The crystal structure of an esterase from the hyperthermophilic microorganism <Emphasis Type="Italic">Pyrobaculum calidifontis</Emphasis> VA1 explains its enantioselectivity

The highly thermostable esterase from the hyperthermophilic archaeon Pyrobaculum calidifontis VA1 (PestE) shows high enantioselectivity (E?>?100) in the kinetic resolution of racemic chiral carboxylic acids, but little selectivity towards acetates of tertiary alcohols (E?=?2–4). To explain these unique properties, its crystal structure has been determined at 2.0 Å resolution. The enzyme is a member of the hormone-sensitive lipase group (group H) of the esterase/lipase superfamily on the basis of the amino acid sequence identity. The PestE structure shows a canonical α/β-hydrolase fold as core domain with a cap structure at the C-terminal end of the β-sheet. A tetramer in the crystal packing is formed of two dimers; the dimeric form is observed in solution. Conserved dimers and even tetramers are found in other group H proteins. The amino acid residues Ser157, His284, and Asp254 form the catalytic triad, which is typically found in α/β-hydrolases. The oxyanion hole is composed of Gly85 and Gly86 within the conserved sequence motif HGGG(M,F,W) (amino acid residues 83–87) and Ala158. With the elucidated structure, experimental results about enantioselectivity towards the two model substrate classes (as exemplified for 3-phenylbutanoic acid ethyl ester and 1,1,1-trifluoro-2-phenylbut-3-yn-2-yl acetate) could be explained by molecular modeling. For both enantiomers of the tertiary alcohol, orientations in two binding pockets were obtained without significant energy differences corresponding to the observed low enantioselectivity due to missing steric repulsions. In contrast, for the carboxylic acid ester, two different orientations with significant energy differences for each enantiomer were found matching the high E values.     

Special Rhodococcus sp. CR-53 esterase Est4 contains a GGG(A)X-oxyanion hole conferring activity for the kinetic resolution of tertiary alcohols

Rhodococci are highly adaptable bacteria, capable to degrade or transform a large number of organic compounds, including recalcitrant or toxic products. However, little information is available on the lipases of the genus Rhodococcus, except for LipR, the first lipase isolated and described from strain Rhodococcus CR-53. Taking into consideration the interest raised by the enzymes produced by actinomycetes, a search for new putative lipases was performed in strain Rhodococcus CR-53. We describe here the isolation, cloning, and characterization of intracellular esterase Est4, a mesophilic enzyme showing preference for short-chain-length acyl groups, without interfacial activation. Est4 displays moderate thermal and pH stability and low tolerance to most tested ions, being inhibited by detergents like sodium dodecyl sulfate and Triton X-100®. Nevertheless, the enzyme shows good long-term stability when stored at 4–20 °C and neutral pH. Amino acid sequence analysis of Est4 revealed a protein of 313 amino acids without a signal peptide, bearing most of the conserved blocks that define bacterial lipase family IV, thus being assigned to this family. Detection of a GGG(A)X oxyanion hole in the enzyme motivated the evaluation of Est4 ability to convert tertiary alcohol esters. The newly discovered esterase Est4 from Rhodococcus CR-53 successfully hydrolyzed the tertiary alcohol esters linalyl acetate, terpinyl acetate, and 1,1,1-trifluoro-2-phenylbut-3-yn-2-yl acetate.     

Identification of novel esterases for the synthesis of sterically demanding chiral alcohols by sequence-structure guided genome mining

Six esterases isolated from sequence and structure-guided genome mining approaches were evaluated for the kinetic resolution of secondary and tertiary alcohols that find application in the fine chemical and pharmaceutical industries. Activity and enantioselectivity with E-values of up to 24 were determined towards a range of sterically demanding tertiary alcohol esters. Excellent enantioselectivity (E > 100) was also achieved in the hydrolysis of a less challenging secondary alcohol ester, menthyl acetate. These results highlight that these approaches can be used for the identification of novel esterases applicable to the preparation of commercially desirable alcohols.     

