The effect of mutations near the T1 copper site on the biochemical characteristics of the small laccase from Streptomyces coelicolor A3(2).

Bacterial laccases show low activities but can be of biotechnological interest due to industrially suitable characteristics such as thermostability and tolerance to alkaline pH. In this study, three separate mutations (M298F, V290N and V290A) were introduced at or near the T1 copper site of the small laccase (SLAC) from Streptomyces coelicolor A3(2) and biochemical properties were assessed in comparison with the native enzyme. The mutation, V290N showed approximately double the activity of SLAC when ABTS was used as substrate while the specific activity of SLAC-M298F was 4-5 times higher than that of SLAC when the assays were performed at ≥70°C. There was no significant difference in activity with 2,6-dimethoxyphenol (2,6-DMP); however, there was a significant shift in the optimal pH from pH 9.5 (SLAC) to 7.5 (SLAC-V290N). Optimal temperature for activity was not significantly altered but thermostability was reduced in all three mutants. The substrate range of the mutant variants remained largely unchanged, with the exception of SLAC-M298F which was unable to oxidise veratryl alcohol. Interestingly, the "typical" laccase inhibitor, sodium azide, had no significant inhibitory effect on the activity of SLAC-M298F, which also exhibited increased resistance to inhibition by sulfhydryl compounds. SLAC-V290N showed higher catalytic efficiency for 2,6-DMP (kcat/Km=2.226mM(-1)s(-1)) and ABTS (kcat/Km=1.874mM(-1)s(-1)) compared to SLAC (kcat/Km=1.615mM(-1)s(-1) for 2,6-DMP and kcat/Km=1.611mM(-1)s(-1) for ABTS). This study has shown that three ligands that are closely associated with the T1 copper in SLAC play a key role in maintaining enzymatic activity. Whilst the introduction of mutations at these sites negated favourable characteristics such as thermostability, several favourable effects were observed. This study has also extended the knowledge base on the biochemical characteristics of SLAC, and its suitability as a template for engineering with the aim of widening its potential range of industrial applications.

Elucidating the structural and conformational factors responsible for the activity and substrate specificity of alkanesulfonate monooxygenase.

The mechanism and substrate specificity of alkanesulfonate monooxygenase (SsuD) was investigated by combining molecular dynamics simulations, docking, and a comprehensive quantitative structure activity relationships (QSAR) analysis. The FMNH(2) dependent monooxygenase undergoes a dynamic conformational change of the active site, passing from a closed to an open state. As a consequence, substrates have access to the active site and the cofactor is then regenerated by the associated oxidoreductase FMN reductase SsuE.. Computational analysis of the interaction of SsuD with FMNH(2) based on molecular docking and multiple 20 ns molecular dynamics simulations pointed out that the conformational change is mainly driven by salt bridge formation between Arg297 and Glu20 or Asp111. A set of substrates accepted by SsuD were described by means of ALMOND chemical descriptors and a partial least square (PLS) mathematical model was constructed. The PLS model correlates the structure of substrates and enzyme activity, namely kinetic properties (k (cat)/K (M)). Therefore, information coming from the PLS analysis goes beyond the simple ability of the enzyme to recognize the substrate, but includes the factors that affect the capacity of the enzyme to reduce the activation energy of the rate determining step of the reaction. The two principal components of the model are able to describe both steric and electronic factors and, more importantly, their interactions. Indeed, interactions of factors appear to affect significantly the ability of SsuD of transforming efficiently a substrate.

Engineering of Candida antarctica lipase B for hydrolysis of bulky carboxylic acid esters.

