dynamics simulations showed more stable overall conformation of the mutant and tighter binding effect between enzyme and substrate. 【Conclusion】The excellent mutant D118V with significantly improved catalytic performance was obtained by site-directed saturation mutagenesis. The results provide an application case and theoretical guidance for the molecular modification of steroid 11α hydroxylase.

Key words: 11α,17α-dihydroxyprogesterone, 11α hydroxylase, site-directed saturation mutagenesis, molecular docking, molecular dynamics simulations

SHI Jing-hui, CHEN Wen-hui, LU Kun, ZHENG Ting-ting, REN Zhi-yuan, BAO Guo-qing, WANG Min, LUO Jian-mei. Site-directed Saturation Mutagenesis to Improve the Catalytic Performance of 11α-hydroxylase from Aspergillus ochraceus[J]. Biotechnology Bulletin, 2024, 40(1): 322-331.

Figures/Tables 6

Table 1 PCR primers for the saturated mutation

引物名称Primer name	引物序列Primer sequence（5'-3'）
D118-F	NNKTCTCATGGTTATATTCCTGGTTTTG
D118-R	ATCAGTAGTTGGAGTTTCAAAATCCATTC
F216-F	NNKGGTGTTGGTGATAAATTGAG
F216-R	AGCCAAAGCAGCATATTGAGAAGAAG
M488-F	NNKACTTATTTGGCTGATCCAAACAC
M488-R	ACCAATATTCAATGGTTGTGGTTTAAAAC

Fig. 1 Distribution of the D118V, F216 and M488 in the docking structure of CYP68J5 with substrate The α-helix, β-folding and random curl of CYP68J5 are colored in bright blue, bright purple and pink, respectively, while the heme, key amino acid sites and substrates are colored in dark red, yellow and green, respectively

Fig. 2 Activity analysis of mutants The black slash indicates that the mutation occurs at the same mutation site

Fig. 3 S. cerevisiae expressing CYP68J5 and its mutants D118V, F216W, M488L, and M488W at 0.5 g/L substrate concentration product formation curves and production intensities（A, B）and 2.0 g/L substrate concentration（C, D）

Fig. 4 2D plot of the intermolecular interactions of CYP68J5 and its the mutant D118V, F216W, M488L, and M488W residues and heme with substrates using Discovery Studio

Fig. 5 Molecular dynamics simulations of CYP68J5 and its elite mutant D118V A: Root mean square deviation; B: root mean square fluctuation; C: radius of gyration; D: solvent accessible surface area; E: number of hydrogen bonds; F: free energy

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