Evidence that elongation of the catalytic loop of the Azotobacter vinelandii rhodanese changed selectivity from sulfur- to phosphate-containing substrates

Recent investigations have shown that the rhodanese domains,ubiquitous structural modules which might represent an exampleof conserved structures with possible functional diversity,are structurally related to the catalytic subunit of Cdc25 phosphataseenzymes. The major difference characterizing the active-siteof the Azotobacter vinelandii rhodanese RhdA, with respect tothe closely related Cdc25s (A, B, C), is that in Cdc25 phosphatasesthe active site loop [His–Cys–(X)₅–Arg] isone residue longer than in RhdA [His–Cys–(X)₄–Arg].According to the hypothesis that the length of the RhdA active-siteloop should play a key role in substrate recognition and catalyticactivity, RhdA scaffold was the starting point for producingmutants with single-residue insertion to generate the catalyticloop HCQTHAHR (in RhdA-Ala) and HCQTHSHR (in RhdA-Ser). Analysesof the catalytic performances of the engineered RhdAs revealedthat elongation of the catalytic loop definitely compromisedthe ability to catalyze sulfur transfer reactions, while itgenerated ‘phosphatase’ enzymes able to interactproductively with the artificial substrate 3-O-methylfluoresceinphosphate. Although this study is restricted to an example ofrhodanese modules (RhdA), it provided experimental evidenceof the hypothesis that a specific mutational event (a single-residueinsertion or deletion in the active-site loop) could changethe selectivity from sulfur- to phosphate-containing substrates(or vice versa). Received February 17, 2003; revised May 30, 2003; accepted June 6, 2003.     

Effect of mutations involving charged residues on the stability of staphylococcal nuclease: a continuum electrostatics study

A continuum electrostatics model is used to calculate the relativestabilities of 117 mutants of staphylococcal nuclease (SNase)involving the mutation of a charged residue to an unchargedresidue. The calculations are based on the crystallographicstructure of the wild-type protein and attempt to take implicitlyinto account the effect of the mutations in the denatured stateby assuming a linear relationship between the free energy changescaused by the mutation in the native and denatured states. Agood correlation (linear correlation coefficient of 0.8) isfound with published experimental relative stabilities of thesemutants. The results suggest that in the case of SNase (i) chargedresidues contribute to the stability of the native state mainlythrough electrostatic interactions, and (ii) native-like electrostaticinteractions may persist in the denatured state. The continuumelectrostatics method is only moderately sensitive to modelparameters and leads to quasi-predictive results for the relativemutant stabilities (error of 2–3 kJ mol^–1 or ofthe order of k_BT), except for mutants in which a charged residueis mutated to glycine. Received March 24, 2003; revised August 11, 2003; accepted September 12, 2003.     

In silico mutations and molecular dynamics studies on a winged bean chymotrypsin inhibitor protein

Winged bean chymotrypsin inhibitor (WCI) has an intruding residueAsn14 that plays a crucial role in stabilizing the reactivesite loop conformation. This residue is found to be conservedin the Kunitz (STI) family of serine protease inhibitors. Tounderstand the contribution of this scaffolding residue on thestability of the reactive site loop, it was mutated in silicoto Gly, Ala, Ser, Thr, Leu and Val and molecular dynamics (MD)simulations were carried out on the mutants. The results ofMD simulations reveal the conformational variability and rangeof motions possible for the reactive site loop of differentmutants. The N-terminus side of the scissile bond, which isclose to a ß-barrel, is conformationally less variable,while the C-terminus side, which is relatively far from anysuch secondary structural element, is more variable and needsstability through hydrogen-bonding interactions. The simulatedstructures of WCI and the mutants were docked in the peptide-bindinggroove of the cognate enzyme chymotrypsin and the ability toform standard hydrogen-bonding interactions at P3, P1 and P2'residues were compared. The results of the MD simulations coupledwith docking studies indicate that hydrophobic residues likeLeu and Val at the 14th position are disruptive for the integrityof the reactive site loop, whereas a residue like Thr, whichcan stabilize the C-terminus side of the scissile bond, canbe predicted at this position. However, the size and chargeof the Asn residue made it most suitable for the best maintenanceof the integrity of the reactive site loop, explaining its conservednature in the family. Received October 17, 2002; revised June 6, 2003; accepted June 19, 2003.     

