His224 Alters the R2 Drug Binding Site and Phe218 Influences the Catalytic Efficiency of the Metallo-β-Lactamase VIM-7

ABSTRACTMetallo-β-lactamases (MBLs) are the causative mechanism for resistance to β-lactams, including carbapenems, in many Gram-negative pathogenic bacteria. One important family of MBLs is the Verona integron-encoded MBLs (VIM). In this study, the importance of residues Asp120, Phe218, and His224 in the most divergent VIM variant, VIM-7, was investigated to better understand the roles of these residues in VIM enzymes through mutations, enzyme kinetics, crystal structures, thermostability, and docking experiments. The tVIM-7-D120A mutant with a tobacco etch virus (TEV) cleavage site was enzymatically inactive, and its structure showed the presence of only the Zn1 ion. The mutant was less thermostable, with a melting temperature (Tm) of 48.5°C, compared to 55.3°C for the wild-type tVIM-7. In the F218Y mutant, a hydrogen bonding cluster was established involving residues Asn70, Asp84, and Arg121. The tVIM-7-F218Y mutant had enhanced activity compared to wild-type tVIM-7, and a slightly higherTm(57.1°C) was observed, most likely due to the hydrogen bonding cluster. Furthermore, the introduction of two additional hydrogen bonds adjacent to the active site in the tVIM-7-H224Y mutant gave a higher thermostability (Tm, 62.9°C) and increased enzymatic activity compared to those of the wild-type tVIM-7. Docking of ceftazidime in to the active site of tVIM-7, tVIM-7-H224Y, and VIM-7-F218Y revealed that the side-chain conformations of residue 224 and Arg228 in the L3 loop and Tyr67 in the L1 loop all influence possible substrate binding conformations. In conclusion, the residue composition of the L3 loop, as shown with the single H224Y mutation, is important for activity particularly toward the positively charged cephalosporins like cefepime and ceftazidime.

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Contribution of a buried aspartate residue towards the catalytic efficiency and structural stability of Bacillus stearothermophilus lactate dehydrogenase

Biochemical Journal ◽

10.1042/bj3000491 ◽

1994 ◽

Vol 300 (2) ◽

pp. 491-499 ◽

Cited By ~ 5

Author(s):

T J Nobbs ◽

A Cortés ◽

J L Gelpi ◽

J J Holbrook ◽

T Atkinson ◽

...

Keyword(s):

Hydrogen Bonding ◽

Lactate Dehydrogenase ◽

Active Site ◽

Bacillus Stearothermophilus ◽

Mutant Enzyme ◽

Hydroxyl Group ◽

Side Chain ◽

General Effect ◽

Carboxylate Group ◽

Wild Type

The X-ray structure of lactate dehydrogenase (LDH) shows the side-chain carboxylate group of Asp-143 to be buried in the hydrophobic interior of the enzyme, where it makes hydrogen-bonding interactions with both the side-chain hydroxyl group of Ser-273 and the main-chain amide group of His-195. This is an unusual environment for a carboxylate side-chain as hydrogen bonding normally occurs with water molecules at the surface of the protein. A charged hydrogen-bonding interaction in the interior of a protein would be expected to be much stronger than a similar interaction on the solvent-exposed exterior. In this respect the side-chain carboxylate group of Asp-143 appears to be important for maintaining tertiary structure by providing a common linkage point between three discontinuous elements of the secondary structure, alpha 1F, beta K and the beta-turn joining beta G and beta H. The contribution of the Asp-143 side-chain to the structure and function of Bacillus stearothermophilus LDH was assessed by creating a mutant enzyme containing Asn-143. The decreased thermal stability of both unactivated and fructose-1,6-diphosphate (Fru-1,6-P2)-activated forms of the mutant enzyme support a structural role for Asp-143. Furthermore, the difference in stability of the wild-type and mutant enzymes in guanidinium chloride suggested that the carboxylate group of Asp-143 contributes at least 22 kJ/mol to the conformational stability of the wild-type enzyme. However, there was no alteration in the amount of accessible tryptophan fluorescence in the mutant enzyme, indicating that the mutation caused a structural weakness rather than a gross conformational change. Comparison of the wild-type and mutant enzyme steady-state parameters for various 2-keto acid substrates showed the mutation to have a general effect on catalysis, with an average difference in binding energy of 11 kJ/mol for the transition-state complexes. The different effects of pH and Fru-1,6-P2 on the wild-type and mutant enzymes also confirmed a perturbation of the catalytic centre in the mutant enzyme. As the side-chain of Asp-143 is not sufficiently close to the active site to be directly involved in catalysis or substrate binding it is proposed that the effects on catalysis shown by the mutant enzyme are induced either by a structural change or by charge imbalance at the active site.(ABSTRACT TRUNCATED AT 400 WORDS)