Candida antarctica lipase B (CALB) is a widely used biocatalyst with high activity and specificity for a wide range of primary and secondary alcohols. However, the range of converted carboxylic acids is more narrow and mainly limited to unbranched fatty acids. To further broaden the biotechnological applications of CALB it is of interest to expand the range of converted carboxylic acid and extend it to carboxylic acids that are branched or substituted in close proximity of the carboxyl group. An in silico library of 2400 CALB variants was built and screened in silico by substrate-imprinted docking, a four step docking procedure. First, reaction intermediates of putative substrates are covalently docked into enzyme active sites. Second, the geometry of the resulting enzyme-substrate complex is optimized. Third, the substrate is removed from the complex and then docked again into the optimized structure. Fourth, the resulting substrate poses are rated by geometric filter criteria as productive or non-productive poses. Eleven enzyme variants resulting from the in silico screening were expressed in Escherichia coli BL21 and measured in the hydrolysis of two branched fatty acid esters, isononanoic acid ethyl ester and 2-ethyl hexanoic acid ethyl esters. Five variants showed an initial increase in activity. The variant with the highest wet mass activity (T138S) was purified and further characterized. It showed a 5-fold increase in hydrolysis of isononanoic acid ethyl ester, but not toward sterically more demanding 2-ethyl hexanoic acid ethyl ester.

A model of the pressure dependence of the enantioselectivity of Candida rugosalipase towards (+/-)-menthol.

Transesterification of (+/-)-menthol using propionic acid anhydride and Candida rugosa lipase was performed in chloroform and water at different pressures (1, 10, 50, and 100 bar) to study the pressure dependence of enantioselectivity E. As a result, E significantly decreased with increasing pressure from E = 55 (1 bar) to E = 47 (10 bar), E = 37 (50 bar), and E = 9 (100 bar). To rationalize the experimental findings, molecular dynamics simulations of Candida rugosa lipase were carried out. Analyzing the lipase geometry at 1, 10, 50, and 100 bar revealed a cavity in the Candida rugosa lipase. The cavity leads from a position on the surface distinct from the substrate binding site to the core towards the active site, and is limited by F415 and the catalytic H449. In the crystal structure of the Candida rugosa lipase, this cavity is filled with six water molecules. The number of water molecules in this cavity gradually increased with increasing pressure: six molecules in the simulation at 1 bar, 10 molecules at 10 bar, 12 molecules at 50 bar, and 13 molecules at 100 bar. Likewise, the volume of the cavity progressively increased from about 1864 A(3) in the simulation at 1 bar to 2529 A(3) at 10 bar, 2526 A(3) at 50 bar, and 2617 A(3) at 100 bar. At 100 bar, one water molecule slipped between F415 and H449, displacing the catalytic histidine side chain and thus opening the cavity to form a continuous water channel. The rotation of the side chain leads to a decreased distance between the H449-N epsilon and the (+)-menthyl-oxygen (nonpreferred enantiomer) in the acyl enzyme intermediate, a factor determining the enantioselectivity of the lipase. Although the geometry of the preferred enantiomer is similar in all simulations, the geometry of the nonpreferred enantiomer gets gradually more reactive. This observation correlates with the gradually decreasing enantioselectivity E.

Rational evolution of a medium chain-specific cytochrome P-450 BM-3 variant.

The single mutant F87A of cytochrome P-450 BM-3 from Bacillus megaterium was engineered by rational evolution to achieve improved hydroxylation activity for medium chain length substrates (C8-C10). Rational evolution combines rational design and directed evolution to overcome the drawbacks of these methods when applied individually. Based on the X-ray structure of the enzyme, eight mutation sites (P25, V26, R47, Y51, S72, A74, L188, and M354) were identified by modeling. Sublibraries created by site-specific randomization mutagenesis of each single site were screened using a spectroscopic assay based on omega-p-nitrophenoxycarboxylic acids (pNCA). The mutants showing activity for shorter chain length substrates were combined, and these combi-libraries were screened again for mutants with even better catalytic properties. Using this approach, a P-450 BM-3 variant with five mutations (V26T, R47F, A74G, L188K, and F87A) that efficiently hydrolyzes 8-pNCA was obtained. The catalytic efficiency of this mutant towards omega-p-nitrophenoxydecanoic acid (10-pNCA) and omega-p-nitrophenoxydodecanoic acid (12-pNCA) is comparable to that of the wild-type P-450 BM-3.

Identification of thermodynamically relevant interactions between EF-Tu and backbone elements of tRNA.