The structural roles of conserved Pro196, Pro197 and His199 in the mechanism of thymidylate synthase

We generated replacement sets for three highly conserved residues,Pro196, Pro197 and His199, that flank the catalytic nucleophile,Cys198. Pro196 and Pro197 have restricted mobility that couldbe important for the structural transitions known to be essentialfor activity. To test this hypothesis we obtained and characterized13 amino acid substitutions for Pro196, 14 for Pro197 and 14for His199. All of the Pro196 and Pro197 variants, except P197R,and four of the His199 variants complemented TS-deficient Escherichiacoli cells, indicating they had at least 1% of wild-type activity.For all His199 mutations, k_cat/K_m for substrate and cofactordecreased more than 40-fold, suggesting that the conserved hydrogenbond network co-ordinated by His199 is important for catalysis.Pro196 can be substituted with small hydrophilic residues withlittle loss in k_cat, but 15- to 23-fold increases in K_m^dUMP.Small hydrophobic substitutions for Pro197 were most active,and the most conservative mutant, P197A, had only a 5-fold lowerk_cat/K_m^dUMP than wild-type TS. Several Pro196 and Pro197 variantswere temperature sensitive. The small effects of Pro196 or Pro197mutations on enzyme kinetics suggest that the conformationalrestrictions encoded by the Pro–Pro sequence are largelymaintained when either member of the pair is mutated. Received February 24, 2003; revised June 19, 2003; accepted June 20, 2003.     

Stabilization of a chitinase from Serratia marcescens by Gly->Ala and Xxx->Pro mutations

This paper describes attempts to increase the kinetic stabilityof chitinase B from Serratia marcescens (ChiB) by the introductionof semi-automatically designed rigidifying mutations of theGlyAla and XxxPro type. Of 15 single mutants, several displayedsignificant increases in thermal stability, whereas most mutantsshowed minor effects. All mutations with non-marginal effectson stability clustered in a limited, surface-exposed regionof the enzyme, indicating that this region is involved in apartial unfolding process that triggers irreversible thermalinactivation (aggregation). A double mutant containing two stabilizingmutations in this region (G188A, A234P) displayed a 10-foldincrease in half-life at 57°C and a 4.2°C increase inapparent T_m. These results show that entropic stabilizationworks well for ChiB and they pinpoint a region whose unfoldingmay be crucial for the kinetic stability of this enzyme. Received June 18, 2003; revised September 3, 2003; accepted September 12, 2003.     

Improving tolerance of Candida antarctica lipase B towards irreversible thermal inactivation through directed evolution

To expand the functionality of lipase B from Candida antarctica(CALB) we have used directed evolution to create CALB mutantswith improved resistance towards irreversible thermal inactivation.Two mutants, 23G5 and 195F1, were generated with over a 20-foldincrease in half-life at 70°C compared with the wild-typeCALB (WT-CALB). The increase in half-life was attributed toa lower propensity of the mutants to aggregate in the unfoldedstate and to an improved refolding. The first generation mutant,23G5, obtained by error-prone PCR, had two amino acid mutations,V210I and A281E. The second generation mutant, 195F1, derivedfrom 23G5 by error-prone PCR, had one additional mutation, V221D.Amino acid substitutions at positions 221 and 281 were determinedto be critical for lipase stability, while the residue at position210 had only a marginal effect. The catalytic efficiency ofthe mutants with p-nitrophenyl butyrate and 6,8-difluoro-4-methylumbelliferyloctanoate was also found to be superior to that of WT-CALB. Received May 8, 2003; revised June 9, 2003; accepted June 23, 2003.     

Vicinal disulfide turns

The formation of a disulfide bond between adjacent cysteineresidues is accompanied by the formation of a tight turn ofthe protein backbone. In nearly 90% of the structures analyzeda type VIII turn was found. The peptide bond between the twocysteines is in a distorted trans conformation, the omega torsionangle ranges from 159 to –133°, with an average valueof 171°. The constrained nature of the vicinal disulfideturn and the pronounced difference observed between the oxidizedand reduced states, suggests that vicinal disulfides may beemployed as a ‘redox-activated’ conformational switch. Received December 16, 2002; revised June 30, 2003; accepted July 30, 2003.     

