Redesign of the coenzyme specificity in L-Lactate dehydrogenase from Bacillus stearothermophilus using site-directed mutagenesis and media engineering

L-Lactate dehydrogenase (LDH) from Bacillus stearothermophilusis a redox enzyme which has a strong preference for NADH overNADPH as coenzyme. To exclude NADPH from the coenzyme-bindingpocket, LDH contains a conserved aspartate residue at position52. However, this residue is probably not solely responsiblefor the NADH specificity. In this report we examine the possibilitiesof altering the coenzyme specificity of LDH by introducing arange of different point mutations in the coenzyme-binding domain.Furthermore, after choosing the mutant with the highest selectivityfor NADPH, we also investigated the possibility of further alteringthe coenzyme specificity by adding an organic solvent to thereaction mixture. The LDH mutant, I51K:D52S, exhibited a 56-foldincreased specificity to NADPH over the wild-type LDH in a reactionmixture containing 15% methanol. Furthermore, the NADPH turnovernumber of this mutant was increased almost fourfold as comparedwith wild-type LDH. To explain the altered coenzyme specificityexhibited by the D52SI51K double mutant, molecular dynamicssimulations were performed.     

The structural consequences of exchanging tryptophan and tyrosine residues in B.stearothermophilus lactate dehydrogenase

A mutant Bacillus stearothermophilus lactate dehydrogenase hasbeen prepared in which all three tryptophan residues in thewild-type enzyme have been replaced by tyrosines. In addition,a tyrosine residue has been mutated to a tryptophan, which actsas a fluorescence probe to monitor protein folding. The mutantenzyme crystallizes in the same crystal form as the wild-type.The crystal structure of the mutant has been determined at 2.8Å resolution. Solution studies have suggested that thereis little effect upon the mutant enzyme as judged by its kineticproperties. Comparison of the crystal structures of the mutantand wild-type enzymes confirms this conclusion, and revealsthat alterations in structure in the region of these mutationsare of a similar magnitude to those observed throughout thestructure, and are not significant when compared with the errorsin atomic positions expected for a structure at this resolution.     

Zinc ions bound to chimeric His4/lactate dehydrogenase facilitate decarboxylation of oxaloacetate

A chemically synthesized DNA linker coding for a peptide fragmentthat contains four histidines was fused in-frame to the 5'-endof the Bacillus stearothermophilus lactate dehydrogenase gene.The gene product, His₄/lactate dehydrogenase, could be purifiedto homogeneity using either immobilized metal (Zn²⁺)-affinitychromatography or affinity chromatography on oxamate agarose.The stability against heat and urea for the modified enzymeswas decreased as compared to the native lactate dehydrogenasebut could be increased if zinc ions were present during thedenaturation. In the presence of zinc ions the His₄/lactatedehydrogenase could catalyse the sequential reaction from oxaloacetateto L-lactate, hence operating as a semi-synthetic bifunctionalenzyme. A small increase in the apparent secondorder rate constant(k_cat/K_m) of the coupled reaction was observed as compared toa corresponding system with native lactate dehydrogenase.     

Site-directed mutagenesis to fine-tune enzyme specificity

We have used a combination of a genetic selection and oligonucleotide-directedmutagenesis to introduce a series of amino add replacementsfor a single residue into Escherichia coliglutaminyl-tRNA synthetase.The mutant enzymes mischarge supFtRNA^Tyr, with glutamine, tovarying degrees depending on the polarity of the side chainintroduced but apparently not depending on the size or shapeof the side chain. These results indicate that repulsive charge-chargeinteractions may be important for specific recognition of nucleicacids by proteins and illustrate how a mutant, derived fromgenetic selection, may be further modified in activity by oligonucleotide-directedmutagenesis.     