A set of 45 different tRNAs, each containing a single deoxynucleotide substitution covering the upper half of the molecule was used in conjunction with a high-throughput ribonuclease protection assay to investigate the thermodynamic role of 2' hydroxyl groups in stabilizing a complex with elongation factor Tu (EF-Tu) from Thermus thermophilus. Five distinct 2' hydroxyl groups were identified where substitution with a proton resulted in an approximately tenfold decrease in the binding affinity. The same five 2' hydroxyl groups reduced the affinity of the interaction with the nearly identical Thermus aquaticus EF-Tu. Four of these 2' hydroxyl groups were observed to form hydrogen bonds in a co-crystal structure of tRNA(Phe) and T. aquaticus EF-Tu, while the fifth 2' hydroxyl group can be associated with an intramolecular hydrogen bond in the tRNA. However, four additional hydrogen bonds to 2' hydroxyl groups observed in the crystal structure show no thermodynamic effect upon disruption. Some of these discrepancies may be reconciled based on the unbound structures of the protein and RNA.

The His gap motif in microbial lipases: a determinant of stereoselectivity toward triacylglycerols and analogs.

Lipases preferably hydrolyze the sn-1 and sn-3 acyl chain of triacylglycerols and sn-2 substituted analogs. Molecular modeling studies of the stereopreference of microbial lipases from Rhizopus oryzae, Rhizomucor miehei, Candida rugosa, and lipase B from Candida antarctica toward the hydrolysis of triacylglycerols and analogs revealed that sterical interactions occurring between the sn-2 substituent and the His gap affect substrate geometry, which can be monitored by a single torsion angle. This torsion angle correlates to the experimentally determined stereopreference and is, therefore, suitable to predict stereopreference by molecular modeling. For a given microbial lipase, stereopreference can be estimated by measuring the distance between the side chains of the His gap residues: a narrow His gap cleft implies sn-3 stereopreference for all investigated substrates; a medium-sized His gap discriminates by flexibility of the substrates: flexible substrates are hydrolyzed in sn-1, while rigid substrates are hydrolyzed in sn-3. A wide open His gap implies sn-1 stereopreference for all substrates. This rule holds for all investigated microbial wild type lipases and mutants.

Stereoselectivity of Pseudomonas cepacia lipase toward secondary alcohols: a quantitative model.

The lipase from Pseudomonas cepacia represents a widely applied catalyst for highly enantioselective resolution of chiral secondary alcohols. While its stereopreference is determined predominantly by the substrate structure, stereoselectivity depends on atomic details of interactions between substrate and lipase. Thirty secondary alcohols with published E values using P. cepacia lipase in hydrolysis or esterification reactions were selected, and models of their octanoic acid esters were docked to the open conformation of P. cepacia lipase. The two enantiomers of 27 substrates bound preferentially in either of two binding modes: the fast-reacting enantiomer in a productive mode and the slow-reacting enantiomer in a nonproductive mode. Nonproductive mode of fast-reacting enantiomers was prohibited by repulsive interactions. For the slow-reacting enantiomers in the productive binding mode, the substrate pushes the active site histidine away from its proper orientation, and the distance d(H(N epsilon) - O(alc)) between the histidine side chain and the alcohol oxygen increases, d(H(N epsilon) - O(alc)) was correlated to experimentally observed enantioselectivity: in substrates for which P. cepacia lipase has high enantioselectivity (E > 100), d(H(N epsilon) - O(alc)) is >2.2 A for slow-reacting enantiomers, thus preventing efficient catalysis of this enantiomer. In substrates of low enantioselectivity (E < 20), the distance d(H(N epsilon) - O(alc)) is less than 2.0 A, and slow- and fast-reacting enantiomers are catalyzed at similar rates. For substrates of medium enantioselectivity (20 < E < 100), d(H(N epsilon) - O(alc)) is around 2.1 A. This simple model can be applied to predict enantioselectivity of P. cepacia lipase toward a broad range of secondary alcohols.

