Tailoring the specificity of a plant cystatin toward herbivorous insect digestive cysteine proteases by single mutations at positively selected amino acid sites

Plant cystatins, similar to other defense proteins, include hypervariable, positively selected amino acid sites presumably impacting their biological activity. Using 29 single mutants of the eighth domain of tomato (Solanum lycopersicum) multicystatin, SlCYS8, we assessed here the potential of site-directed mutagenesis at positively selected amino acid sites to generate cystatin variants with improved inhibitory potency and specificity toward herbivorous insect digestive cysteine (Cys) proteases. Compared to SlCYS8, several mutants (22 out of 29) exhibited either improved or lowered potency against different model Cys proteases, strongly suggesting the potential of positively selected amino acids as target sites to modulate the inhibitory specificity of the cystatin toward Cys proteases of agronomic significance. Accordingly, mutations at positively selected sites strongly influenced the inhibitory potency of SlCYS8 against digestive Cys proteases of the insect herbivore Colorado potato beetle (Leptinotarsa decemlineata). In particular, several variants exhibited improved potency against both cystatin-sensitive and cystatin-insensitive digestive Cys proteases of this insect. Of these, some variants also showed weaker activity against leaf Cys proteases of the host plant (potato [Solanum tuberosum]) and against a major digestive Cys protease of the two-spotted stinkbug Perillus bioculatus, an insect predator of Colorado potato beetle showing potential for biological control. Overall, these observations suggest the usefulness of site-directed mutagenesis at positively selected amino acid sites for the engineering of recombinant cystatins with both improved inhibitory potency toward the digestive proteases of target herbivores and weaker potency against nontarget Cys proteases in the host plant or the environment.     

Large improvement in the thermal stability of a tetrameric malate dehydrogenase by single point mutations at the dimer-dimer interface

The stability of tetrameric malate dehydrogenase from the green phototrophic bacterium Chloroflexus aurantiacus (CaMDH) is at least in part determined by electrostatic interactions at the dimer-dimer interface. Since previous studies had indicated that the thermal stability of CaMDH becomes lower with increasing pH, attempts were made to increase the stability by removal of (excess) negative charge at the dimer-dimer interface. Mutation of Glu165 to Gln or Lys yielded a dramatic increase in thermal stability at pH 7.5 (+23.6 -- + 23.9 degrees C increase in apparent t(m)) and a more moderate increase at pH 4.4 (+4.6 -- + 5.4 degrees C). The drastically increased stability at neutral pH was achieved without forfeiture of catalytic performance at low temperatures. The crystal structures of the two mutants showed only minor structural changes close to the mutated residues, and indicated that the observed stability effects are solely due to subtle changes in the complex network of electrostatic interactions in the dimer-dimer interface. Both mutations reduced the concentration dependency of thermal stability, suggesting that the oligomeric structure had been reinforced. Interestingly, the two mutations had similar effects on stability, despite the charge difference between the introduced side-chains. Together with the loss of concentration dependency, this may indicate that both E165Q and E165K stabilize CaMDH to such an extent that disruption of the inter-dimer electrostatic network around residue 165 no longer limits kinetic thermal stability.     

Directed mutagenesis confirms the functional importance of positively selected sites in polygalacturonase inhibitor protein

Polygalacturonase inhibitor proteins (PGIPs) protect plants against invasion by diverse microbial and invertebrate enemies that use polygalacturonase (PG) to breach the plant cell wall. Directed mutagenesis has identified specific natural mutations conferring novel defensive capability in green bean PGIP against a specific fungal PG. These same sites are identified as positively selected by phylogenetic codon-substitution models, demonstrating the utility of such models for connecting retrospective comparative analyses with contemporary, ecologically relevant variation.     