Alteration of the amino acid substrate specificity of clostridial glutamate dehydrogenase by site-directed mutagenesis of an active-site lysine residue

Two residues, K89 and S380, thought to interact with the -carboxylgroup of the substrate L-glutamate, have been altered by site-directedmutagenesis of clostridial glutamate dehydrogenase (GDH). Thesingle mutants K89L and S380V and the combined double mutantK89L/S380V were constructed. All three mutants were satisfactorilyoverproduced in soluble form. However, only the K89L mutantwas retained by the dye column normally used in purifying thewild-type enzyme. All three mutant enzymes were purified tohomogeneity and tested for substrate specificity with 24 aminoacids. The single mutant S380V showed no detectable activity.The alternative single mutant K89L showed an activity towardsL-glutamate that was decreased nearly 2000-fold compared withwild-type enzyme, whereas the activities towards the monocarboxylicsubstrates -aminobutyrate and norvaline were increased 2- to3-fold. A similar level of activity was obtained with methionine(0.005 U/mg) and norleucine (0.012 U/mg), neither of which giveany activity with the wild-type enzyme under the same conditions.The double mutant showed decreased activity with all substratescompared with the wild-type GDH. In view of its novel activities,the K89L mutant was investigated in greater detail. A strictlylinear relationship between reaction velocity and substrateconcentration was observed up to 80 mM L-methionine and 200mM L-norleucine, implying very high K_m values. Values of k_cat/K_m,for L-methionine and L-norleucine were 6.7x10^–2 and 0.15s^–1M^–1, respectively. Measurements with dithiobisnitrobenzoicacid showed that the mutant enzymes all reacted with a stoichiometryof one -SH group per subunit and all showed protection by coenzyme,indicating essentially unimpaired coenzyme binding. With glutamateor 2-oxoglutarate as substrate the K_m values for the vestigialactivity in the mutant enzyme preparations were strikingly closeto the wild-type K_m values. Both for wild-type GDH and K89L,L-glutamate gave competitive product inhibition of 2-oxoglutaratereduction but did not inhibit the reduction of 2-oxocaproatecatalysed by K89L enzyme. This suggests that the low levelsof glutamate/2-oxoglutarate activity shown by the mutant enzymeare due to trace contamination. Since stringent precautionswere taken, it appears possible that this reflects the levelof reading error during overexpression of the mutant proteins.CD measurements indicate that the S380V mutant has an alteredconformation, whereas the K89L enzyme gave an identical CD spectrumto that of wild-type GDH; the spectrum of the double mutantwas similar, although somewhat altered in intensity. The resultsconfirm the key role of K89 in dicarboxylate recognition byGDH.     

A novel random mutagenesis approach using human mutagenic DNA polymerases to generate enzyme variant libraries

The in vitro MutaGen procedure is a new random mutagenesis method based on the use of low-fidelity DNA polymerases. In the present study, this technique was applied on a 2 kb gene encoding amylosucrase, an attractive enzyme for the industrial synthesis of amylose-like polymers. Mutations were first introduced during a single replicating step performed by mutagenic polymerases pol beta and pol eta. Three large libraries (>10(5) independent clones) were generated (one with pol beta and two with pol eta). The sequence analysis of randomly chosen clones confirmed the potential of this strategy for the generation of diversity. Variants generated by pol beta were 4-7-fold less mutated than those created with pol eta, indicating that our approach enables mutation rate control following the DNA polymerase employed for mutagenesis. Moreover, pol beta and pol eta provide different and complementary mutation spectra, allowing a wider sequence space exploration than error-prone PCR protocols employing Taq polymerase. Interestingly, some of the variants generated by pol eta displayed unusual modifications, including combinations of base substitutions and codon deletions which are rarely generated using other methods. By taking advantage of the mutation bias of naturally highly error-prone DNA polymerases, MutaGen thus appears as a very useful tool for gene and protein randomisation.     