Structural basis of substrate selectivity of Δ-pyrroline-5-carboxylate dehydrogenase (ALDH4A1): Semialdehyde chain length

The enzyme Δ¹-pyrroline-5-carboxylate (P5C) dehydrogenase (aka P5CDH and ALDH4A1) is an aldehyde dehydrogenase that catalyzes the oxidation of γ-glutamate semialdehyde to l-glutamate. The crystal structures of mouse P5CDH complexed with glutarate, succinate, malonate, glyoxylate, and acetate are reported. The structures are used to build a structure-activity relationship that describes the semialdehyde carbon chain length and the position of the aldehyde group in relation to the cysteine nucleophile and oxyanion hole. Efficient 4- and 5-carbon substrates share the common feature of being long enough to span the distance between the anchor loop at the bottom of the active site and the oxyanion hole at the top of the active site. The inactive 2- and 3-carbon semialdehydes bind the anchor loop but are too short to reach the oxyanion hole. Inhibition of P5CDH by glyoxylate, malonate, succinate, glutarate, and l-glutamate is also examined. The K_i values are 0.27 mM for glyoxylate, 58 mM for succinate, 30 mM for glutarate, and 12 mM for l-glutamate. Curiously, malonate is not an inhibitor. The trends in K_i likely reflect a trade-off between the penalty for desolvating the carboxylates of the free inhibitor and the number of compensating hydrogen bonds formed in the enzyme-inhibitor complex.     

Combined docking and molecular dynamics simulations to enlighten the capacity of Pseudomonas cepacia and Candida antarctica lipases to catalyze quercetin acetylation

A combined docking and molecular dynamics protocol was applied to investigate quercetin binding modes within the catalytic cavity of Candida antarctica lipase B (CALB) and Pseudomonas cepacia lipase (PCL), aiming to explain the difference of specificity of these enzymes in acetylation reaction. For both lipases, docking of quercetin yielded two families of conformers with either the quercetin A or B-ring pointing towards the catalytic residues. Molecular dynamics (MD) calculations were subsequently performed on several complexes of each family. MD trajectories were analyzed focusing on the orientation of the acyl donor bound to the catalytic serine towards the oxyanion hole residues and the proximity of quercetin hydroxyl groups to the catalytic residues. Results showed that with CALB, the acetate was not correctly positioned within the oxyanion hole whatever the orientation of quercetin, suggesting that no product could be obtained. With PCL, the acetate remained within the oxyanion hole during all MD trajectories. Depending on quercetin orientation, either the 7-OH group or the 3, 5, 3′, 4′-OH groups came alternatively near the catalytic residues, suggesting that all of them could be acylated. The capacity of models to explain the regioselectivity of the reaction was discussed. Key residues and interactions involved in quercetin binding modes were identified and related to the reaction feasibility.     

Molecular dynamic study of subtilisin Carlsberg in aqueous and nonaqueous solvents

Using molecular dynamics simulations, we have obtained an important insight into the structural and dynamical changes exerted by a nonaqueous solvent on the serine protease subtilisin Carlsberg. Our findings show that the structural properties of the subtilisin–acetonitrile (MeCN) system were sensitive to the amount of water present at the protein surface. A decrease or lack of water promoted the enzyme–MeCN interaction, which increased structural changes of the enzyme primarily at the surface loops. This effect caused variations on the secondary and tertiary structure of the protein and induced the opening of a pathway for the solvent to the protein core. Also, disturbance of the oxyanion hole was observed due to changes in the orientation in the Asn-155 side chain. The disruption of the oxyanion hole and the changes of the tertiary structure should affect the optimal activity of the enzyme.     

Exploring the Enantioselective Mechanism of Halohydrin Dehalogenase from Agrobacterium radiobacter AD1 by Iterative Saturation Mutagenesis

Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) shows great potential in producing valuable chiral epoxides and β-substituted alcohols. The wild-type (WT) enzyme displays a high R-enantiopreference toward most aromatic substrates, whereas no S-selective HheC has been reported to date. To obtain more enantioselective enzymes, seven noncatalytic active-site residues were subjected to iterative saturation mutagenesis (ISM). After two rounds of screening aspects of both activity and enantioselectivity (E), three outstanding mutants (Thr134Val/Leu142Met, Leu142Phe/Asn176His, and Pro84Val/Phe86Pro/Thr134Ala/Asn176Ala mutants) with divergent enantioselectivity were obtained. The two double mutants displayed approximately 2-fold improvement in R-enantioselectivity toward 2-chloro-1-phenylethanol (2-CPE) without a significant loss of enzyme activity compared with the WT enzyme. Strikingly, the Pro84Val/Phe86Pro/Thr134Ala/Asn176Ala mutant showed an inverted enantioselectivity (from an E_R of 65 [WT] to an E_S of 101) and approximately 100-fold-enhanced catalytic efficiency toward (S)-2-CPE. Molecular dynamic simulation and docking analysis revealed that the phenyl side chain of (S)-2-CPE bound at a different location than that of its R-counterpart; those mutations generated extra connections for the binding of the favored enantiomer, while the eliminated connections reduced binding of the nonfavored enantiomer, all of which could contribute to the observed inverted enantiopreference.     