Destabilization of pea lectin by substitution of a single amino acid in a surface loop

Legume lectins are considered to be antinutritional factors (ANF) in the animal feeding industry. Inactivation of ANF is an important element in processing of food. In our study on the stability ofPisum sativum L. lectin (PSL), a conserved hydrophobic amino acid (Val¹⁰³) in a surface loop was replaced with alanine. The mutant lectin, PSL V103A, showed a decrease in unfolding temperature (T _m ) by some 10 °C in comparison with wild-type (wt) PSL, and the denaturation energy (H) is only about 55% of that of wt PSL. Replacement of an adjacent amino acid (Phe¹⁰⁴) with alanine did not result in a significant difference in stability in comparison with wt PSL. Both mutations did not change the sugarbinding properties of the lectin, as compared with wt PSL and with PSL from pea seeds, at ambient temperatures. The double mutant, PSL V103A/F104A, was produced inEscherichia coli, but could not be isolated in an active (i.e. sugar-binding) form. Interestingly, the mutation in PSL V103A reversibly affected sugar-binding at 37 °C, as judged from haemagglutination assays. These results open the possibility of production of lectins that are activein planta at ambient temperatures, but are inactive and possibly non-toxic at 37 °C in the intestines of mammals.     

Proceedings of the SMBE Tri-National Young Investigators' Workshop 2005. Control of the false discovery rate applied to the detection of positively selected amino acid sites

In this article, we consider the probabilistic identification of amino acid positions that evolve under positive selection as a multiple hypothesis testing problem. The null hypothesis "H0,s: site s evolves under a negative selection or under a neutral process of evolution" is tested at each codon site of the alignment of homologous coding sequences. Standard hypothesis testing is based on the control of the expected proportion of falsely rejected null hypotheses or type-I error rate. As the number of tests increases, however, the power of an individual test may become unacceptably low. Recent advances in statistics have shown that the false discovery rate--in this case, the expected proportion of sites that do not evolve under positive selection among those that are estimated to evolve under this selection regime--is a quantity that can be controlled. Keeping the proportion of false positives low among the significant results generally leads to an increase in power. In this article, we show that controlling the false detection rate is relevant when searching for positively selected sites. We also compare this new approach to traditional methods using extensive simulations.     

Alteration of a single amino acid changes the substrate specificity of dihydroflavonol 4-reductase

Many plant species exhibit a reduced range of flower colors due to the lack of an essential gene or to the substrate specificity of a biosynthetic enzyme. Petunia does not produce orange flowers because dihydroflavonol 4-reductase (DFR) from this species, an enzyme involved in anthocyanin biosynthesis, inefficiently reduces dihydrokaempferol, the precursor to orange pelargonidin-type anthocyanins. The substrate specificity of DFR, however, has not been investigated at the molecular level. By analyzing chimeric DFRs of Petunia and Gerbera, we identified a region that determines the substrate specificity of DFR. Furthermore, by changing a single amino acid in this presumed substrate-binding region, we developed a DFR enzyme that preferentially reduces dihydrokaempferol. Our results imply that the substrate specificity of DFR can be altered by minor changes in DFR.     

Positive selection for single amino acid change promotes substrate discrimination of a plant volatile-producing enzyme

We used a combined evolutionary and experimental approach tobetter understand enzyme functional divergence within the SABATHgene family of methyltransferases (MTs). These enzymes catalyzethe formation of a variety of secondary metabolites in plants,many of which are volatiles that contribute to floral scentand plant defense such as methyl salicylate and methyl jasmonate.A phylogenetic analysis of functionally characterized membersof this family showed that salicylic acid methyltransferase(SAMT) forms a monophyletic lineage of sequences found in severalflowering plants. Most members of this lineage preferentiallymethylate salicylic acid (SA) as compared with the structurallysimilar substrate benzoic acid (BA). To investigate if positiveselection promoted functional divergence of this lineage ofenzymes, we performed a branch-sites test. This test showedstatistically significant support (P < 0.05) for positiveselection in this lineage of MTs (d_N/d_S = 10.8). A high posteriorprobability (pp = 0.99) identified an active site methionineas the only site under positive selection in this lineage. Toinvestigate the potential catalytic effect of this positivelyselected codon, site-directed mutagenesis was used to replaceMet with the alternative amino acid (His) in a Datura wrightiifloral–expressed SAMT sequence. Heterologous expressionof wild-type and mutant D. wrightii SAMT in Escherichia colishowed that both enzymes could convert SA to methyl salicylateand BA to methyl benzoate. However, competitive feeding withequimolar amounts of SA and BA showed that the presence of Metin the active site of wild-type SAMT resulted in a >10-foldhigher amount of methyl salicylate produced relative to methylbenzoate. The Met156His-mutant exhibited little differentialpreference for the 2 substrates because nearly equal amountsof methyl salicylate and methyl benzoate were produced. Evolutionof the ability to discriminate between the 2 substrates by SAMTmay be advantageous for efficient production of methyl salicylate,which is important for pollinator attraction as well as pathogenand herbivore defense. Because BA is a likely precursor forthe biosynthesis of SA, SAMT might increase methyl salicylatelevels directly by preferential methylation and indirectly byleaving more BA to be converted into SA.     

