The structure-function relationship of the lipases from Pseudomonas aeruginosa and Bacillus subtilis

Within the BRIDGE T-project on lipases we investigate the structure-functionrelationships of the lipases from Bacillus subtilis and Pseudomonasaeruginosa. Construction of an overproducing Bacillus. strainallowed the purification of > 100 mg lipase from 30 l culturesupernatant. After testing a large variety of crystallizationconditions, the Bacillus lipase gave crystals of reasonablequality in PEG-4000 (38-45%), Na₂SO₄ and octyl-ß-glucosideat 22°C, pH 9.0. A 2.5 Å; dataset has been obtainedwhich is complete from 15 to 2.5 A resolution. P.aeruginosawild-type strain PAC1R was fermented using conditions of maximumlipase production. More than 90% of the lipase was cell boundand could be solubilized by treatment of the cells with TritonX-100. This permitted the purification of 50 mg lipase. So far,no crystals of sufficient quality were obtained. Comparisonof the model we built for the Pseudomonas lipase, on the basisof sequences and structures of various hydrolases which werefound to possess a common folding pattern (/ß hydrolasefold), with the X-ray structure of the P.glumae lipase revealedthat it is possible to correctly build the structure of thecore of a protein even in the absence of obvious sequence homologywith a protein of known 3-D structure.     

Redesigning the active site of Geotrichum candidum lipase

Attempts to engineer enzymes with unique catalytic propertieshave largely focused on altering the existing specificitiesby reshaping the substrate binding pockets. Few experimentshave aimed at modifying the configuration of the residues essentialfor catalysis. The difference in the topological location ofthe triad acids of Geotrichum candidum lipase (GCL) and thecatalytic domain of human pancreatic lipase (HPL), despite greatsimilarities in their topologies and 3-D structures, suggestthat these are related enzymes whose catalytic triads have beenrearranged in the course of evolution (Schrag et aL, 1992).In this study we prepared a double mutant GCL in which the catalytictriad acid is shifted to the position equivalent to the locationof the triad acid of HPL. The double mutant maintains 10% ofthe wild type activity against triglycerides and the fluorogenicester 4-methylumbelliferyl-oleate. The only significant differencesbetween the 3-D structures of the double mutant and wild typeGCL are at the mutated sites. Even the water structure in theregion of the triad is unchanged. The hydrogen bonding patternof the catalytic triad of the double mutant is very similarto that of pancreatic lipase. The acid of the double mutantis stabilized by only two hydrogen bonds, whereas three hydrogenbonds are observed in the wild type enzyme. These results stronglysupport the hypothesis that the pancreatic Upases are evolutionaryswitchpoints between the two observed arrangements of the catalytictriads supported by the /ß hydrolase fold and suggestthat this fold provides a stable protein core for engineeringenzymes with unique catalytic properties.     

The 3.0 A crystal structure of xylose isomerase from Streptomyces olivochromogenes

The crystal structure of xylose isomerase [E.C. 5.3.1.5 [EC] ] fromStreptomyces olivochromogenes has been determined to 3.0 Åresolution. The crystals belong to space group P22₁2₁ with unitcell parameters a = 98.7, b = 93.9, c = 87.7. The asymmetricunit contains half of a tetrameric molecule of 222 symmetry.The two-fold axis relating the two molecules in the asymmetricunit is close to where a crystallographic two-fold would beif the space group were 1222. This causes the diffraction patternto have strong 1222 pseudo-symmetry, so all data were collectedin this pseudo-space group. Since the sequence of this enzymehas not been reported, a polyalanine backbone has been fittedto the electron density. Xylose isomerase has two domains: theN-terminal domain is an eight-stranded /ß barrel of299 residues. The C-terminal domain is a large loop of 50 residueswhich is involved in inter-molecular contacts. Comparison ofxylose isomerase with the archetypical /ß barrel protein,triose phosphate isomerase, reveals that the proteins overlapbest when the third (ß) strand of xylose isomeraseis superimposed on the first (ß) strand of triosephosphate isomerase. This same overlap has also been found betweenthe muconate lactonising enzyme and triose phosphate isomerase[Goldman et al. (1987) J. Mol. Biol., in press].     