Engineering a chimeric pyrroloquinoline quinone glucose dehydrogenase: improvement of EDTA tolerance, thermal stability and substrate specificity

An engineered Escherichia coli PQQ glucose dehydrogenase (PQQGDH)with improved enzymatic characteristics was constructed by substitutingand combining the gene-encoding protein regions responsiblefor EDTA tolerance, thermal stability and substrate specificity.The protein region responsible for complete EDTA tolerance inAcinetobacter calcoaceticus, which is recognized as the indicatorof high stability in co-factor binding, was elucidated. Theregion is located between 32 and 59% from the N-terminus ofA.calcoaceticus PQQGDH(A27 region) and also corressponds tothe same position from 32 to 59% from the N-terminus in E.coliPQQGDH, though E.coli PQQGDH is EDTA sensitive. We previouslyreported that the C-terminal 3% region of A.calcoaceticus (A3region) played an important role in the increase of thermalstability, and that His775Asn substitution in E.coli PQQGDHresulted in an increase in the substrate specificity of E.coliPQQGDH towards glucose. Based on these findings, chimeric and/ormutated PQQGDHs, E97A3 H775N, E32A27E41 H782N, E32A27E38A3 andE32A27E38A3 H782N were constructed to investigate the compatibilityof two protein regions and one amino acid substitution. His775substitution to Asn corresponded to His782 substitution to Asn(H782N) in chimeric enzymes harbouring the A27 region. Sinceall the chimeric PQQGDHs harbouring the A27 region were EDTAtolerant, the A27 region was found to be compatible with theother region and substituted amino acid responsible for theimprovement of enzymatic properties. The contribution of theA3 region to thermal stability complemented the decrease inthe thermal stability due to the His775 or His782 substitutionto Asn. E32A27E38A3 H782N, which harbours all the above mentionedthree regions, showed improved EDTA tolerance, thermal stabilityand substrate specificity. These results suggested a strategyfor the construction of a semi-artificial enzyme by substitutingand combining the gene-encoding protein regions responsiblefor the improvement of enzyme characteristics. The characteristicsof constructed chimeric PQQGDH are discussed based on the predictedmodel, ß-propeller structure.     

A general method for relieving substrate inhibition in lactate dehydrogenases

The mutation S163L in human heart lactate dehydrogenase removessubstrate inhibition while only modestly reducing the turnoverrate for pyruvate. Since this is the third enzyme to show thisbehaviour, we suggest that the S163L mutation is a general methodfor the removal of substrate inhibition in L-LDH enzymes. Engineeringsuch enzymatic properties has clear industrial applicationsin the use of these enzymes to produce enantiomerically pure-hydroxy acids. The mutation leads to two principal effects.(1) Substrate inhibition is caused by the formation of a covalentadduct between pyruvate and the oxidized form of the cofactor.The inability of S163L mutants to catalyse the formation ofthis inhibitory adduct is demonstrated. However, NMR experimentsshow that the orientation of the nicotinamide ring in the mutantNAD⁺ binary complex is not perturbed. (2) The mutation alsoleads to a large increase in the K_M for pyruvate. The kineticand binding properties of S163L LDH mutants are accounted forby a mechanism which invokes a non-productive, bound form ofthe cofactor. Molecular modelling suggests a structure for thisnon-productive enzyme–NADH complex.     

Directed evolution by accumulating tailored mutations: thermostabilization of lactate oxidase with less trade-off with catalytic activity

We assumed that adverse effects posed by introducing multiple mutations could be decomposed into those of each of the component mutations and that the risk could be reduced by the accumulation of mutations that were finely tuned for directed improvement of a specific property. We propose here a directed evolution strategy for improving a specific property with less effect on other ones. This strategy is composed of fine-tuning of mutations and their accumulation by our original mutation-assembling method. In this study, we selected lactate oxidase (LOX) as a model enzyme, because its directed evolution had showed a trade-off between thermostability and catalytic activity. Mutation profiling at each of the sites found by error-prone PCR revealed a strong inverse relationship between the two properties. Thermostable mutations with less effect on catalytic activity were selected at each site and accumulated with ideal combinations by our method. The resultant multiple mutants exhibited 5- to 10-fold superior catalytic activity and comparable thermostability with those created by accumulating thermostable mutations, which were not tuned for catalytic activity. This result demonstrates that the accumulation of fine-tuned mutations is an advantageous approach to reduce the risk of adverse effects posed by accumulating multiple mutations.     

