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Tuck Seng Wong

Publications and source records attributed to Tuck Seng Wong.

10 recordsLinked to original sources
PubMed2007

Are transversion mutations better? A Mutagenesis Assistant Program analysis on P450 BM-3 heme domain.

Directed evolution represents a versatile tool to tailor enzyme properties to needs in industrial applications and to understand structure-function relationships. Genetic diversity is commonly generated using error-prone PCR. Exploration of sequence space by random mutagenesis strongly favors transitions when enzyme-based mutagenesis methods are employed (Wong, T. S., Zhurina, D., Schwaneberg, U., Comb. Chem. High Throughput Screen. 2006, 9, 271-288). The genetic code has been organized in a manner that limits chemical diversity when a single transition mutation occurs in a codon (Wong, T. S., Roccatano, D., Schwaneberg, U., Biocatal. Biotransformation 2006, in press). Are transitions more beneficial than transversions for adapting biocatalysts to non-natural process conditions? In a statistical analysis performed with the Mutagenesis Assistant Program (MAP), we compared the consequences of transition and transversion bias on amino acid substitution patterns of the P450 BM-3 heme domain. For the analysis, we used a recently introduced benchmarking system consisting of a protein structure indicator, an amino acid diversity indicator with a codon diversity coefficient, and a chemical diversity indicator. A detailed analysis for the P450 BM-3 heme domain showed that an ideal transversion bias generates more diverse amino acid substitution patterns with a significantly different chemical composition than an ideal transition bias. Emphasis is given on the theoretical analysis with a brief discussion on potential implication of transition and transversion bias in directed evolution experiments.

Algorithms↗
PubMed2006

Toward understanding the inactivation mechanism of monooxygenase P450 BM-3 by organic cosolvents: a molecular dynamics simulation study.

Cytochrome P450 BM-3 from Bacillus megaterium is an extensively studied enzyme for industrial applications. A major focus of current protein engineering research is directed to improving the catalytic performance of P450 BM-3 toward nonnatural substrates of industrial importance in the presence of organic solvents or cosolvents. For the latter reason, it is important to study the effect of organic cosolvent molecules on the structure and dynamics of the enzyme, in particular, the effect of cosolvent molecules on the active site's structure and dynamics. In this paper, we have studied, using molecular dynamics (MD) simulations, the F87A mutant of P450 BM-3 in the presence of DMSO as cosolvent, to understand the role of the F87A substitution for its catalytic activity. This mutant exhibits an altered regioselectivity and substrate specificity compared with wild-type; however, it has lower tolerance toward DMSO. The simulation results offer an explanation for the DMSO sensitivity of the F87A mutant. Our simulation results show that the F87 side chain prevents the disturbance of the water molecule bound to the heme iron by DMSO molecules. The absence of the phenyl ring in F87A mutant promotes interactions of the DMSO molecule with the heme iron resulting in water displacement by DMSO at the catalytic heme center.

Bacillus megaterium↗
PubMed2006

The diversity challenge in directed protein evolution.

Over the past decade, we have witnessed a bloom in the field of evolutive protein engineering which is fueled by advances in molecular biology techniques and high-throughput screening technology. Directed protein evolution is a powerful algorithm using iterative cycles of random mutagenesis and screening for tailoring protein properties to our needs in industrial applications and for elucidating proteins' structure function relationships. This review summarizes, categorizes and discusses advantages and disadvantages of random mutagenesis methods used for generating genetic diversity. These random mutagenesis methods have been classified into four main categories depending on the method employed for nucleotide substitutions: enzyme based methods (Category I), synthetic chemistry based methods (Category II), whole cell methods (Category III) and combined methods (Category I-II, I-III and II-III). The basic principle of each method is discussed and varied mutagenic conditions are summarized in Tables and compared (benchmarked) to each other in terms of: mutational bias, controllable mutation frequency, ability to generate consecutive nucleotide substitutions and subset diversity, dependency on gene length, technical simplicity/robustness and cost-effectiveness. The latter comparison shows how highly-biased and limited current diversity creating methods are. Based on these limitations, strategies for generating diverse mutant libraries are proposed and discussed (RaMuS-Flowchart; KISS principle). We hope that this review provides, especially for researchers just entering the field of directed evolution, a guide for developing successful directed evolution strategies by selecting complementary methods for generating diverse mutant libraries.

Base Composition↗
PubMed2006

A filter paper-based assay for laboratory evolution of hydrolases and dehydrogenases.

Industrially important enzyme classes such as hydrolases and dehydrogenases are often not amenable to laboratory evolution methods due to a lack of sensitive and reliable high-throughput screening (HTS) systems. We developed a conceptually novel and technically simple high-throughput screening system based on detection of volatile aldehydes with the sensitive reagent Purpald (4-amino-3-hydrazino-5-mercapto-1,2,4-triazole). The aldehyde detection takes place on a filter-paper that is pre-soaked with Purpald and covers the microtiter plate. The filter paper-based Purpald assay separates aldehyde detection from biocatalytical conversion and thereby avoids interferences from biological materials with assay components. This screening principle allows, to our knowledge, for the first time to determine the synthetic activity of hydrolases such as lipases and esterases in organic solvents in a 96-well whole-cell format. Its simplicity and cost-effectiveness make the reported HTS system suitable as fast pre-screen in laboratory evolution experiments and for semi-quantitative assays of improved mutants.

Alcohol Dehydrogenase↗
PubMed2005

A statistical analysis of random mutagenesis methods used for directed protein evolution.