Interspecific luciferase {beta} subunit hybrids between Vibrio harveyi, Vibrio fischeri and Photobacterium leiognathi

Bacterial luciferase (EC 1.14.143) is a heterodimer composedof and ß-chains encoded by luxA and luxB, respectively.Although some interspecific combinations of these subunits leadto active enzyme, others do not. The ß subunits ofVibrio fischeri and Photobacterium leiognathi form active enzymewith the subunits of V.fischeri, P.leiognathi and Vibrio harveyi,while the ß subunit from V.harveyi only complementsthe subunit of V.harveyi. Inactivity is caused by a lack ofdimerization of the ß subunit of V.harveyi with the subunits of V.fischeri and P.leiognathi. These observationsserved as the basis for a search to discover which segment ofthe ß polypeptide confers the ability to dimerizewith the subunits of V.fischeri and P.leiognathi. Intragenicß subunit hybrids were made between V.harveyi, V.fischeriand P.leiognathi. Unique restriction sites were introduced intothe respective luxB genes to divide them into four roughly equalsegments. In all, 78 hybrids were constructed by in vitro techniques.The N-terminal segment of the peptide contains the signals thatdifferentiate between the ß subunits of V.fischeriand P.leiognathi and the ß subunit of V.harveyi, andallow the former to dimerize with their a subunits. The secondsegment has no major effect on enzyme activity but does exhibitsome context effects. Important interactions were found betweenthe third and fourth segments of the polypeptide with respectto enzymatic activity.     

Role of electrostatic interaction in the stability of the hexamer of constitutive phycocyanin from Fremyella diplosiphon

The role of electrostatic interactions between the charges carriedby the titratable groups in the different aggregates of constitutivephycocyanin from Fremyella diplosiphon has been studied by usinga simple theoretical approach based on the modified Tanford- Kirkwood model. The electrostatic potential has also beencalculated by means of a numerical solution of the linearizedPoisson - Boltzmann equations using the finite-differences technique.The pH dependence of the electrostatic contribution to freeenergy suggests an electrostatic stabilization of the - andß-subunits as well as of the (ß)-monomerover a broad pH interval. The charge distributions in the individual-and ß-subunlts produce electrostatic complementarityand promote the assembly of the subunits to the (ß)-monomer,as well as of the monomers to the larger trimeric and hexamericaggregates. Trimer -trimer electrostatic interactions exhibitstrong pH dependence, predicting an association/dissociationequilibrium with a midpoint at pH 6. The electrostatic trimer-trimerinteractions correspond to the steric fit, suggesting that electrostaticinteractions may initially help to orient the trimers duringaggregation. The distribution of the electrostatic potentialof the monomers and of the higher aggregates suggests that itplays an important role also in phycocyanin-linker protein binding.     

Functional roles and subsite locations of Leu177, Trp178 and Asn182 of Aspergillus awamori glucoamylase determined by site-directed mutagenesis

Fungal glucoamylases contain four conserved regions. One regionfrom the Aspergillus niger enzyme contains three key carboxylicacid residues, the general acid catalytic group, Glu179, alongwith Asp176 and Glu180. Three site-directed mutations, Leu177– His, Trp178 – Arg and Asn182 – Ala, wereconstructed near these acidic groups to reveal the functionof other conserved residues in this region. Leu177 and Trp178are strictly conserved among fungal glucoamylases, while anamide, predominantly Asn, always occurs at position 182. Substitutionsof Leu177 or Trp178 cause significant decreases in k_cat withthe substrates tested. Similar increases in activation energiesobtained with Leu177 – His with both -(1,4)- and -(1,6)-linkedsubstrates indicate Leu177 is located in subsite 1. K_M valuesobtained with the Trp178 – Arg mutation increase for an-(1,6)-linked substrate, but not for -(1,4)-linked substrates.Calculated differences in activation energy between substratesindicate Trp178 interacts specifically with subsite 2. The Asn182 Ala mutation did not change k_cat or K_M values, indicating thatAsn182 is not crucial for activity. These results support amechanism for glucoamylase catalytic activity consisting ofa fast substrate binding step followed by a conformational changeat subsite 1 to stabilize the transition state complex.     