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Effects of environment on flavin reactivity in morphinone reductase: analysis of enzymes displaying differential charge near the N-1 atom and C-2 carbonyl region of the active-site flavin

Biochemical Journal ◽

10.1042/bj3590315 ◽

2001 ◽

Vol 359 (2) ◽

pp. 315-323 ◽

Cited By ~ 5

Author(s):

Daniel H. CRAIG ◽

Terez BARNA ◽

Peter C. E. MOODY ◽

Neil C. BRUCE ◽

Stephen K. CHAPMAN ◽

...

Keyword(s):

Active Site ◽

Positive Charge ◽

Hydride Transfer ◽

Reduced Form ◽

Side Chain ◽

Wild Type ◽

Reduction Potentials ◽

In The Wild ◽

Mutant Forms ◽

Positively Charged

The side chain of residue Arg238 in morphinone reductase (MR) is located close to the N-1/C-2 carbonyl region of the flavin isoalloxazine ring. During enzyme reduction negative charge develops in this region of the flavin. The positioning of a positively charged side chain in the N-1/C-2 carbonyl region of protein-bound flavin is common to many flavoprotein enzymes. To assess the contribution made by Arg238 in stabilizing the reduced flavin in MR we isolated three mutant forms of the enzyme in which the position of the positively charged side chain was retracted from the N-1/C-2 carbonyl region (Arg238 → Lys), the positive charge was removed (Arg238 → Met) or the charge was reversed (Arg238 → Glu). Each mutant enzyme retains flavin in its active site. Potentiometric studies of the flavin in the wild-type and mutant forms of MR indicate that the flavin semiquinone is not populated to any appreciable extent. Reduction of the flavin in each enzyme is best described by a single Nernst function, and the values of the midpoint reduction potentials (E12) for each enzyme fall within the region of −247±10mV. Stopped-flow studies of NADH binding to wild-type and mutant MR enzymes reveal differences in the kinetics of formation and decay of an enzyme–NADH charge-transfer complex, reflecting small perturbations in active-site geometry. Reduced rates of hydride transfer in the mutant enzymes are attributed to altered geometrical alignment of the nicotinamide coenzyme with FMN rather than major perturbations in reduction potential, and this is supported by an observed entropy–enthalpy compensation effect on the hydride transfer reaction throughout the series of enzymes. The data indicate, in contrast with dogma, that the presence of a positively charged side chain close to the N-1/C-2 carbonyl region of the flavin in MR is not required to stabilize the reduced flavin. This finding may have general implications for flavoenzyme catalysis, since it has generally been assumed that positive charge in this region has a stabilizing effect on the reduced form of flavin.

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Bioinformatics Based Understanding of Effect of Mutations in the Human β Tubulin Outside Drug Binding Sites and its Significance in Drug Resistance

The Open Bioinformatics Journal ◽

10.2174/1875036201811010029 ◽

2018 ◽

Vol 11 (1) ◽

pp. 29-37

Author(s):

Selvaa Kumar C ◽

Debjani Dasgupta ◽

Nikhil Gadewal

Keyword(s):