Specificity grafting of human antibody frameworks selected from a phage display library: generation of a highly stable humanized anti-CD22 single-chain Fv fragment

A prerequisite for the enrichment of antibodies screened fromphage display libraries is their stable expression on a phageduring multiple selection rounds. Thus, if stringent panningprocedures are employed, selection is simultaneously drivenby antigen affinity, stability and solubility. To take advantageof robust pre-selected scaffolds of such molecules, we graftedsingle-chain Fv (scFv) antibodies, previously isolated froma human phage display library after multiple rounds of in vitropanning on tumor cells, with the specificity of the clinicallyestablished murine monoclonal anti-CD22 antibody RFB4. We showthat a panel of grafted scFvs retained the specificity of themurine monoclonal antibody, bound to the target antigen withhigh affinity (6.4–9.6 nM), and exhibited exceptionalbiophysical stability with retention of 89–93% of theinitial binding activity after 6 days of incubation in humanserum at 37°C. Selection of stable human scaffolds withhigh sequence identity to both the human germline and the rodentframeworks required only a small number of murine residues tobe retained within the human frameworks in order to maintainthe structural integrity of the antigen binding site. We expectthis approach may be applicable for the rapid generation ofhighly stable humanized antibodies with low immunogenic potential. Received June 10, 2003; accepted August 27, 2003.     

Adaptation of a thermophilic enzyme, 3-isopropylmalate dehydrogenase, to low temperatures

Random mutagenesis coupled with screening of the active enzymeat a low temperature was applied to isolate cold-adapted mutantsof a thermophilic enzyme. Four mutant enzymes with enhancedspecific activities (up to 4.1-fold at 40°C) at a moderatetemperature were isolated from randomly mutated Thermus thermophilus3-isopropylmalate dehydrogenase. Kinetic analysis revealed twotypes of cold-adapted mutants, i.e. k_cat-improved and K_m-improvedtypes. The k_cat-improved mutants showed less temperature-dependentcatalytic properties, resulting in improvement of k_cat (up to7.5-fold at 40°C) at lower temperatures with increased K_mvalues mainly for NAD. The K_m-improved enzyme showed higheraffinities toward the substrate and the coenzyme without significantchange in k_cat at the temperatures investigated (30–70°C).In k_cat-improved mutants, replacement of a residue was foundnear the binding pocket for the adenine portion of NAD. Twoof the mutants retained thermal stability indistinguishablefrom the wild-type enzyme. Extreme thermal stability of thethermophilic enzyme is not necessarily decreased to improvethe catalytic function at lower temperatures. The present strategyprovides a powerful tool for obtaining active mutant enzymesat lower temperatures. The results also indicate that it ispossible to obtain cold-adapted mutant enzymes with high thermalstability.     

An empirical approach for structure-based prediction of carbohydrate-binding sites on proteins

A computer program system was developed to predict carbohydrate-bindingsites on three-dimensional (3D) protein structures. The programssearch for binding sites by referring to the empirical rulesderived from the known 3D structures of carbohydrate–proteincomplexes. A total of 80 non-redundant carbohydrate–proteincomplex structures were selected from the Protein Data Bankfor the empirical rule construction. The performance of theprediction system was tested on 50 known complex structuresto determine whether the system could detect the known bindingsites. The known monosaccharide-binding sites were detectedamong the best three predictions in 59% of the cases, whichcovered 69% of the polysaccharide-binding sites in the targetproteins, when the performance was evaluated by the overlapbetween residue patches of predicted and known binding sites. Received April 24, 2003; revised June 2, 2003; accepted June 10, 2003.     

Immobilized oxidoreductase as an additive for refolding inclusion bodies: application to antibody fragments

Three foldases—the apical domain of GroEL (mini-chaperone)and two oxidoreductases (DsbA and DsbC) from Escherichia coli—werestudied in refolding a protein with immunoglobulin fold (immunoglobulin-foldedprotein) that had been produced as inclusion bodies in E.coli.The foldases promoted the refolding of single-chain antibodyfragments from denaturant-solubilized and reduced inclusionbodies in vitro, and also effectively functioned as alternativesfor labilizing agent and oxidizing reagent in the stepwise dialysissystem. Immobilization of the oxidoreductases enhanced refoldingand recovery of functional single-chain antibody in the dialysissystem, suggesting that immobilized oxidoreductases can be usedas an effective additive for refolding immunoglobulin-foldedproteins in vitro. Received April 7, 2003; revised June 5, 2003; accepted June 6, 2003.     