Modular mutagenesis of a TEM-barrel enzyme: the crystal structure of a chimeric E.coli TIM having the eighth {beta}{alpha}-unit replaced by the equivalent unit of chicken TIM

Abstract The crystal structure of a hybrid Escherichia coli triosephosphateisomerase (TIM) has been determined at 2.8 Å resolution.The hybrid TIM (ETIM8CHI) was constructed by replacing the eighthß-unit of E.coli TIM with the equivalent unit of chickenTIM. This replacement involves 10 sequence changes. One of thechanges concerns the mutation of a buried alanine (Ala232 instrand 8) into a phenylalanine. The ETIM8CHI structure showsthat the A232F sequence change can be incorporated by a side-chainrotation of Phe224 (in helix 7). No cavities or strained dihedralsare observed in ETIM8CHI in the region near position 232, whichis in agreement with the observation that ETIM8CHI and E.coliTIM have similar stabilities. The largest CA (C-alpha atom)movements, 3 Å, are seen for the C-terminal end of helix8 (associated with the outward rotation of Phe224) and for theresidues in the loop after helix 1 (associated with sequencechanges in helix 8). From the structure it is not clear whythe k_cat of ETIM8CHI is 10 times lower than in wild type E.coliTIM     

Pseudomonas glumae lipase: increased proteolytic stabifity by protein engineering

The feasibility of stabilizing proteins towards proteolyticdegradation was explored by engineering the primary proteolyticcleavage site(s). This novel approach does not require informationon the 3-D structure of the native enzyme. As a model system,the extracellular lipase of Pseudomonas glumae was chosen, whichis sensitive towards degradation by subtilisin-tvpe proteases.The primary proteolytic cleavage in the lipase appeared to belocated between amino acids serine 153 and histidine 154. SincesubtUisins are known to show a preference towards amino acidresidues surrounding the scissile bond, non-preferred aminoadds were introduced in this area. Two concepts were tested:the introduction of arginine or glutainate residues (chargeconcept) and the introduction of proline residues (proUne concept).Although the mutant Upases produced according to either of theseconcepts were still cleaved in the same area, they showed aconsiderably increased stability towards proteolytic degradation.     

The predicted secondary structure of the G-type glutamineamidotransferase is compatible with TEM-barrel topology

Glutamine amidotransferase (GAT) subunits or domains catalyzean important partial reaction in many complex biosynthetic reactions.The structure of one member of the F-type GATs is known, butthe structure of the unrelated G-type is still unknown. Becausemany protein sequences are available for anthranilate synthasecomponent II (product of the trpG gene), we have predicted itsaverage secondary structure by a joint prediction method [Niermannand Kirschner (1991a) Protein Engng, 4, 359–370]. Thepredicted eight ß-strands and seven -helices followan 8-fold cyclic repetition of a ß-strand-loop--helix-loopmodule with helix 7 missing. This pattern of secondary structuresuggests that the G-type GAT domain has an 8-fold ß-barreltopology, as found first in triose phosphate isomerase (TIM-barrel).This model is supported by the location of known catalyticallyessential residues in loops between (ß-strands and-helices. Evidence from published sequencing and mutationalstudies on selected members of the GAT superfamily (carbamoylphosphate, imidazoleglycerol phosphate, GMP and CTP synthases)support both the secondary structure prediction and the TIM-barreltopology.     

Structure-function relationships in the catalytic and starch binding domains of glucoamylase

Sixteen primary sequences from five sub-families of fungal,yeast and bacterial glucoamylases were related to structuralinformation from the model of the catalytic domain of Aspergillusawamori var. X100 glucoamylase obtained by protein crystallography.This domain is composed of thirteen -belices, with five conservedregions defining the active site. Interactions between methyl-maltoside and active site residues were modelled, and the importanceof these residues on the catalytic action of different glucoamylaseswas shown by their presence in each primary sequence. The overallstructure of the starch binding domain of some fungal glucoamylaseswas determined based on homology to the Cterminal domains ofBacillus cyclodextrin glucosyltransferases. Crystallographyindicated that this domain contains 6–8 ß-strandsand homology allowed the attribution of a disulfide bridge inthe glucoamylase starch binding domain. Glucoamylase residuesThr525, Asn530 and Trp560, homologous to Bacillus stearothermophiluscyclodextrin glucosyltransferase residues binding to maltosein the Cterminal domain, could be involved in raw-starch binding.The structure and length of the linker region between the catalyticand starch binding domains in fungal glucoamylases can varysubstantially, a further indication of the functional independenceof the two domains.     