Variability patterns and positively selected sites at the gametophytic self-incompatibility pollen SFB gene in a wild self-incompatible Prunus spinosa (Rosaceae) population

Current models for the generation of new gametophytic self-incompatibility specificities require that neutral variability segregates within specificity classes. Furthermore, one of the models predicts greater ratios of nonsynonymous to synonymous substitutions in pollen than in pistil specificity genes. All models assume that new specificities arise by mutation only. To test these models, 21 SFB (the pollen S-locus) alleles from a wild Prunus spinosa (Rosaceae) population were obtained. For seven of these, the corresponding S-haplotype was also characterized. The SFB data set was also used to identify positively selected sites. Those sites are likely to be the ones responsible for defining pollen specificities. Of the 23 sites identified as being positively selected, 21 are located in the variable (including a new region described here) and hypervariable regions. Little variability is found within specificity classes. There is no evidence for selective sweeps being more frequent in pollen than in pistil specificity genes. The S-RNase and the SFB genes have only partially correlated evolutionary histories. None of the models is compatible with the variability patterns found in the SFB and the S-haplotype data.     

The inhibition of a plant proteinase by lysine copolymers is modulated by the hydrophobicity of the interposed amino acid side chains

Summary One Lys/Phe copolymer and two series of copolymers of lysine with either alanine or tyrosine have been used as inhibitors of a plant proteinase that is known to be inhibited by polycationic inhibitors. The copolymers differ in the hydrophobicity of the non-lysine amino acid residue and the single members of each series differ from each other in their degree of polymerization and in their charge density, i.e., the frequency of occurrence of the lysine residue in the synthetic polyamino acid chain. All the tested copolymers show cooperative inhibition, with a Hill coefficient higher than 1. CD measurements indicate that the inhibition is realized through a conformational change of the enzyme molecule. Both the enzyme inhibition and the conformational change are supported by aspecific electrostatic binding between the positively charged groups of the lysine moiety and the negatively charged groups of the enzyme surface. In each series the inhibitory power increases with the charge density, while at the same charge density the inhibitory efficiency depends on the hydrophobicity of the side chain of the non-lysine amino acid in the order Phe>Tyr>Ala.     

Characterization of the acidic and basic limbs of a bell-shaped pH profile in the inhibitory activity of bromelain inhibitor VI

Bromelain inhibitor VI (BI-VI) is a cysteine proteinase inhibitor from pineapple stem and a unique two-chain inhibitor composed of two distinct domains. BI-VI's inhibitory activity toward the target enzyme bromelain is maximal at pH 4 and shows a bell-shaped pH profile with pKa values of about 2.5 and 5.3. This pH profile is quite different from that of bromelain, which is optimally active around pH 7. In the present article, to characterize the acidic limb, we first expressed the recombinant inhibitors designed to lose two putative hydrogen bonds of Ser7(NH)-Asp28(beta-CO2H) and Lys38(NH)-Asp51(beta-CO2H) and confirmed the existence of the hydrogen bonds by two-dimensional nuclear magnetic resonance (NMR). Moreover, it was revealed that these hydrogen bonds are not the essential electrostatic factor and some ionizable groups would be responsible for the acidic limb in the pH-inhibition profile. On the other hand, to characterize the basic limb, we examined the pH-dependent inhibition using the cysteine proteinase papain, some of whose properties differ from those of bromelain, and compared the data with the corresponding data for bromelain. The result suggests that the basic limb would be affected by some electrostatic factors, probably some carboxyl groups in the target proteinase.     