Change in the crystal packing of soybean {beta}-amylase mutants substituted at a few surface amino acid residues

In spite of the high similarity of amino acid sequence and three-dimensionalstructure between soybean ß-amylase (SBA) and sweetpotato ß-amylase (SPB), their quaternary structureis quite different, being a monomer in SBA and a tetramer inSPB. Because most of the differences in amino acid sequencesare found in the surface region, we tested the tetramerizationof SBA by examining mutations of residues located at the surface.We designed the SBA tetramer using the SPB tetramer structureas a model and calculating the change of accessible surfacearea (ASA) for each residue in order to select sites for themutation. Two different mutant genes encoding SB3 (D374Y/L481R/P487D)and SB4 (K462S added to SB3), were constructed for expressionin Escherichia coli and the recombinant proteins were purified.They existed as a monomer in solution, but gave completely differentcrystals from the native SBA. The asymmetric unit of the mutantscontains four molecules, while that of native SBA contains one.The interactions of the created interfaces revealed that therewere more intermolecular interactions in the SB3 than in theSB4 tetramer. The substituted charged residues on the surfaceare involved in interactions with adjacent molecules in a differentway, forming a new crystal packing pattern. Received June 9, 2003; revised September 11, 2003; accepted September 16, 2003.     

High thermal stability of 3-isopropylmalate dehydrogenase from Thermus thermophilus resulting from low {Delta}{Delta}Cp of unfolding

To characterize the thermal stability of 3-isopropylmalate dehydrogenase(IPMDH) from an extreme thermophile, Thermus thermophilus, urea-inducedunfolding of the enzyme and of its mesophilic counterpart fromEscherichia coli was investigated at various temperatures. Theunfolding curves were analyzed with a three-state model forE.coli IPMDH and with a two-state model for T.thermophilus IPMDH,to obtain the free energy change     

Synthesis of a novel histidine analogue and its efficient incorporation into a protein in vivo

Proteins containing unnatural amino acids have immense potentialin biotechnology and medicine. We prepared several histidineanalogues including a novel histidine analogue, ß-(1,2,3-triazol-4-yl)-DL-alanine.These histidine analogues were assayed for translational activityin histidine-auxotrophic Escherichia coli strain UTH780. Weobserved that several histidine analogues, including our novelhistidine analogue, were efficiently incorporated into the proteinin vivo; however, other analogues were rejected. These resultssuggest that the hydrogen atom at a specific position seriouslyaffects incorporation. Received April 10, 2003; revised June 20, 2003; accepted July 22, 2003.     

Improvement of binding of Puumala virus neutralization site resembling peptide with a second-generation phage library

We have previously selected a peptide insert FPCDRLSGYWERGIPSPCVRrecognizing the Puumala virus (PUUV) G2-glycoprotein-specificneutralizing monoclonal antibody (MAb) 1C9 with K_d of 2.85x10^–8from a random peptide library X₂CX₁₄CX₂ expressed on the pIIIprotein of the filamentous phage fd-tet. We have now createda second-generation phage-displayed peptide library in whicheach amino acid of the peptide was mutated randomly to anotherwith a certain probability. Peptides were selected for higheraffinity for MAb 1C9 and for a common binding motif for MAb4G2 having an overlapping epitope with MAb 1C9 in G2 glycoprotein.The resulting peptides were synthesized as spots on cellulosemembrane. Amino acid changes which improved the reactivity ofthe peptides to MAb 1C9 were combined in the peptide ATCDKLFGYYERGIPLPCALwith K_d of 1.49x10^–9 in biosensor measurements. Our resultsshow that the binding properties of peptides, the affinity andthe specificity can be improved and the binding specificitydetermining amino acids and structural factors can be analyzedby combining binding assays with synthetic peptides on membranewith the use of second-generation phage display libraries. Received October 18, 2002; revised April 16, 2003; accepted May 26, 2003.     

PROSPECT II: protein structure prediction program for genome-scale applications

A new method for fold recognition is developed and added tothe general protein structure prediction package PROSPECT (http://compbio.ornl.gov/PROSPECT/).The new method (PROSPECT II) has four key features. (i) We havedeveloped an efficient way to utilize the evolutionary informationfor evaluating the threading potentials including singletonand pairwise energies. (ii) We have developed a two-stage threadingstrategy: (a) threading using dynamic programming without consideringthe pairwise energy and (b) fold recognition considering allthe energy terms, including the pairwise energy calculated fromthe dynamic programming threading alignments. (iii) We havedeveloped a combined z-score scheme for fold recognition, whichtakes into consideration the z-scores of each energy term. (iv)Based on the z-scores, we have developed a confidence index,which measures the reliability of a prediction and a possiblestructure–function relationship based on a statisticalanalysis of a large data set consisting of threadings of 600query proteins against the entire FSSP templates. Tests on severalbenchmark sets indicate that the evolutionary information andother new features of PROSPECT II greatly improve the alignmentaccuracy. We also demonstrate that the performance of PROSPECTII on fold recognition is significantly better than any othermethod available at all levels of similarity. Improvement inthe sensitivity of the fold recognition, especially at the superfamilyand fold levels, makes PROSPECT II a reliable and fully automatedprotein structure and function prediction program for genome-scaleapplications. Received March 20, 2003; revised June 28, 2003; accepted July 8, 2003.     