We have developed a statistical method named MAP (mutagenesis assistant program) to equip protein engineers with a tool to develop promising directed evolution strategies by comparing 19 mutagenesis methods. Instead of conventional transition/transversion bias indicators as benchmarks for comparison, we propose to use three indicators based on the subset of amino acid substitutions generated on the protein level: (1) protein structure indicator; (2) amino acid diversity indicator with a codon diversity coefficient; and (3) chemical diversity indicator. A MAP analysis for a single nucleotide substitution was performed for four genes: (1) heme domain of cytochrome P450 BM-3 from Bacillus megaterium (EC 1.14.14.1); (2) glucose oxidase from Aspergillus niger (EC 1.1.3.4); (3) arylesterase from Pseudomonas fluorescens (EC 3.1.1.2); and (4) alcohol dehydrogenase from Saccharomyces cerevisiae (EC 1.1.1.1). Based on the MAP analysis of these four genes, 19 mutagenesis methods have been evaluated and criteria for an ideal mutagenesis method have been proposed. The statistical analysis showed that existing gene mutagenesis methods are limited and highly biased. An average amino acid substitution per residue of only 3.15-7.4 can be achieved with current random mutagenesis methods. For the four investigated gene sequences, an average fraction of amino acid substitutions of 0.5-7% results in stop codons and 4.5-23.9% in glycine or proline residues. An average fraction of 16.2-44.2% of the amino acid substitutions are preserved, and 45.6% (epPCR method) are chemically different. The diversity remains low even when applying a non-biased method: an average of seven amino acid substitutions per residue, 2.9-4.7% stop codons, 11.1-16% glycine/proline residues, 21-25.8% preserved amino acids, and 55.5% are amino acids with chemically different side-chains. Statistical information for each mutagenesis method can further be used to investigate the mutational spectra in protein regions regarded as important for the property of interest.

Amino Acid Substitution↗
PubMed2005

Structural and dynamic properties of cytochrome P450 BM-3 in pure water and in a dimethylsulfoxide/water mixture.

Solvent molecules play an important role for the structural and dynamical properties of proteins. A major focus of current protein engineering is the development of enzymes that are catalytically active in the presence of organic solvents. The monooxygenase P450 BM-3 is one of the best-studied enzymes and promising for industrial applications but with limited activity in the presence of organic solvents or cosolvents. To gain insights into the structural and dynamical properties of the heme domain of this enzyme in solution, molecular dynamics simulations in pure water and in a 14% DMSO/water mixture were performed. The results of the simulations show overall similar structural fluctuations in both solvent systems, with no indication of partial or global unfolding. In 14% DMSO, the regions comprising the helices E, F, and the EF loop (implicated in controlling the entry to the active site channel) undergo a large shift. Significant changes were also observed near the active site access channel at the residue R47. During the simulation, no DMSO molecule penetrated the active site. However, a significant accumulation of DMSO molecules close to the substrate-binding site and to the Flavin Mononucleotide (FMN) reductase domain interface was observed.

Bacillus megaterium↗
PubMed2004

Sequence saturation mutagenesis (SeSaM): a novel method for directed evolution.

Sequence saturation mutagenesis (SeSaM) is a conceptually novel and practically simple method that truly randomizes a target sequence at every single nucleotide position. A SeSaM experiment can be accomplished within 2-3 days and comprises four steps: generating a pool of DNA fragments with random length, 'tailing' the DNA fragments with universal base using terminal transferase at 3'-termini, elongating DNA fragments in a PCR to the full-length genes using a single-stranded template and replacing the universal bases by standard nucleotides. Random mutations are created at universal sites due to the promiscuous base-pairing property of universal bases. Using enhanced green fluorescence protein as the model system and deoxyinosine as the universal base, we proved by sequencing 100 genes the concept of the SeSaM method and achieved a random distribution of mutations with the mutational bias expected for deoxyinosine.

DNA↗
PubMed2004

Laboratory evolution of cytochrome p450 BM-3 monooxygenase for organic cosolvents.

Cytochrome p450 BM-3 (EC 1.14.14.1) catalyzes the hydroxylation and/or epoxidation of a broad range of substrates, including alkanes, alkenes, alcohols, fatty acids, amides, polyaromatic hydrocarbons, and heterocycles. For many of these notoriously water-insoluble compounds, p450 BM-3's K(m) values are in the millimolar range. Polar organic cosolvents are therefore added to increase substrate solubility and achieve high catalytic efficiency. Using p450 BM-3 as a catalyst for these important transformations requires that we improve its ability to tolerate the cosolvents. By directed evolution, we improved the activity of p450 BM-3 in the presence of dimethylsulfoxide (DMSO) and tetrahydrofuran (THF), achieving increases in specific activity up to 10-fold in 2% (v/v) THF and 6-fold in 25% (v/v) DMSO. The engineered p450 BM-3's are also significantly more resistant to acetone, acetonitrile, dimethylformamide, and ethanol as cosolvents in the reaction.

Bacillus megaterium↗
PubMed2003

Protein engineering in bioelectrocatalysis.

Electrochemistry of redox proteins is a broadly applicable technology with important applications in biosensors, biofuel cells and chemical syntheses. Escalating attention in this area is driven by remarkable progress in designing efficient interfaces for transferring electrons between electrode surfaces and redox proteins. Research in interface design is slowly shifting from modifying electrode surfaces towards the engineering of redox proteins. Protein engineering, which encompasses rational design, directed evolution and combined methods, offers many powerful methods and strategies for improving the electron transfer properties of redox proteins.

Catalysis↗