Mapping contacts between Escherichia coli alanyl tRNA synthetase and 2' hydroxyls using a complete tRNA molecule.

A dual-specific derivative of yeast tRNA(Phe) is described whose features facilitate structure-function studies of tRNAs. This tRNA has been made in three different bimolecular forms that allow modifications to be easily introduced into any position within the molecule. A set of deoxynucleotide substituted versions of this tRNA has been created and used to examine contacts between tRNA and Escherichia coli alanyl-tRNA synthetase, an enzyme previously shown to interact with 2'-hydroxyls in the acceptor stem of the tRNA. Because the present experiments used a full-length tRNA, several contacts were identified that had not been previously found using microhelix substrates. Contacts at similar sites in the T-loop are seen in the cocrystal structure of tRNA(Ser) and Thermus thermophilus seryl-tRNA synthetase.

Intact aminoacyl-tRNA is required to trigger GTP hydrolysis by elongation factor Tu on the ribosome.

GTP hydrolysis by elongation factor Tu (EF-Tu) on the ribosome is induced by codon recognition. The mechanism by which a signal is transmitted from the site of codon-anticodon interaction in the decoding center of the 30S ribosomal subunit to the site of EF-Tu binding on the 50S subunit is not known. Here we examine the role of the tRNA in this process. We have used two RNA fragments, one which contains the anticodon and D hairpin domains (ACD oligomer) derived from tRNA(Phe) and the second which comprises the acceptor stem and T hairpin domains derived from tRNA(Ala) (AST oligomer) that aminoacylates with alanine and forms a ternary complex with EF-Tu. GTP. While the ACD oligomer and the ternary complex containing the Ala-AST oligomer interact with the 30S and 50S A site, respectively, no rapid GTP hydrolysis was observed when both were bound simultaneously. The presence of paromomycin, an aminoglycoside antibiotic that binds to the decoding site and stabilizes codon-anticodon interaction in unfavorable coding situations, did not increase the rate of GTP hydrolysis. These results suggest that codon recognition as such is not sufficient for GTPase activation and that an intact tRNA molecule is required for transmitting the signal created by codon recognition to EF-Tu.

Stereoselectivity of Mucorales lipases toward triradylglycerols--a simple solution to a complex problem.

The lipases from Rhizopus and Rhizomucor are members of the family of Mucorales lipases. Although they display high sequence homology, their stereoselectivity toward triradylglycerols (sn-2 substituted triacylglycerols) varies. Four different triradylglycerols were investigated, which were classified into two groups: flexible substrates with rotatable O'-C1' ether or ester bonds adjacent to C2 of glycerol and rigid substrates with a rigid N'-C1' amide bond or a phenyl ring in sn-2. Although Rhizopus lipase shows opposite stereopreference for flexible and rigid substrates (hydrolysis in sn-1 and sn-3, respectively), Rhizomucor lipase hydrolyzes both groups of triradylglycerols preferably in sn-1. To explain these experimental observations, computer-aided molecular modeling was applied to study the molecular basis of stereoselectivity. A generalized model for both lipases of the Mucorales family highlights the residues mediating stereoselectivity: (1) L258, the C-terminal neighbor of the catalytic histidine, and (2) G266, which is located in a loop contacting the glycerol backbone of a bound substrate. Interactions with triradylglycerol substrates are dominated by van der Waals contacts. Stereoselectivity can be predicted by analyzing the value of a single substrate torsion angle that discriminates between sn-1 and sn-3 stereopreference for all substrates and lipases investigated here. This simple model can be easily applied in enzyme and substrate engineering to predict Mucorales lipase variants and synthetic substrates with desired stereoselectivity.

T7 RNA polymerase produces 5' end heterogeneity during in vitro transcription from certain templates.

