Enhanced catalytic activities and modified substrate preferences for taxoid 10β-O-acetyl transferase mutants by engineering catalytic histidine residues

You, Linfeng; Huang, Jiajun; Wei, Tao; Lin, Shu-Ling; Jiang, Bing‐Hua; Guo, Li‐Qiong; Lin, Jizhen

doi:10.1007/s10529-018-2573-9

Cited by 7 publications

(2 citation statements)

References 18 publications

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“…Molecular docking also revealed the critical catalytic role of His162 in DBAT catalysis, and alanine scanning identified 4 residues that led to a complete loss of activity when mutated to alanine (H162A, R363A, G361A, and I164A). Two of these residues (H162 and R363) and an additional active site residue (D166) were further investigated by another group by a similar computational strategy supported by site directed mutagenesis (You et al, 2018). Postulating the benefits of histidine residues in the DBAT catalytic pocket, these residues were mutated to histidine, leading to D166H, R363H, H162A/R363H, and D166H/R363H that demonstrated superior catalytic activities (15-, 26-, 3-, and 60times improvements compared to wild type DBAT, respectively) (Table 2).…”

Section: Semi-rational Designing Of Dbatmentioning

confidence: 99%

Recent Advances in Metabolic Engineering, Protein Engineering, and Transcriptome-Guided Insights Toward Synthetic Production of Taxol

Mutanda

et al. 2021

Front. Bioeng. Biotechnol.

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The diterpenoid paclitaxel (Taxol®) is a blockbuster anticancer agent that was originally isolated from the Pacific yew (Taxus brevifolia) five decades ago. Despite the wealth of information gained over the years on Taxol research, there still remains supply issues to meet increasing clinical demand. Although alternative Taxol production methods have been developed, they still face several drawbacks that cause supply shortages and high production costs. It is highly desired to develop biotechnological production platforms for Taxol, however, there are still gaps in our understanding of the biosynthetic pathway, catalytic enzymes, regulatory and control mechanisms that hamper production of this critical drug by synthetic biology approaches. Over the past 5 years, significant advances were made in metabolic engineering and optimization of the Taxol pathway in different hosts, leading to accumulation of taxane intermediates. Computational and experimental approaches were leveraged to gain mechanistic insights into the catalytic cycle of pathway enzymes and guide rational protein engineering efforts to improve catalytic fitness and substrate/product specificity, especially of the cytochrome P450s (CYP450s). Notable breakthroughs were also realized in engineering the pathway in plant hosts that are more promising in addressing the challenging CYP450 chemistry. Here, we review these recent advances and in addition, we summarize recent transcriptomic data sets of Taxus species and elicited culture cells, and give a bird’s-eye view of the information that can be gleaned from these publicly available resources. Recent mining of transcriptome data sets led to discovery of two putative pathway enzymes, provided many lead candidates for the missing steps and provided new insights on the regulatory mechanisms governing Taxol biosynthesis. All these inferences are relevant to future biotechnological production of Taxol.

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Section: Semi-rational Designing Of Dbatmentioning

confidence: 99%

Recent Advances in Metabolic Engineering, Protein Engineering, and Transcriptome-Guided Insights Toward Synthetic Production of Taxol

Mutanda

et al. 2021

Front. Bioeng. Biotechnol.

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“…Among them, residues H162, D166 and R363, located in the catalytic pocket of the enzyme, were important for DBAT activity; and residues S31 and D34 from motif SXXD, D372 and G376 from motif DFGWG were important for acylation. Based on the above results, You et al [ 75 ] redesigned the active sites of enzyme DBAT (H162A/R363H, D166H, R363H and D166H/R363H), which displayed 3, 15, 26 and 60 times higher catalytic activities than that of the WT, respectively, and these mutants could transfer acetyl group from unnatural acetyl donor (e.g., vinyl acetate, sec-butyl acetate, isobutyl acetate, amyl acetate and isoamyl acetate) to 10-DAB. These studies also provide a reference for the comprehensive elucidation of taxane acylation mechanism and the synthesis and regulation of taxol in vitro and in vivo.…”

Section: Acylation Reactions Mediated By Taxus Actsmentioning

confidence: 95%

Recent Research Progress in Taxol Biosynthetic Pathway and Acylation Reactions Mediated by Taxus Acyltransferases

Wang

Zhuang

et al. 2021

Molecules

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Taxol is one of the most effective anticancer drugs in the world that is widely used in the treatments of breast, lung and ovarian cancer. The elucidation of the taxol biosynthetic pathway is the key to solve the problem of taxol supply. So far, the taxol biosynthetic pathway has been reported to require an estimated 20 steps of enzymatic reactions, and sixteen enzymes involved in the taxol pathway have been well characterized, including a novel taxane-10β-hydroxylase (T10βOH) and a newly putative β-phenylalanyl-CoA ligase (PCL). Moreover, the source and formation of the taxane core and the details of the downstream synthetic pathway have been basically depicted, while the modification of the core taxane skeleton has not been fully reported, mainly concerning the developments from diol intermediates to 2-debenzoyltaxane. The acylation reaction mediated by specialized Taxus BAHD family acyltransferases (ACTs) is recognized as one of the most important steps in the modification of core taxane skeleton that contribute to the increase of taxol yield. Recently, the influence of acylation on the functional and structural diversity of taxanes has also been continuously revealed. This review summarizes the latest research advances of the taxol biosynthetic pathway and systematically discusses the acylation reactions supported by Taxus ACTs. The underlying mechanism could improve the understanding of taxol biosynthesis, and provide a theoretical basis for the mass production of taxol.

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Optimization of Baccatin III Production by Cross-Linked Enzyme Aggregate of Taxoid 10β-O-Acetyltransferase

et al. 2019

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Enhanced catalytic activities and modified substrate preferences for taxoid 10β-O-acetyl transferase mutants by engineering catalytic histidine residues

Abstract: Taxoid 10β-O-acetyl transferase mutants with redesigned active sites displayed increased catalytic activities and modified substrate preferences, indicating their possible application in the enzymatic synthesis of baccatin III and taxol.

Cited by 7 publications

References 18 publications

Recent Advances in Metabolic Engineering, Protein Engineering, and Transcriptome-Guided Insights Toward Synthetic Production of Taxol

Recent Advances in Metabolic Engineering, Protein Engineering, and Transcriptome-Guided Insights Toward Synthetic Production of Taxol

Recent Research Progress in Taxol Biosynthetic Pathway and Acylation Reactions Mediated by Taxus Acyltransferases

Optimization of Baccatin III Production by Cross-Linked Enzyme Aggregate of Taxoid 10β-O-Acetyltransferase

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