Mutations in the stereospecificity pocket and at the entrance of the active site of Candida antarctica lipase B enhancing enzyme enantioselectivity

Two different parts of Candida antarctica lipase B (stereospecificity pocket at the bottom of the active site and hydrophobic tunnel leading to the active site) were redesigned by single- or double-point mutations, in order to better control and improve enzyme enantioselectivity toward secondary alcohols. Single-point isosteric mutations of Ser47 and Thr42 situated in the stereospecificity pocket gave rise to variants with doubled enantioselectivity toward pentan-2-ol, in solid/gas reactor. Besides, the width and shape of the hydrophobic tunnel leading to the active site was modified by producing the following single-point mutants: Ile189Ala, Leu278Val and Ala282Leu. For each of these variants a significant modification of enantioselectivity was observed compared to wild-type enzyme, indicating that discrimination of the enantiomers by the enzyme could also arise from their different accessibilities from the enzyme surface to the catalytic site.     

Key residues for controlling enantioselectivity of Halohydrin dehalogenase from Arthrobacter sp. strain AD2, revealed by structure-guided directed evolution

Halohydrin dehalogenase from Agrobacterium radiobacter AD1 (HheC) is a valuable tool in the preparation of R enantiomers of epoxides and β-substituted alcohols. In contrast, the halohydrin dehalogenase from Arthrobacter sp. AD2 (HheA) shows a low S enantioselectivity toward most aromatic substrates. Here, three amino acids (V136, L141, and N178) located in the two neighboring active-site loops of HheA were proposed to be the key residues for controlling enantioselectivity. They were subjected to saturation mutagenesis aimed at evolving an S-selective enzyme. This led to the selection of two outstanding mutants (the V136Y/L141G and N178A mutants). The double mutant displayed an inverted enantioselectivity (from S enantioselectivity [E(S)] = 1.7 to R enantioselectivity [E(R)] = 13) toward 2-chloro-1-phenylethanol without compromising enzyme activity. Strikingly, the N178A mutant showed a large enantioselectivity improvement (E(S) > 200) and a 5- to 6-fold-enhanced specific activity toward (S)-2-chloro-1-phenylethanol. Further analysis revealed that those mutations produced some interference for the binding of nonfavored enantiomers which could account for the observed enantioselectivities. Our work demonstrated that those three active-site residues are indeed crucial in modulating the enantioselectivity of HheA and that a semirational design strategy has great potential for rapid creation of novel industrial biocatalysts.     

Facile modulation of enantioselectivity of thermophilic Geobacillus zalihae lipase by regulating hydrophobicity of its Q114 oxyanion

Site-directed mutagenesis of the oxyanion-containing amino acid Q114 in the recombinant thermophilic T1 lipase previously isolated from Geobacillus zalihae was performed to elucidate its role in the enzyme⿿s enantioselectivity and reactivity. Substitution of Q114 with a hydrophobic methionine to yield mutant Q114M increased enantioselectivity (3.2-fold) and marginally improved reactivity (1.4-fold) of the lipase in catalysing esterification of ibuprofen with oleyl alcohol. The improved catalytic efficiency of Q114L was concomitant with reduced flexibility in the active site while the decreased enantioselectivity of Q114L could be directly attributed to diminished electrostatic repulsion of the substrate carboxylate ion that rendered partial loss in steric hindrance and thus enantioselectivity. The highest E-values for both Q114L (E-value 14.6) and Q114M (E-value 48.5) mutant lipases were attained at 50 °C, after 12⿿16 h, with a molar ratio of oleyl alcohol to ibuprofen of 1.5:1 and at 2.0% (w/v) enzyme load without addition of molecular sieves. Pertinently, site-directed mutagenesis on the Q114 oxyanion of T1 resulted in improved enantioselectivity and such approach may be applicable to other lipases of the same family. We demonstrated that electrostatic repulsion phenomena could affect flexibility/rigidity of the enzyme-substrate complex, aspects vital for enzyme activity and enantioselectivity of T1.     