An approach to protein homology modelling based on an ensemble of NMR structures: application to the Sox-5 HMG-box protein

A new approach has been developed to reduce multiple proteinstructures obtained from NMR structure analysis to a smallernumber of representative structures which still reflect thestructural diversity of the data sets. The method, based onthe clustering of similar structures, has been tested in thehomology model building of the structure of Sox-5, a sequence-specificDNA-binding protein belonging to the high mobility group (HMG)nuclear proteins family. Sox (SRY box) genes are the autosomalgenes related to the sex-determining SRY, Y chromosomal gene.The Sox-5 protein, encoded by one of the SRY-related genes,displays a 29% sequence identity with the HMG1 B-box domainwhose structure, determined previously by NMR, has been usedin our study to predict the structure of Sox-5. Two independentensembles of HMG1 structures, each represented by closely relatedcoordinate sets, were used. Nine representative structures forHMG1 were subsequently selected as starting points for the modellingof Sox-5. The model of the protein shows close similarity tothe HMG1 fold, with differences at the secondary structure levellocated mainly in a-helices 1 and 3. A left-handed, three residueper turn polyproline II helix, forming a conserved polyprolineII/-helix supersecondary motif, was identified in the N-terminalregion of Sox-5 and other HMG boxes.     

The {alpha}/{beta} hydrolase fold

We have identified a new protein fold—the /ßhydrolase fold—that is common to several hydrolytic enzymesof widely differing phylogenetic origin and catalytic function.The core of each enzyme is similar: an /ß sheet, notbarrel, of eight ß-sheets connected by -helices. Theseenzymes have diverged from a common ancestor so as to preservethe arrangement of the catalytic residues, not the binding site.They all have a catalytic triad, the elements of which are borneon loops which are the best-conserved structural features inthe fold. Only the histidine in the nucleophile-histidine-acidcatalytic triad is completely conserved, with the nucleophileand acid loops accommodating more than one type of amino acid.The unique topological and sequence arrangement of the triadresidues produces a catalytic triad which is, in a sense, amirror-image of the serine protease catalytic triad. There arenow four groups of enzymes which contain catalytic triads andwhich are related by convergent evolution towards a stable,useful active site: the eukaryotic serine proteases, the cysteineproteases, subtilisins and the /ß hydrolase fold enzymes.     

Chimeras of human extracellular and intracellular superoxide dismutases. Analysis of structure and function of the individual domains

Human extracellular superoxide dismutase (hEC-SOD) is a secretedtetrameric protein involved in protection against oxygen freeradicals. Since EC-SOD is too large a protein for structuraldetermination by multi-dimensional NMR and attempts to crystallizethe protein for X-ray structural determination have failed,the three-dimensional structure of hEC-SOD is unknown. By fusionprotein techniques we have previously shown that an amphipathic-helix in the N-terminal domain of hEC-SOD is essential forthe tetramer interaction. However, the central domain, whichis homologous to intracellular hCuZnSOD, has also been proposedto be involved in the tetramer formation. Despite great efforts,the production of recombinant hEC-SOD in prokaryotic systemsor simple eukaryotes (such as yeast) has failed. This lack ofsuccess has greatly complicated large-scale production and geneticengineering of the protein. In the study reported here, we constructedtwo chimeras comprising the N- or the N- and C-terminal domainsfrom hEC-SOD fused to hCuZnSOD, called FusNCZ and PseudoEC-SOD,respectively. We show that these proteins can be produced inlarge quantities in Escherichia coli, that they can be purifiedwith high yields and that the characteristics of PseudoEC-SODclosely resemble those of hEC-SOD. Further, we extended ourstudies of the nature of the subunit interaction by investigatingthe involvement of the central domain.     