Drug Resistance ◽

Hydrogen Bonding ◽

Amino Acid ◽

Binding Site ◽

Drug Binding ◽

Amino Acid Substitutions ◽

Wild Type ◽

Hydrogen Bonding Pattern ◽

Drug Binding Site ◽

Β Tubulin

Background: Human β tubulin displays resistance to drugs like Taxol and Vinblastine due to amino acids substitutions within and outside the drug binding site. Objective: This study focuses on the effect of amino acid substitutions outside the drug binding site on drug resistance. Amino acid substitution like R306C (mut2) is associated with Taxol resistance and D197N (mut1) and K350N (mut3) are associated with Vinblastine resistance. However, the mechanism of resistance has not been understood yet. This study has attempted to investigate the mechanism of resistance. Methods: SWISSMODEL server was used to model the wild and the mutant β subunits which were later considered for protein-protein and protein-ligand docking using HADDOCK and AutoDock 1.5.6 software respectively. Dimer mutants were generated using Swisspdbviewer. POCASA 1.1 server was used to calculate the overall effect of substitution on pocket volume and the effect of substitution on domain mobility was explored using GROMACS software. Results: From sequence perspective, amino acid replacement in all three positions viz. D197N (mut1), R306C (mut2) and K350N (mut3) were found to have a deleterious effect on the stability of the protein. This study was further confirmed through structural analysis. Change in hydrogen bonding pattern was observed within the site of substitution in modeled mut1 and mut3 which is known to be specifically involved in Vinblastine interaction. In mut2 associated with Taxol binding, the hydrogen bonding pattern remained unaltered. All three mutants showed better protein-protein (β-β) interactions compared to the wild-type. Pocket size analysis in β subunit revealed that Taxol binding site increased in size after substitution in mut2 compared to the wild-type. However, the size of the Vinblastine binding site in the dimer interface remained the same before and after the substitution in wild and the mutants. Wild-type (β monomer and αβ dimer) associated with Taxol and Vinblastine, respectively showed better drug interaction compared to their mutants. Conclusion: This study throws light on the mechanism of drug resistance due to amino acid substitutions outside the drug binding site. It was found that amino acid substitution outside the drug site enhanced protein-protein interaction between the β-β subunits.

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Mutation of a single amino acid converts the human water channel aquaporin 5 into an anion channel

AJP Cell Physiology ◽

10.1152/ajpcell.00129.2013 ◽

2013 ◽

Vol 305 (6) ◽

pp. C663-C672 ◽

Cited By ~ 12

Author(s):

Xue Qin ◽

Walter F. Boron

Keyword(s):

Water Channel ◽

Anion Channel ◽

Single Amino Acid ◽

Side Chain ◽

Wild Type ◽

Aquaporin 5 ◽

Anion Conductance ◽

Electrode Voltage ◽

Microelectrode Measurements ◽

Positively Charged

Aquaporin 6 (AQP6) is unique among mammalian AQPs in being an anion channel with negligible water permeability. However, the point mutation Asn60Gly converts AQP6 from an anion channel into a water channel. In the present study of human AQP5, we mutated Leu51 (corresponding to residue 61 in AQP6), the side chain of which faces the central pore. We evaluated function in Xenopus oocytes by two-electrode voltage clamp, video measurements of osmotic H2O permeability ( Pf), microelectrode measurements of surface pH (pHS) to assess CO2 permeability, and surface biotinylation. We found that AQP5-L51R does not exhibit the H2O or CO2 permeability of the wild-type protein but instead has a novel p-chloromercuribenzene sulfonate (pCMBS)-sensitive current. The double mutant AQP5-L51R/C182S renders the conductance insensitive to pCMBS, demonstrating that the current is intrinsic to AQP5. AQP5-L51R has the anion permeability sequence I− > NO3− ≅ NO2− > Br− > Cl− > HCO3− > gluconate. Of the other L51 mutants, L51T (polar uncharged) and L51V (nonpolar) retain H2O and CO2 permeability and do not exhibit anion conductance. L51D and L51E (negatively charged) have no H2O or CO2 permeability. L51K (positively charged) has an intermediate H2O and CO2 permeability and anion conductance. L51H is unusual in having a relatively low CO2 permeability and anion conductance, but a moderate Pf. Thus, positively charged mutations of L51 can convert AQP5 from a H2O/CO2 channel into an anion channel. However, the paradoxical effect of L51H is consistent with the hypothesis that CO2, in part, takes a pathway different from H2O through AQP5.

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P219L substitution in human D-amino acid oxidase impacts the ligand binding and catalytic efficiency

The Journal of Biochemistry ◽

10.1093/jb/mvaa083 ◽

2020 ◽

Vol 168 (5) ◽

pp. 557-567

Author(s):

Wanitcha Rachadech ◽

Yusuke Kato ◽

Rabab M Abou El-Magd ◽

Yuji Shishido ◽

Soo Hyeon Kim ◽

...