MD simulations reveal alternate conformations of the oxyanion hole in the Zika virus NS2B/NS3 protease

Recent crystallography studies have shown that the binding site oxyanion hole plays an important role in inhibitor binding, but can exist in two conformations (active/inactive). We have undertaken molecular dynamics (MD) calculations to better understand oxyanion hole dynamics and thermodynamics. We find that the Zika virus (ZIKV) NS2B/NS3 protease maintains a stable closed conformation over multiple 100-ns conventional MD simulations in both the presence and absence of inhibitors. The S1, S2, and S3 pockets are stable as well. However, in two of eight simulations, the A132-G133 peptide bond in the binding pocket of S1' spontaneously flips to form a 3₁₀-helix that corresponds to the inactive conformation of the oxyanion hole, and then maintains this conformation until the end of the 100-ns conventional MD simulations without inversion of the flip. This conformational change affects the S1' pocket in ZIKV NS2B/NS3 protease active site, which is important for small molecule binding. The simulation results provide evidence at the atomic level that the inactive conformation of the oxyanion hole is more favored energetically when no specific interactions are formed between substrate/inhibitor and oxyanion hole residues. Interestingly, however, transition between the active and inactive conformation of the oxyanion hole can be observed by boosting the valley potential in accelerated MD simulations. This supports a proposed induced-fit mechanism of ZIKV NS2B/NS3 protease from computational methods and provides useful direction to enhance inhibitor binding predictions in structure-based drug design.     

Residue Val237 is critical for the enantioselectivity of Penicillium expansum lipase

The shape of the hydrophobic tunnel leading to the active site of Penicillium expansum lipase (PEL) was redesigned by single-point mutations, in order to better understand enzyme enantioselectivity towards naproxen. A variant with a valine-to-glycine substitution at residue 237 exhibited almost no enantioselectivity (E = 1.1) compared with that (E = 104) of wild-type PEL. The function of the residue, Val237, in the hydrophobic tunnel was further analyzed by site-directed mutagenesis. For each of these variants a significant decrease of enantioselectivity (E < 7) was observed compared with that of wild-type enzyme. Further docking result showed that Val237 plays the most important role in stabilizing the correct orientation of (R)-naproxen. Overall, these results indicate that the residue Val237 is the key amino acid residue maintaining the enantioselectivity of the lipase.     

Enzymatic activity of the Staphylococcus aureus SplB serine protease is induced by substrates containing the sequence Trp-Glu-Leu-Gln

Proteases are of significant importance for the virulence of Staphylococcus aureus. Nevertheless, their subset, the serine protease-like proteins, remains poorly characterized. Here presented is an investigation of SplB protease catalytic activity revealing that the enzyme possesses exquisite specificity and only cleaves efficiently after the sequence Trp-Glu-Leu-Gln. To understand the molecular basis for such selectivity, we solved the three-dimensional structure of SplB to 1.8 Å. Modeling of substrate binding to the protease demonstrated that selectivity relies in part on a canonical specificity pockets-based mechanism. Significantly, the conformation of residues that ordinarily form the oxyanion hole, an essential structural element of the catalytic machinery of serine proteases, is not canonical in the SplB structure. We postulate that within SplB, the oxyanion hole is only formed upon docking of a substrate containing the consensus sequence motif. It is suggested that this unusual activation mechanism is used in parallel with classical determinants to further limit enzyme specificity. Finally, to guide future development, we attempt to point at likely physiological substrates and thus the role of SplB in staphylococcal physiology.     