Design of four-helix bundle protein as a candidate for HIV vaccine

To be efficient, a synthetic vaccine should contain differentT and B cell epitopes of human immunodeficiency virus (HIV)antigens, and the B epitope regions in the vaccine and in theHIV should be conformationally similar. We have suggested previouslythe construction of vaccines in the form of a protein with apredetermined tertiary structure, namely a four--helix bundle.Antigenic determinants of cellular and humoral immunity areblocks for the vaccine design. From experimentally studied HIV-1T and B cell epitopes, we constructed a sequence of a four-helixprotein TBI (T and B cell epitopes containing immunogen). Thegene of the protein was synthesized and the protein was producedin C600 Escherichia coli cells under recA promoter from Proteusmirabelis. CD spectroscopy of the protein demonstrated that30% of amino acid residues adopt an -helical conformation. Miceimmunized with TBI have shown both humoral and cellular immuneresponses to HIV-1. The obtained data show that the design ofTBI was successful. The synthesized gene structure makes possiblefurther reconstruction and improvement of the protein vaccinestructure.     

Affinity maturation of a Taq DNA polymerase specific affibody by helix shuffling

The possibility of increasing the affinity of a Taq DNA polymerasespecific binding protein (affibody) was investigated by an -helixshuffling strategy. The primary affibody was from a naive combinatoriallibrary of the three-helix bundle Z domain derived from staphylococcalprotein A. A hierarchical library was constructed through selectivere-randomization of six amino acid positions in one of the two-helices of the domain, making up the Taq DNA polymerase bindingsurface. After selections using monovalent phage display technology,second generation variants were identified having affinities(K_D) for Taq DNA polymerase in the range of 30–50 nM asdetermined by biosensor technology. Analysis of binding dataindicated that the increases in affinity were predominantlydue to decreased dissociation rate kinetics. Interestingly,the affinities observed for the second generation Taq DNA polymerasespecific affibodies are of similar strength as the affinitybetween the original protein A domain and the Fc domain of humanimmunoglobulin G. Further, the possibilities of increasing theapparent affinity through multimerization of affibodies wasdemonstrated for a dimeric version of one of the second generationaffibodies, constructed by head-to-tail gene fusion. As comparedwith its monomeric counterpart, the binding to sensor chip immobilizedTaq DNA polymerase was characterized by a threefold higher apparentaffinity, due to slower off-rate kinetics. The results showthat the binding specificity of the protein A domain can bere-directed to an entirely different target, without loss ofbinding strength.     

High-level expression, purification, kinetic characterization and crystallization of protein farnesyltransferase -subunit C-terminal mutants

Protein farnesyltransferase (FPT) is a 97 000 Da heterodimericenzyme that catalyzes post-translational farnesylation of manycellular regulatory proteins including p21 Ras. To facilitatethe construction of site-directed mutants, a novel translationallycoupled, two-cistron Escherichia coli expression system forrat FPT has been developed. This expression system enabled yieldsof >5 mg of purified protein per liter of E.coli cultureto be obtained. The E.coli-derived FPT demonstrated an activitycomparable to that of protein isolated from other sources. Thereported expression system was used to construct three ß-subunitC-terminal truncation mutants, 5, 10 and 14, which were designedto eliminate a lattice interaction between the ß-subunitC-terminus of one molecule and the active site of a symmetry-relatedmolecule. Steady-state kinetic analyses of these mutants showedthat deletion up to 14 residues at the C-terminus did not reducethe value of k_cat; however, K_m values for both peptide and FPPincreased 2–3-fold. A new crystalline form of FPT was obtainedfor the 10 C-terminal mutant grown in the presence of the substrateanalogs acetyl-Cys-Val-Ile-Met-COOH peptide and -hydroxyfarnesylphosphonicacid. The crystals diffract to beyond 2.0 Å resolution.The refined structure clearly shows that both substrate analogsadopt extended conformations within the FPT active site cavity.     