Keyword(s):

Amino Acid ◽

Conformational Changes ◽

Active Site ◽

Structural Changes ◽

Catalytic Efficiency ◽

Acid Oxidase ◽

Wild Type ◽

Amino Acid Oxidase ◽

Adenine Ring ◽

The Impact

Abstract Human D-amino acid oxidase (DAO) is a flavoenzyme that is implicated in neurodegenerative diseases. We investigated the impact of replacement of proline with leucine at Position 219 (P219L) in the active site lid of human DAO on the structural and enzymatic properties, because porcine DAO contains leucine at the corresponding position. The turnover numbers (kcat) of P219L were unchanged, but its Km values decreased compared with wild-type, leading to an increase in the catalytic efficiency (kcat/Km). Moreover, benzoate inhibits P219L with lower Ki value (0.7–0.9 µM) compared with wild-type (1.2–2.0 µM). Crystal structure of P219L in complex with flavin adenine dinucleotide (FAD) and benzoate at 2.25 Å resolution displayed conformational changes of the active site and lid. The distances between the H-bond-forming atoms of arginine 283 and benzoate and the relative position between the aromatic rings of tyrosine 224 and benzoate were changed in the P219L complex. Taken together, the P219L substitution leads to an increase in the catalytic efficiency and binding affinity for substrates/inhibitors due to these structural changes. Furthermore, an acetic acid was located near the adenine ring of FAD in the P219L complex. This study provides new insights into the structure–function relationship of human DAO.

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Roles of key active-site residues in flavocytochrome P450 BM3

Biochemical Journal ◽

10.1042/bj3390371 ◽

1999 ◽

Vol 339 (2) ◽

pp. 371-379 ◽

Cited By ~ 118

Author(s):

Michael A. NOBLE ◽

Caroline S. MILES ◽

Stephen K. CHAPMAN ◽

Dominikus A. LYSEK ◽

Angela C. MACKAY ◽

...

Keyword(s):

Electron Transfer ◽

Fatty Acid ◽

Fatty Acid Oxidation ◽

Active Site ◽

Side Chain ◽

Acid Oxidation ◽

Dodecanoic Acid ◽

Wild Type ◽

Active Site Residues ◽

P450 Bm3

The effects of mutation of key active-site residues (Arg-47, Tyr-51, Phe-42 and Phe-87) in Bacillus megaterium flavocytochrome P450 BM3 were investigated. Kinetic studies on the oxidation of laurate and arachidonate showed that the side chain of Arg-47 contributes more significantly to stabilization of the fatty acid carboxylate than does that of Tyr-51 (kinetic parameters for oxidation of laurate: R47A mutant, Km 859 µM, kcat 3960 min-1; Y51F mutant, Km 432 µM, kcat 6140 min-1; wild-type, Km 288 µM, kcat 5140 min-1). A slightly increased kcat for the Y51F-catalysed oxidation of laurate is probably due to decreased activation energy (ΔG‡) resulting from a smaller ΔG of substrate binding. The side chain of Phe-42 acts as a phenyl ‘cap ’ over the mouth of the substrate-binding channel. With mutant F42A, Km is massively increased and kcat is decreased for oxidation of both laurate (Km 2.08 mM, kcat 2450 min-1) and arachidonate (Km 34.9 µM, kcat 14620 min-1; compared with values of 4.7 µM and 17100 min-1 respectively for wild-type). Amino acid Phe-87 is critical for efficient catalysis. Mutants F87G and F87Y not only exhibit increased Km and decreased kcat values for fatty acid oxidation, but also undergo an irreversible conversion process from a ‘fast ’ to a ‘slow ’ rate of substrate turnover [for F87G (F87Y)-catalysed laurate oxidation: kcat ‘fast ’, 760 (1620) min-1; kcat ‘slow ’, 48.0 (44.6) min-1; kconv (rate of conversion from fast to slow form), 4.9 (23.8) min-1]. All mutants showed less than 10% uncoupling of NADPH oxidation from fatty acid oxidation. The rate of FMN-to-haem electron transfer was shown to become rate-limiting in all mutants analysed. For wild-type P450 BM3, the rate of FMN-to-haem electron transfer (8340 min-1) is twice the steady-state rate of oxidation (4100 min-1), indicating that other steps contribute to rate limitation. Active-site structures of the mutants were probed with the inhibitors 12-(imidazolyl)dodecanoic acid and 1-phenylimidazole. Mutant F87G binds 1-phenylimidazole > 10-fold more tightly than does the wild-type, whereas mutant Y51F binds the haem-co-ordinating fatty acid analogue 12-(imidazolyl)dodecanoic acid > 30-fold more tightly than wild-type.

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The Role of Lysine Residues 42, 43, and 44 in the Activation Peptide Region of TAFI in Its Activation by the Thrombin-Thrombomodulin Complex.

Blood ◽

10.1182/blood.v114.22.3187.3187 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3187-3187

Author(s):

Chengliang Wu ◽

Paul Y Kim ◽

Reg Manuel ◽

Ann Gils ◽

Paul Declerck ◽

...

