Engineering the cytokinin-glucoside specificity of the maize β-d-glucosidase Zm-p60.1 using site-directed random mutagenesis

Filipi, Tomáš; Mazura, Pavel; Janda, Lubomír; Kiran, Nagavalli Subbanna; Brzobohatý, Břetislav

doi:10.1016/j.phytochem.2011.10.008

Cited by 10 publications

(16 citation statements)

References 48 publications

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“…These results strongly suggest that a specific combination of amino acids in this region is important for both specificity and structural stability. This hypothesis has been confirmed by experiments involving random mutagenesis close to W373K, and the identification of a variant (P372T/W373K/M376L) with partially rescued catalytic activity and changed specificity [8].…”

Section: Introductionmentioning

confidence: 74%

“…However, in our Zm-p60.1 engineering project we initially used NNM (A/C/G/T, A/C/G/T, A/C/) codon randomization and functional screening of E. coli clones [8]. NNM randomization provides similar performance to NNK, but the resulting libraries do not contain variants with Trp or Met residues at the varied positions.…”

Section: Resultsmentioning

confidence: 99%

“…On the basis of our previous work we expected to find one or a few functional variants of the weakly catalyzing mutant W373K [7], [8]. However, most clones proved to be active, for example 20 of 21 clones initially obtained displayed various levels of β-glucosidase activity.…”

Section: Resultsmentioning

confidence: 99%

“…The protein variants were expressed and purified as previously reported [8] and described in Supplemental Results (File S4). Protein concentration was determined by the Bradford Protein Assay with BSA as the calibration standard (Bio-Rad Laboratories, Hercules, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

“…One of the findings of the cited studies is that W373 plays an important role in stabilizing the aglycone part of the substrate in the entrance of the active site [5], [8], [9], [10]. Residues in corresponding positions often play similar roles in other glucosidases [11], although the amino acid composition is not preserved in all cases [12].…”

Section: Introductionmentioning

confidence: 99%

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Combining Rational and Random Strategies in β-Glucosidase Zm-p60.1 Protein Library Construction

et al. 2014

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Saturation mutagenesis is a cornerstone technique in protein engineering because of its utility (in conjunction with appropriate analytical techniques) for assessing effects of varying residues at selected positions on proteins’ structures and functions. Site-directed mutagenesis with degenerate primers is the simplest and most rapid saturation mutagenesis technique. Thus, it is highly appropriate for assessing whether or not variation at certain sites is permissible, but not necessarily the most time- and cost-effective technique for detailed assessment of variations’ effects. Thus, in the presented study we applied the technique to randomize position W373 in β-glucosidase Zm-p60.1, which is highly conserved among β-glucosidases. Unexpectedly, β-glucosidase activity screening of the generated variants showed that most variants were active, although they generally had significantly lower activity than the wild type enzyme. Further characterization of the library led us to conclude that a carefully selected combination of randomized codon-based saturation mutagenesis and site-directed mutagenesis may be most efficient, particularly when constructing and investigating randomized libraries with high fractions of positive hits.

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Section: Introductionmentioning

confidence: 74%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Combining Rational and Random Strategies in β-Glucosidase Zm-p60.1 Protein Library Construction

et al. 2014

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An automated method to evaluate the enzyme kinetics of β‐glucosidases

et al. 2016

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Enzyme kinetic measurements are important for the characterization and engineering of biocatalysts, with applications in a wide range of research fields. The measurement of initial reaction velocity is usually slow and laborious, which motivated us to explore the possibilities for automating this process. Our model enzyme is the maize b-glucosidase Zm-p60.1. Zm-p60.1 plays a significant role in plant growth and development by regulating levels of the active plant hormone cytokinin. Zm-p60.1 belongs to a wide group of hydrolytic enzymes. Members of this group hydrolyze several different types of glucosides, releasing glucose as a secondary product. Enzyme kinetic measurements using artificial substrates are well established, but burdensome and timeconsuming. Thus, they are a suitable target for process automation. Simple optical methods for enzyme kinetic measurements using natural substrates are often impossible given the optical properties of the enzymatic reaction products. However, we have developed an automated method based on glucose detection, as glucose is released from all substrates of glucosidase reactions. The presented method can obtain 24 data points from up to 15 substrate concentrations to precisely describe the enzyme kinetics. The combination of an automated liquid handling process with assays that have been optimized for measuring the initial hydrolysis velocity of b-glucosidases yields two distinct methods that are faster, cheaper, and more accurate than the established protocols.Keywords: enzyme kinetics; glycoside hydrolases; lab automation; b-glucosidase; glucose; fluorescence Abbreviations used: pNP, para-nitrophenol; tZOG, trans-zeatin-O-b-d-glucoside; tZ, trans-zeatin; GO, glucose oxidase; HRP, horseradish peroxidase; CP, citrate-phosphate buffer (50 mM, pH 5.5); EDTA, ethylenediaminetetraacetic acid Additional Supporting Information may be found in the online version of this article.The automation of laboratory procedures is often desirable in research to increase efficiency and reduce costs. Two automated methods were optimized for enzyme kinetic studies using artificial and natural substrates. The automation of the liquid handling system demonstrated improved performance and reduced reagent consumption when compared to manual pipetting. The presented methods are especially suitable for the high-throughput characterization of enzymes and their mutants.

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Retargeting a maize β-glucosidase to the vacuole – Evidence from intact plants that zeatin-O-glucoside is stored in the vacuole

et al. 2012

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Engineering the cytokinin-glucoside specificity of the maize β-d-glucosidase Zm-p60.1 using site-directed random mutagenesis

Cited by 10 publications

References 48 publications

Combining Rational and Random Strategies in β-Glucosidase Zm-p60.1 Protein Library Construction

Combining Rational and Random Strategies in β-Glucosidase Zm-p60.1 Protein Library Construction

An automated method to evaluate the enzyme kinetics of β‐glucosidases

Retargeting a maize β-glucosidase to the vacuole – Evidence from intact plants that zeatin-O-glucoside is stored in the vacuole

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