Over-production of 5-enolpyruvylshikimate-3-phosphate synthase in Escherichia coli: use of the T7 promoter

5-Enolpyruvylshikimate-3-phosphate (EPSP) synthase, the productof the Escherichia coli aroA gene, has been overproduced inE.coli BL21(DE3) under the control of the T7 gene 10 promoterand ribosome binding site, to a level of {small tilde}50% oftotal cell protein. EPSP synthase is the primary target of thepost-emergence herbicide, glyphosate, commonly known as Roundup^TM.A simple two step purification is described, which results in99% pure homogeneous protein (as determined by PAGE). The integrityof the protein has been compared with previously characterizedprotein from .E.coli AB2829(pKD501) by determination of itskinetic parameters, N-terminal protein and DNA sequences, aminoacid analysis and ¹³C-NMR spectroscopy. This new overproducingstrain readily provides the gram quantities of highly pure proteinrequired for NMR studies of the active site and the developmentof novel time-resolved solid-state NMR techniques currentlyunderway in this laboratory.     

Characterization of the DNA binding domain of the mouse IRF-2 protein

The DNA binding domain of the interferon regulatory factor-2protein (IRF-2) has been produced and characterized, -chymotrypsindigestion of the purified IRF-2 protein bound to a syntheticbinding site yields a peptide fragment of 14 K in molecularweight. N-terminal analysis of this peptide fragment showedthat its sequence is the same as that of the intact IRF-2. Apeptide fragment of {small tilde} 14 K, IRF-2(113), which correspondsto the N-terminal 113 amino acids of the intact IRF-2 protein,has been expressed in a functional form in Escherichia coli.The first methionine was processed during the expression andthe purified IRF-2(113) thus contains 112 amino acids. DNaseI footprinting and gel retardation assaying showed that IRF-2(113)binds to a synthetic DNA having the consensus binding site andto the upstream regulatory sequence of the IFN-ß geneas intact IRF-2 does. These results showed that this peptidefragment, IRF-2(113), may be a good material for investigationof the DNA binding domain of IRF-2 and of the DNA–proteininteraction.     

Recognition of transmembrane {alpha}-helical segments with environmental profiles

A method for assessing the environmental properties of membrane-spanning-helical peptides in proteins has beenproposed. The algorithmemploys a set of environmental preference parameters derivedfor amino acid residues based on the analysis of the 3-D structuresof membrane domains in bacteriorhodopsin and photoreaction centersRhodopseudomonasviridis and Rhodobacter sphaeroides. The resulting3-D–1-D scores for transmembrane segments are significantlydifferent from those derived for -helices in globular proteins.The parameters obtained havebeen used to construct environmentalprofiles for membrane -helices in bacteriorhodopsin and photoreactioncenters. The profiles successfully recognize their own sequencesin several specially designed large databases. The method hasbeenapplied to several membrane proteins with unknown spatial structures.Most of their membrane-spanning peptides were efficiently recognizedby the profiles. The predicted environment of the residues inthe membrane segments fits the experimental data well. The approachis independent of any homology data and can be employed to delineatethe membrane segments of a protein with environmental characteristicsclose to those of bacteriorhodopsin and photoreaction centers.The alignment of these segments with the reference profilesprovides a considerable amount of data about their lipid andprotein exposure.     

Spectroscopic investigation of structure in octarellin (a de novo protein designed to adopt the {alpha}/{beta}-barred packing)

We present here a spectroscopic structural characterizationof octarellin, a recently reported de novo protein modelledon /ß-barrel proteins [K. Go raj, A.Renard and J.A.Martial(1990) Protein Engng, 3, 259–266]. Infrared and Ramanspectra analyses of octarellin‘s secondary structure revealthe expected percentage of -helices (30%) and a higher ß-sheetcontent (40%) than predicted from the design. When the Ramanspectra obtained with octarellin and native triosephosphateisomerase (a natural /ß-barrel) are compared, similarpercentages of secondary structures are found. Thermal denaturationof octarellin monitored by CD confirms that its secondary structuresare quite stable, whereas its native-like tertiary fold is not.Tyrosine residues, predicted to be partially hidden from solvent,are actually exposed as revealed by Raman and UV absorptionspectra. We conclude that the attempted /ß-barrelconformation in octarellin may be loosely packed. The criteriaused to design octarellin are discussed and improvements suggested.