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Exploring Alternative Taxol Sources: Biocatalysis of 7-β-Xylosyl-10- Deacetyltaxol and Application for Taxol Production
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2020
Wan-Cang Liu, Bing-Juan Li, Ting Gong, Ping Zhu
Enzyme activity generally declines if the amino acids involved in the substrate binding or catalytic sites are subjected to l-alanine scanning mutagenesis (mutated to Ala). However, this activity may be maintained or even increased if other amino acid residues are mutated to l-alanine (Kong et al., 2014). To improve the catalytic efficiency of the 10-deacetylbaccatin III-10-O-acetyltransferase (DBAT) of Taxus towards DT, DBAT was firstly subjected to l-alanine scanning mutagenesis (Fig. 2.5). The following amino acid residues within the 5 Å zone around the substrate were chosen for l-alanine scanning mutagenesis: Pro37, Gly38, Arg40, Glu41, Phe44, Phe160, His162, Ile164, Cys165, Phe301, Ser351, Asn353, Gly359, Gly361, Arg363, Ser396 and Phe400. Results showed that DBATH162A had no measurable enzyme activity against 10-DAB or DT, which was consistent with the function of this residue as a potential catalytic base. Similarly, each of the DBATR363A, DBATG361A and DBATI164A mutations lost virtually all activity against 10-DAB or DT, which also implied that these residues are potential substrate binding or catalytic sites. DBATF44A activity was nearly undetectable against DT and significantly declined against 10-DAB, which means that this residue may be involved in catalysis especially to the unnatural substrate DT. Other mutations, such as DBATG359A, DBATF400A, DBATP37A and DBATF160A, also negatively affected enzyme activities, but the impact of each mutation on 10-DAB and DT were quite different. However, DBATG38A and DBATF301A activities against DT significantly increased compared with that of the wild-type control, leading to 1.6 (DBATF301A) and 1.45 (DBATG38A) times more active than the wild-type, respectively. However, their activities against 10-DAB were apparently degraded (DBATF301A) or slightly decreased (DBATG38A).
Residue-specific free energy analysis in ligand bindings to JAK2
Published in Molecular Physics, 2018
Yifan Zhou, Xiao Liu, Youzhi Zhang, Long Peng, John Z. H. Zhang
In protein–ligand binding, only a few key residues make substantial contributions to enzyme- binding free energy. Those residues are typically identified by alanine scanning (AA) approach and are called hot-spots in protein–protein binding. Varieties of algorithms have been developed to predict protein–ligand binding free energy including knowledge-based methods, empirical formula-based methods and molecule dynamics simulation-based methods [20–23]. Among them, the most popular method is the MM/PB(GB)SA method for free energy calculations [24,25]. In the present paper, we apply a recently proposed interaction entropy (IE) approach [26], combined with computational alanine scanning (AS) approach to discover hot-spots in inhibitors’ binding to JAK2 by computing quantitatively residue-specific contributions to the total free energies in JAK2 binding to inhibitors. Finally, the residue-specific binding free energies are added together to give a reliable estimate of total binding free energies (AS-IE) that are compared with the experimentally measured binding affinities.