Keyword(s):

Regression Analysis ◽

Kinetic Parameters ◽

Catalytic Efficiency ◽

Molecular Surface ◽

Surface Patch ◽

Charged Surface ◽

Wild Type ◽

Lysine Residues ◽

Activation Peptide ◽

Positively Charged

Abstract Abstract 3187 Poster Board III-124 Thrombin-activatable fibrinolysis inhibitor (TAFI) is a 60 kDa plasma protein that is activated to the enzyme TAFIa, by a single cleavage at Arg92 by thrombin, plasmin or trypsin. TAFIa is a carboxypeptidase B-like enzyme that attenuates fibrinolysis. Thrombomodulin (TM) is a cofactor which increases the overall efficiency of thrombin-mediated TAFI activation by 1250-fold. Thus, the thrombin-TM complex is believed to be the physiological TAFI activator. The minimal structure of TM required for efficient TAFI activation contains the EGF-like domains 3 through 6. New structure models have postulated that the C-loop of TM EGF-like domain 3 has a negatively charged molecular surface that could interact with several positively charged surface patches on TAFI. One positively charged surface patch of TAFI consists of the three consecutive lysine residues at positions 42, 43, and 44, which are unique to the TAFI activation peptide as no corresponding residues exist in rattus, bovine or human tissue procarboxypeptidases A and B. More interestingly, all three lysine residues are conserved in human, rattus, murine and canine TAFI, but not for bovine TAFI which only has a single lysine residue at position 42. We previously reported that when the three lysine residues are substituted by alanine residues (K42/43/44A), compared to the wild-type, the catalytic efficiencies for TAFI activation by thrombin-TM complex decreased 8-fold. In order to identify which residue(s) are key for TAFI activation by the thrombin-TM complex, combinations of mutations of the three lysine residues were constructed and expressed. TAFI wild-type or mutants were activated by thrombin for 10 minutes in the absence or presence of TM at varying levels. At this point, the levels of TAFIa formed were measured by adding the synthetic substrate AAFR containing PPAck and measuring the absorbance change at 349nm. The rates were used to determine the kinetic parameters of TAFI activation. The non-linear regression analysis with the NONLIN module of SYSTAT returned best fit values along with their asymptotic standard errors (A.S.E) for the kinetic parameters of TAFI activation (kcat, Km, and Kd). The value of Kd (the dissociation constant for the thrombin-TM interaction) is assumed to be the same for wild-type TAFI and the mutants, because all reactions have this interaction in common. The regression analysis yielded Kd = 22.4 ± 1.3 nM for this interaction. This value agrees favourably with a value of 22 nM measured directly and reported previously. The kcat values (1/sec) ranged from 1.06 ± 0.18 (K44A) to 1.19 ± 0.18 (K43A). The value for wild-type TAFI was 1.50 ± 0.63 (1/sec). Km values ranged from 1.14 ± 0.73 μM (WT) to 3.01 ± 2.17 μM (K42A). The kcat / Km ratios (1/sec/μM), which provides the best indication of overall catalytic efficiency, ranged from 1.43 ± 0.27 (WT) to 0.43 ± 0.17 (K42A). When the three lysine residues are individually substituted by alanine residues (K42A, K43A, and K44A), compared to the wild-type, their catalytic efficiencies (kcat / Km) for TAFI activation by the thrombin-TM complex decreased 3.3-fold for K42A, 1.83-fold for K43A, and 1.96-fold for K44A. When Lys43 and Lys44 are substituted by alanine residues simultaneously (K43/44A), its catalytic efficiency decreased 3.3-fold. Together, our data show that each of these lysine residues on the activation peptide of TAFI may contribute partially to the interactions of TAFI with the thrombin-TM complex that are needed for efficient activation. In addition, the effects of the mutations may be additive. Disclosures No relevant conflicts of interest to declare.

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Three factors that modulate the activity of class D β-lactamases and interfere with the post-translational carboxylation of Lys70

Biochemical Journal ◽

10.1042/bj20101122 ◽

2010 ◽

Vol 432 (3) ◽

pp. 495-506 ◽

Cited By ~ 29

Author(s):

Lionel Vercheval ◽

Cédric Bauvois ◽

Alexandre di Paolo ◽

Franck Borel ◽

Jean-Luc Ferrer ◽

...