Identification of amino acid residues responsible for the alpha5 subunit binding selectivity of L-655,708, a benzodiazepine binding site ligand at the GABA(A) receptor

L-655,708 is a ligand for the benzodiazepine site of the gamma-aminobutyric acid type A (GABA(A)) receptor that exhibits a 100-fold higher affinity for alpha5-containing receptors compared with alpha1-containing receptors. Molecular biology approaches have been used to determine which residues in the alpha5 subunit are responsible for this selectivity. Two amino acids have been identified, alpha5Thr208 and alpha5Ile215, each of which individually confer approximately 10-fold binding selectivity for the ligand and which together account for the 100-fold higher affinity of this ligand at alpha5-containing receptors. L-655,708 is a partial inverse agonist at the GABA(A) receptor which exhibited no functional selectivity between alpha1- and alpha5-containing receptors and showed no change in efficacy at receptors containing alpha1 subunits where amino acids at both of the sites had been altered to their alpha5 counterparts (alpha1Ser205-Thr,Val212-Ile). In addition to determining the binding selectivity of L-655,708, these amino acid residues also influence the binding affinities of a number of other benzodiazepine (BZ) site ligands. They are thus important elements of the BZ site of the GABA(A) receptor, and further delineate a region just N-terminal to the first transmembrane domain of the receptor alpha subunit that contributes to this binding site.     

Breaking the stereo barrier of amino acid attachment to tRNA by a single nucleotide

Aminoacyl-tRNA synthetases are responsible for attaching amino acid residues to the tRNA 3'-end. The two classes of synthetases approach tRNA as mirror images, with opposite but symmetrical stereochemistries that allow the class I enzymes to attach amino acid residues to the 2'-hydroxyl group of the terminal ribose, whereas, the class II enzymes attach amino acid residues to the 3'-hydroxyl group. However, we show here that the attachment of cysteine to tRNA(Cys) by the class I cysteinyl-tRNA synthetase (CysRS) is flexible; the enzyme is capable of using either the 2' or 3'-hydroxyl group as the attachment site. The molecular basis for this flexibility was investigated. Introduction of the nucleotide U73 of tRNA(Cys) into tRNA(Val) was found to confer the flexibility. While valylation of the wild-type tRNA(Val) by the class I ValRS was strictly dependent on the terminal 2'-hydroxyl group, that of the U73 mutant of tRNA(Val) occurred at either the 2' or 3'-hydroxyl group. Thus, the single nucleotide U73 of tRNA has the ability to break the stereo barrier of amino acid attachment to tRNA, by mobilizing the 2' and 3'-hydroxyl groups of A76 in flexible geometry with respect to the tRNA acceptor stem.     

Engineering the substrate specificity of Escherichia coli asparaginase. II. Selective reduction of glutaminase activity by amino acid replacements at position 248

The use of Escherichia coli asparaginase II as a drug for the treatment of acute lymphoblastic leukemia is complicated by the significant glutaminase side activity of the enzyme. To develop enzyme forms with reduced glutaminase activity, a number of variants with amino acid replacements in the vicinity of the substrate binding site were constructed and assayed for their kinetic and stability properties. We found that replacements of Asp248 affected glutamine turnover much more strongly than asparagine hydrolysis. In the wild-type enzyme, N248 modulates substrate binding to a neighboring subunit by hydrogen bonding to side chains that directly interact with the substrate. In variant N248A, the loss of transition state stabilization caused by the mutation was 15 kJ mol(-1) for L-glutamine compared to 4 kJ mol(-1) for L-aspartic beta-hydroxamate and 7 kJ mol(-1) for L-asparagine. Smaller differences were seen with other N248 variants. Modeling studies suggested that the selective reduction of glutaminase activity is the result of small conformational changes that affect active-site residues and catalytically relevant water molecules.     