A long insertion reverts the functional effect of a substitution in acetylcholinesterase

Proteins are thought to undertake single substitutions, deletionsand insertions to explore the fitness landscape. Nevertheless,the ways in which these different kind of mutations act togetherto alter a protein phenotype remain poorly described. We introducedincrementally the single substitution W290A and a 26 amino acidlong insertion at the 297 location in the Nippostrongylus brasiliensisacetylcholinesterase B sequence and analysed in vitro the inducedchanges in the hydrolysis rate of three hemi-substrates: pirimicarb,paraoxon methyl and omethoate. The substitution decreased thehydrolysis rate of the three hemi-substrates. The insertiondid not influence this kinetic alteration induced by the substitutionfor the former hemi-substrate, but reverted it for the two others.These results show that two different kinds of mutations caninteract together to influence the direction of a protein’sadaptative walk on the fitness landscape. Received January 28, 2003; revised April 25, 2003; accepted June 6, 2003.     

A systematic approach for yielding a potential pool of enzymes: practical case for chiral resolution of (R,S)-ketoprofen ethyl ester

A systematic approach for the selection of potential biocatalystsfrom a natural source was developed and then a practical applicationwas addressed. The approach that involves systematically combinedconventional screening methods and current tools comprises thefollowing consecutive steps: strain enrichment for activityscreening, identification of positive strains, choosing wholegenome-sequenced strains as candidates, gathering informationabout responsible enzymes, bioinformatic analyses and gene mining,probing genetic molecules and then functional expression. Thetarget compound (R,S)-ketoprofen ethyl ester was to be resolvedinto an enantiomer, and a potential esterase from Pseudomonasfluorescens KCTC 1767 was prepared by the proposed procedure.The enzyme had a high activity and also strict selectivity forthe enantiomer (S)-ketoprofen and was suitable therefore asa biocatalyst for practical use. The result achieved by thecombined approach could not easily be obtained using other approacheswith typical procedures. Hence the approach proposed here shouldbe of considerable use for the screening of potential enzymes,particularly for enzymes with desired activity to unnaturalsubstrates, from conditionally expressed and/or repressed proteinsthat are distributed widely in natural pools under normal conditions. Received July 4, 2002; revised January 20, 2003; accepted April 4, 2003.     

Differential effects of zinc on functionally distinct human growth hormone mutations

Human growth hormone (hGH) binds and activates lactogenic receptorsby a sequential receptor dimerization mechanism. The affinityfor the first lactogenic receptor is increased due to one zincmolecule linking hGH residues H18 and E174, located in helices1 and 4, respectively, with two adjacent residues in the lactogenicreceptor (D187 and H188). Two functionally unique groups ofmutant hGHs have been identified. Addition of 25 µM zincto lactogenic bioassays differentially affects mutant activitiesbased on which group they belong to. One mutation (G120R) islocated within the binding surface of hGH that interacts withthe second lactogenic receptor. In the presence of endogenouszinc, G120R reduces the maximal activity of hGH without alteringeither the agonist or antagonist phases of the bell-shaped dose–responsecurve. Addition of zinc to this assay further reduces the activityof this protein. In contrast, mutations within a hydrophobicmotif in hGH that functionally couples the two lactogenic receptorbinding sites decrease the sensitivity (right-shift) of theagonist phase of the dose–response curve without similarlyaffecting the antagonist phase. The addition of zinc to theselactogenic assays increases the sensitivity (left-shifts) ofthe dose–response curves, largely negating the effectof these mutations. The effects of zinc differentiate betweenmutations within these two distinct functional motifs by limitingthe pool of potential conformations that are available for bindingwithin either of the receptor binding sites of this ligand. Received April 10, 2003; revised June 7, 2003; accepted June 8, 2003.