The use of T7 RNA polymerase to prepare large quantities of RNA of a particular sequence has greatly facilitated the study of both the structure and function of RNA. Generally, it has been believed that the products of this technique are highly homogeneous in sequence, with only a few noted exceptions. We have carefully examined the transcriptional products of several tRNAs that vary in their 5' end sequence and found that, for those molecules that begin with multiple, consecutive guanosines, the transcriptional products are far from homogenous. Although a template beginning with GCG showed no detectable 5' end heterogeneity, two tRNA templates designed to have either four or five consecutive guanosines at their 5' ends had more than 30% of their total transcriptional products extended by at least one untemplated nucleotide at their 5' end. By simply reducing the number of consecutive guanosines, the heterogeneity was reduced significantly. The presence of this 5' end heterogeneity in combination with the 3' end heterogeneity common to T7 transcriptions results in a mixture of RNA molecules even after rigorous size purification.

Rational design of Rhizopus oryzae lipase with modified stereoselectivity toward triradylglycerols.

The binding site of sn-1(3)-regioselective Rhizopus oryzae lipase (ROL) has been engineered to change the stereoselectivity of hydrolysis of triacylglycerol substrates and analogs. Two types of prochiral triradylglycerols were considered: 'flexible' substrates with ether, benzylether or ester groups, and 'rigid' substrates with amide or phenyl groups, respectively, in the sn-2 position. The molecular basis of sn-1(3) stereoselectivity of ROL was investigated by modeling the interactions between substrates and ROL, and the model was confirmed by experimental determination of the stereoselectivity of wild-type and mutated ROL. For the substrates, the following rules were derived: (i) stereopreference of ROL toward triradylglycerols depends on the substrate structure. Substrates with 'flexible' sn-2 substituents are preferably hydrolyzed at sn-1, 'rigid' substrates at sn-3. (ii) Stereopreference of ROL toward triradylglycerols can be predicted by analyzing the geometry of the substrate docked to ROL: if the torsion angle phiO3-C3 of glycerol is more than 150 degrees, the substrate will preferably be hydrolyzed in sn-1, otherwise in sn-3. For ROL, the following rules were derived: (i) residue 258 affects stereoselectivity by steric interactions with the sn-2 substituent rather than polar interactions. To a lower extent, stereoselectivity is influenced by mutations further apart (L254) from residue 258. (ii) With 'rigid' substrates, increasing the size of the binding site (mutations L258A and L258S) shifts stereoselectivity of hydrolysis toward sn-1, decreasing its size (L258F and L258F/L254F) toward sn-3.

A new assay for tRNA aminoacylation kinetics.

An improved quantitative assay for tRNA aminoacylation is presented based on charging of a nicked tRNA followed by separation of an aminoacylated 3'-fragment on an acidic denaturing polyacrylamide gel. Kinetic parameters of tRNA aminoacylation by Escherichia coli AlaRS obtained by the new method are in excellent agreement with those measured by the conventional method. This assay provides several advantages over the traditional methods of measuring tRNA aminoacylation: (1) the fraction of aminoacyl-tRNA is measured directly; (2) data can be obtained at saturating amino acid concentrations; and (3) the assay is significantly more sensitive.

Anatomy of lipase binding sites: the scissile fatty acid binding site.

Shape and physico-chemical properties of the scissile fatty acid binding sites of six lipases and two serine esterases were analyzed and compared in order to understand the molecular basis of substrate specificity. All eight serine esterases and lipases have similar architecture and catalytic mechanism of ester hydrolysis, but different substrate specificities for the acyl moiety. Lipases and esterases differ in the geometry of their binding sites, lipases have a large, hydrophobic scissile fatty acid binding site, esterases like acetylcholinesterase and bromoperoxidase have a small acyl binding pocket, which fits exactly to their favorite substrates. The lipases were subdivided into three sub-groups: (1) lipases with a hydrophobic, crevice-like binding site located near the protein surface (lipases from Rhizomucor and Rhizopus); (2) lipases with a funnel-like binding site (lipases from Candida antarctica, Pseudomonas and mammalian pancreas and cutinase); and (3) lipases with a tunnel-like binding site (lipase from Candida rugosa). The length of the scissile fatty acid binding site varies considerably among the lipases between 7.8 A in cutinase and 22 A in Candida rugosa and Rhizomucor miehei lipase. Location and properties of the scissile fatty acid binding sites of all lipases of known structure were characterized. Our model also identifies the residues which mediate chain length specificity and thus may guide protein engineering of lipases for changed chain length specificity. The model was supported by published experimental data on the chain length specificity profile of various lipases and on mutants of fungal lipases with changed fatty acid chain length specificity.