Keyword(s):

Active Site ◽

Chloride Ions ◽

Side Chain ◽

Wild Type ◽

Post Translational Modification ◽

Rate Limiting Step ◽

Class D ◽

The Individual ◽

Rate Limiting

The activity of class D β-lactamases is dependent on Lys70 carboxylation in the active site. Structural, kinetic and affinity studies show that this post-translational modification can be affected by the presence of a poor substrate such as moxalactam but also by the V117T substitution. Val117 is a strictly conserved hydrophobic residue located in the active site. In addition, inhibition of class D β-lactamases by chloride ions is due to a competition between the side chain carboxylate of the modified Lys70 and chloride ions. Determination of the individual kinetic constants shows that the deacylation of the acyl–enzyme is the rate-limiting step for the wild-type OXA-10 β-lactamase.

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Homodimeric Hexaprenyl Pyrophosphate Synthase from the Thermoacidophilic Crenarchaeon Sulfolobus solfataricus Displays Asymmetric Subunit Structures

Journal of Bacteriology ◽

10.1128/jb.187.23.8137-8148.2005 ◽

2005 ◽

Vol 187 (23) ◽

pp. 8137-8148 ◽

Cited By ~ 22

Author(s):

Han-Yu Sun ◽

Tzu-Ping Ko ◽

Chih-Jung Kuo ◽

Rey-Ting Guo ◽

Chia-Cheng Chou ◽

...

Keyword(s):

Active Site ◽

Hydrophobic Interactions ◽

Sulfolobus Solfataricus ◽

Side Chain ◽

Farnesyl Pyrophosphate Synthase ◽

Dimer Interface ◽

Active Site Cavity ◽

Flexible Region ◽

Chain Conformations ◽

Temperature Factors

ABSTRACT Hexaprenyl pyrophosphate synthase (HexPPs) from Sulfolobus solfataricus catalyzes the synthesis of trans-C30-hexaprenyl pyrophosphate (HexPP) by reacting two isopentenyl pyrophosphate molecules with one geranylgeranyl pyrophosphate. The crystal structure of the homodimeric C30-HexPPs resembles those of other trans-prenyltransferases, including farnesyl pyrophosphate synthase (FPPs) and octaprenyl pyrophosphate synthase (OPPs). In both subunits, 10 core helices are arranged about a central active site cavity. Leu164 in the middle of the cavity controls the product chain length. Two protein conformers are observed in the S. solfataricus HexPPs structure, and the major difference between them occurs in the flexible region of residues 84 to 100. Several helices (αI, αJ, αK, and part of αH) and the associated loops have high-temperature factors in one monomer, which may be related to the domain motion that controls the entrance to the active site. Different side chain conformations of Trp136 in two HexPPs subunits result in weaker hydrophobic interactions at the dimer interface, in contrast to the symmetric π-π stacking interactions of aromatic side chains found in FPPs and OPPs. Finally, the three-conformer switched model may explain the catalytic process for HexPPs.

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Improving 10-deacetylbaccatin III-10-β-O-acetyltransferase catalytic fitness for Taxol production

Nature Communications ◽

10.1038/ncomms15544 ◽

2017 ◽

Vol 8 (1) ◽

Cited By ~ 23

Author(s):

Bing-Juan Li ◽

Hao Wang ◽

Ting Gong ◽

Jing-Jing Chen ◽

Tian-Jiao Chen ◽

...

Keyword(s):

Anticancer Drug ◽

Active Site ◽

Three Dimensional ◽

Catalytic Efficiency ◽

Dimensional Structure ◽

Wild Type ◽

One Pot ◽

Natural Concentration ◽

Taxol Production

Abstract The natural concentration of the anticancer drug Taxol is about 0.02% in yew trees, whereas that of its analogue 7-β-xylosyl-10-deacetyltaxol is up to 0.5%. While this compound is not an intermediate in Taxol biosynthetic route, it can be converted into Taxol by de-glycosylation and acetylation. Here, we improve the catalytic efficiency of 10-deacetylbaccatin III-10-O-acetyltransferase (DBAT) of Taxus towards 10-deacetyltaxol, a de-glycosylated derivative of 7-β-xylosyl-10-deacetyltaxol to generate Taxol using mutagenesis. We generate a three-dimensional structure of DBAT and identify its active site using alanine scanning and design a double DBAT mutant (DBATG38R/F301V) with a catalytic efficiency approximately six times higher than that of the wild-type. We combine this mutant with a β-xylosidase to obtain an in vitro one-pot conversion of 7-β-xylosyl-10-deacetyltaxol to Taxol yielding 0.64 mg ml−1 Taxol in 50 ml at 15 h. This approach represents a promising environmentally friendly alternative for Taxol production from an abundant analogue.

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