Proposed amino acid sequence and the 1.63 A X-ray crystal structure of a plant cysteine protease,ervatamin B: some insights into the structural basis of its stability and substrate specificity

The crystal structure of a cysteine protease ervatamin B, isolated from the medicinal plant Ervatamia coronaria, has been determined at 1.63 A. The unknown primary structure of the enzyme could also be traced from the high-quality electron density map. The final refined model, consisting of 215 amino acid residues, 208 water molecules, and a thiosulfate ligand molecule, has a crystallographic R-factor of 15.9% and a free R-factor of 18.2% for F > 2sigma(F). The protein belongs to the papain superfamily of cysteine proteases and has some unique properties compared to other members of the family. Though the overall fold of the structure, comprising two domains, is similar to the others, a few natural substitutions of conserved amino acid residues at the interdomain cleft of ervatamin B are expected to increase the stability of the protein. The substitution of a lysine residue by an arginine (residue 177) in this region of the protein may be important, because Lys --> Arg substitution is reported to increase the stability of proteins. Another substitution in this cleft region that helps to hold the domains together through hydrogen bonds is Ser36, replacing a conserved glycine residue in the others. There are also some substitutions in and around the active site cleft. Residues Tyr67, Pro68, Val157, and Ser205 in papain are replaced by Trp67, Met68, Gln156, and Leu208, respectively, in ervatamin B, which reduces the volume of the S2 subsite to almost one-fourth that of papain, and this in turn alters the substrate specificity of the enzyme.     

Increase in activation rate of Pro‐Tk‐subtilisin by a single nonpolar‐to‐polar amino acid substitution at the hydrophobic core of the propeptide domain

Tk‐subtilisin (Gly70‐Gly398) is a subtilisin homolog from Thermococcus kodakarensis. Active Tk‐subtilisin is produced from its inactive precursor, Pro‐Tk‐subtilisin (Gly1‐Gly398), by autoprocessing and degradation of the propeptide (Tk‐propeptide, Gly1‐Leu69). This activation process is extremely slow at moderate temperatures owing to high stability of Tk‐propeptide. Tk‐propeptide is stabilized by the hydrophobic core. To examine whether a single nonpolar‐to‐polar amino acid substitution at this core affects the activation rate of Pro‐Tk‐subtilisin, the Pro‐Tk‐subtilisin derivative with the Phe17→His mutation (Pro‐F17H), Tk‐propeptide derivative with the same mutation (F17H‐propeptide), and two active‐site mutants of Pro‐F17H (Pro‐F17H/S324A and Pro‐F17H/S324C) were constructed. The crystal structure of Pro‐F17H/S324A was nearly identical to that of Pro‐S324A, indicating that the mutation does not affect the structure of Pro‐Tk‐subtilisin. The refolding rate of Pro‐F17H/S324A and autoprocessing rate of Pro‐F17H/S324C were also nearly identical to those of their parent proteins (Pro‐S324A and Pro‐S324C). However, the activation rate of Pro‐F17H greatly increased when compared with that of Pro‐Tk‐subtilisin, such that Pro‐F17H is efficiently activated even at 40°C. The far‐UV circular dichroism spectrum of F17H‐propeptide did not exhibit a broad trough at 205–230 nm, which is observed in the spectrum of Tk‐propeptide. F17H‐propeptide is more susceptible to chymotryptic degradation than Tk‐propeptide. These results suggest that F17H‐propeptide is unfolded in an isolated form and is therefore rapidly degraded by Tk‐subtilisin. Thus, destabilization of the hydrophobic core of Tk‐propeptide by a nonpolar‐to‐polar amino acid substitution is an effective way to increase the activation rate of Pro‐Tk‐subtilisin.