Design of high-activity mutants of human butyrylcholinesterase against (-)-cocaine: structural and energetic factors affecting the catalytic efficiency

The present study was aimed to explore the correlation between the protein structure and catalytic efficiency of butyrylcholinesterase (BChE) mutants against (-)-cocaine by modeling the rate-determining transition state (TS1), i.e., the transition state for the first step of chemical reaction process, of (-)-cocaine hydrolysis catalyzed by various mutants of human BChE in comparison with the wild type. Molecular modeling of the TS1 structures revealed that mutations on certain nonactive site residues can indirectly affect the catalytic efficiency of the enzyme against (-)-cocaine through enhancing or weakening the overall hydrogen bonding between the carbonyl oxygen of (-)-cocaine benzoyl ester and the oxyanion hole of the enzyme. Computational insights and predictions were supported by the catalytic activity data obtained from wet experimental tests on the mutants of human BChE, including five new mutants reported for the first time. The BChE mutants with at least ～1000-fold improved catalytic efficiency against (-)-cocaine compared to the wild-type BChE are all associated with the TS1 structures having stronger overall hydrogen bonding between the carbonyl oxygen of (-)-cocaine benzoyl ester and the oxyanion hole of the enzyme. The combined computational and experimental data demonstrate a reasonable correlation relationship between the hydrogen-bonding distances in the TS1 structure and the catalytic efficiency of the enzyme against (-)-cocaine.     

Asymmetric transesterification of secondary alcohols catalyzed by feruloyl esterase from Humicola insolens

A new asymmetric transesterification of secondary alcohols catalyzed by feruloyl esterase from Humicola insolens has been found. Although alcohols are not the natural substrates for this enzyme, a high R enantioselectivity was observed. Stereochemical studies showed that variations in substrate structure lead to strong variations in enantioselectivity. The highest enantioselectivities are obtained when the beta-carbon of the secondary alcohol is tertiary or quaternary.     

Improved enantioselective conversion of styrene epoxides and meso-epoxides through epoxide hydrolases with a mutated nucleophile-flanking residue

In epoxide hydrolase from Agrobacterium radiobacter (EchA), phenylalanine 108 flanks the nucleophilic aspartate and forms part of the substrate-binding pocket. The influence of mutations at this position on the activity and enantioselectivity of the enzyme was investigated. Screening for improved enantioselectivity towards para-nitrophenyl glycidyl ether (pNPGE) using spectrophotometric progress curve analysis yielded five different mutants with 3- to 7-fold improved enantioselectivity. The increase in enantioselectivity was in most cases the result of an enhanced catalytic efficiency toward the preferred enantiomer. Several mutations at position F108 resulted in a higher activity toward cis-disubstituted meso-epoxides, which were converted to a single product enantiomer. Mutant F108C converted cis-2,3-epoxybutane to (2R,3R)-2,3-butanediol of >99% ee with a 7-fold improved activity, and mutant F108A hydrolyzed cyclohexene oxide to (1R,2R)-1,2-cyclohexanediol of >99% ee with a more than 150-fold higher activity than wild-type enzyme. It is concluded that single amino acid substitutions in the active site of epoxide hydrolase can result in enzyme variants with catalytic properties that are suitable for preparative scale production of (S)-epoxides and chiral vicinal diols in high yield and with excellent ee.     

A novel cold-adapted esterase from Salinisphaera sp. P7-4: gene cloning, overproduction, and characterization

Salinisphaera sp. P7-4 was isolated from the intestine of silver whiting, Sillago japonicas caught in the Pacific Ocean, and the esterase gene was cloned using the shotgun method. The amino acid sequence deduced from the nucleotide sequence (951 bp) corresponded to a protein of 316 amino acid residues with a molecular weight of 34,443. The esterase had 46 and 44% identities with the esterase enzymes of Ralstonia eutropha JMP134 and Rhodopseudomonas palustris HaA2, respectively. The primary structure of P7-4 esterase showed the conserved catalytic triad (Ser, Asp, His), consensus pentapeptide GXSXG, and oxyanion hole sequence (HG). The protein P7-4 was successfully expressed in Escherichia coli in a biologically active form. The enzyme showed high catalytic activity at low temperatures (5-25° C) with an activation energy of 2.18 kcal/mol. This result indicated that the esterase from Salinisphaera sp. P7-4 is a new cold-adapted enzyme. The enzyme preferentially hydrolyzed acyl-group chains with short chain lengths of ≤10 carbon. Metal ions such as Cd2(+), Co2(+), Cu2(+), Hg2(+), Ni2(+) and Zn2(+) inhibited enzymatic activity. Additionally, EDTA has no effect on its activity, whereas inhibition was observed with PMSF, a serine hydrolase inhibitor.     