A toolbox of recombinant lipases for industrial applications.

We created a toolbox of recombinant, microbial lipases, which allows us in combination with a lipase database to choose among the overexpressed lipases the most appropriate for a specific application and to improve it further via mutagenesis. By systematic comparison of geometry and properties of the scissile fatty acid binding site of five representative lipases of each family of structurally homologous lipases, three subgroups can be defined. Hence, efficient expression systems for the functional production of large amounts of microbial lipases, representing different lipase subgroups, were developed. In particular, recombinant lipases from Bacillus thermocatenulatus and Pseudomonas cepacia were functionally overexpressed in E. coli. The lipase genes from Geotrichum candidum CMICC 335426 and Rhizopus oryzae were overexpressed in Pichia pastoris. Due to an unusual codon usage that prevents heterologous expression, the LIP1 gene (1647 nt) of Candida rugosa was completely synthesized and overexpressed in Pichia pastoris.

Conformational transition of an alpha-helix studied by molecular dynamics.

Molecular dynamics simulations were performed on a 20-residue polyalanine helix and a spontaneous transition from a kinked to a straight conformation was observed. The kinetics of the transition was analyzed within the framework of the Kramers model for chemical reactions and within a random walk model. The Kramers model which is based on diffusion along a one-dimensional reaction pathway and the crossing of an energy barrier was found to be inadequate. Instead, a random walk model based on diffusion in the high-dimensional phase space of the system was found to be compatible with the data. The high dimensionality of the phase space permits the system to circumvent high energy barriers and diffuse rapidly at about constant energy, but decelerates the reaction since in the labyrinth of pathways the transition state is reached rarely.

Collective vibrations of an alpha-helix. A molecular dynamics study.

The internal dynamics of a 20-residue polyalanine helix was investigated by molecular dynamics simulations. Special attention was paid to the collective vibrations of the helix backbone. The stretch and bend vibrations could be assigned unambiguously to oscillations with periods of 1.4 and 4.3 ps, respectively. The influence of the environment on the dynamics of the collective vibrations was studied by coupling the helix to a heat bath and by adding water molecules. In the presence of water, the stretch vibration becomes more strongly dampened, but still exists as a vibration , while the bend vibration becomes overdamped and degenerates into a relaxation process. The results are compared with available experimental data.

Procyclin gene expression and loss of the variant surface glycoprotein during differentiation of Trypanosoma brucei.

In the mammalian host, the unicellular flagellate Trypanosoma brucei is covered by a dense surface coat that consists of a single species of macromolecule, the membrane form of the variant surface glycoprotein (mfVSG). After uptake by the insect vector, the tsetse fly, bloodstream-form trypanosomes differentiate to procyclic forms in the fly midgut. Differentiation is characterized by the loss of the mfVSG coat and the acquisition of a new surface glycoprotein, procyclin. In this study, the change in surface glycoprotein composition during differentiation was investigated in vitro. After triggering differentiation, a rapid increase in procyclin-specific mRNA was observed. In contrast, there was a lag of several hours before procyclin could be detected. Procyclin was incorporated and uniformly distributed in the surface coat. The VSG coat was subsequently shed. For a single cell, it took 12-16 h to express a maximum level of procyclin at the surface while the loss of the VSG coat required approximately 4 h. The data are discussed in terms of the possible molecular arrangement of mfVSG and procyclin at the cell surface. Molecular modeling data suggest that a (Asp-Pro)2 (Glu-Pro)22-29 repeat in procyclin assumes a cylindrical shape 14-18 nm in length and 0.9 nm in diameter. This extended shape would enable procyclin to interdigitate between the mfVSG molecules during differentiation, exposing epitopes beyond the 12-15-nm-thick VSG coat.