Crystal structure and characterization of esterase Est25 mutants reveal improved enantioselectivity toward (S)-ketoprofen ethyl ester

Esterases comprise a group of enzymes that catalyze the cleavage and synthesis of ester bonds. They are important in biotechnological applications owing to their enantioselectivity, regioselectivity, broad substrate specificity, and the fact that they do not require cofactors. In a previous study, we isolated the esterase Est25 from a metagenomic library. Est25 showed catalytic activity toward the (R,S)-ketoprofen ethyl ester but had low enantioselectivity toward the (S)-ketoprofen ethyl ester. Because (S)-ketoprofen has stronger anti-inflammatory effects and fewer side effects than (R)-ketoprofen, enantioselectivity of this esterase is important. In this study, we generated Est25 mutants with improved enantioselectivity toward the (S)-ketoprofen ethyl ester; improved enantioselectivity of mutants was established by analysis of their crystal structures. The enantioselectivity of mutants was influenced by substitution of Phe72 and Leu255. Substituting these residues changed the size of the binding pocket and the entrance hole that leads to the active site. The enantioselectivity of Est25 (E = 1.1 ± 0.0) was improved in the mutants F72G (E = 1.9 ± 0.2), L255W (E = 16.1 ± 1.1), and F72G/L255W (E = 60.1 ± 0.5). Finally, characterization of Est25 mutants was performed by determining the optimum reaction conditions, thermostability, effect of additives, and substrate specificity after substituting Phe72 and Leu255.

     

Highly efficient synthesis of chiral alcohols with a novel NADH-dependent reductase from Streptomyces coelicolor

An NADH-dependent reductase (ScCR) from Streptomyces coelicolor was discovered by genome mining for carbonyl reductases. ScCR was overexpressed in Escherichia coli BL21, purified to homogeneity and its catalytic properties were studied. This enzyme catalyzed the asymmetric reduction of a broad range of prochiral ketones including aryl ketones, α- and β-ketoesters, with high activity and excellent enantioselectivity (>99% ee) towards β-ketoesters. Among them, ethyl 4-chloro-3-oxobutanoate (COBE) was efficiently converted to ethyl (S)-4-chloro-3-hydroxybutanoate ((S)-CHBE), an important pharmaceutical intermediate, in water/toluene biphasic system. As much as 600 g/L (3.6 M) of COBE was asymmetrically reduced within 22 h using 2-propanol as a co-substrate for NADH regeneration, resulting in a yield of 93%, an enantioselectivity of >99% ee, and a total turnover number (TTN) of 12,100. These results indicate the potential of ScCR for the industrial production of valuable chiral alcohols.     

High-cell-density fermentation and pilot-scale biocatalytic studies of an engineered yeast expressing the heterologous glycoside hydrolase of 7-β-xylosyltaxanes

The glycoside hydrolase of 7-β-xylosyltaxanes (designated as LXYL-P1-2) is encoded by Lxyl-p1-2 isolated from Lentinula edodes. This hydrolase specifically removes C-7 xylose from 7-β-xylosyltaxanes to form 7-β-hydroxyltaxanes, which can be used for the semi-synthesis of paclitaxel or its analogues. In our present study, we established a high-cell-density fermentation of the recombinant Pichia pastoris harboring the Lxyl-p1-2 gene. Moreover, we further optimized the fermentation conditions, including the initial cell density and the dissolved oxygen level in the induction phase. Under optimized conditions, the biomass of 312.3 g/l (wet cell weight, WCW) was obtained, and the biomass activity of the recombinant enzyme reached 6.55 × 10⁴ U/g (WCW). The freeze-dried cells (32 g/l) were used to convert 7-β-xylosyltaxanes (10 g/l, 7-β-xylosyl-10-deacetyltaxol = 62.12 %) in a 5-l reaction volume, and a bioconversion rate about 80 % was achieved. The product purification was performed by ethyl acetate, silica gel chromatography, and preparative HPLC (prep-HPLC), yielding 15.13 g of 10-deacetyltaxol, 3.07 g of 10-deacetylcephalomanine, and 3.47 g of 10-deacetyltaxol C, respectively. In addition, the average recovery rate was around 70 %. Our work provided a foundation for the industrial utilization of the recombinant enzyme on the semi-synthesis of paclitaxel using 7-β-xylosyltaxanes.