Use of site-directed mutagenesis to probe the role of Cys149 in the formation of charge-transfer transition in glyceraldehyde-3-phosphate dehydrogenase

Oligonucleotide-directed mutagenesis was employed to producemutants of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH)of Escherichia coli and Bacillus stearothermophilus. Three differentmutant proteins—His¹⁷⁶ — Asn, Cys¹⁴⁹ — Ser,Cys¹⁴⁹ — Gly—were isolated from one or both of theenzymes. The study of the properties of these mutants has shownthat Cys¹⁴⁹ is clearly responsible for the information of acharge-transfer transition, named the Racker band, observedduring the NAD⁺ binding to apoGAPDH. This result excludes asimilarity between the Racker band and the charge-transfer transitionobserved following the alkylation of GAPDH by 3-chloroacetylpyridine-adenine dinucleotide.     

Multiple interactions of lysine-128 of Escherichia coli glutamate dehydrogenase revealed by site-directed mutagenesis studies

A highly conserved lysine at position 128 of Escherichia coliglutamate dehydrogenase (GDH) has been altered by sitedirectedmutagenesis of the gdhA gene. Chemical modification studieshave previously shown the importance of this residue for catalyticactivity. We report the properties of mutants in which lysine-128has been changed to histidine (K128H) or arginine (K128R). Bothmutants have substantially reduced catalytic centre activitiesand raised pH optima for activity. K128H also has increasedrelative activity with amino acid substrates other than glutamate,especially L-norvaline. These differences, together with alterationsin K_m values, K_d values for NADPH and K₁ values for D-glutamate,imply that lysine-128 is intimately involved in either director indirect interactions with all the substrates and also incatalysis. These multiple interactions of lysine-128 explainthe diverse effects of chemical modifications of the correspondinglysine in homologous GDHs. In contrast, lysine-27, another highlyreactive residue in bovine GDH, is not conserved in all of thesequenced NADP-specific GDHs and is therefore not likely tobe involved in catalysis.     

Site-directed mutagenesis of glyceraldehyde-3-phosphate dehydrogenase reveals the role of residue Ser148

A mutant of Bacillus stearothermophilus D-glyceraldehyde-3-phosphatedehydrogenase, Ser¹⁴⁸ – Ala, was produced byoligonucleotide-directedmutagenesis. The study of the catalytic properties of this mutanthas shown that this mutation significantly affects the Michaelisconstant of inorganic phosphate and to a lesser extent thatof 1,3-diphosphoglycerate and D-glyceraldehyde-3-phosphate.This result is consistent with model-building studies whichshow that, for the phosphorylation step of catalysis, inorganicphosphate must bind to the anion recognition site designatedP_i with the C(3) phosphate of the acyl-enzyme intermediate inthe alternative anion site P_s. Studies of the enantiomeric specificityusing D- and L-glyceraldehyde as substrates show that the hydroxylgroup of Ser¹⁴⁸, combined with the presence of the C(3) phosphateof the substrate, enhances stereospecificity as well as catalysis.However, the stereospecific effect cannot be a consequence ofthe direct interaction of Ser¹⁴⁸ with the C(2)-hydroxyl of thesubstrate. The changed K_m for glyceraldehyde-3-phosphate suggeststhat the initial step of hemithioacetal formation may take placewith its C(3) phosphate bound in the P_i site. This supportsthe molecular mechanism proposed by Moody (1984). Therefore,catalysis could be enhanced through interactions of the serinehydroxyl group not only with inorganic phosphate but also withthe C(3) phosphate of glyceraldehyde-3-phosphate.     

Single amino acid substitutions can further increase the stability of a thermophilic L-lactate dehydrogenase

Lactate dehydrogenases are of considerable interest as stereospecificcatalysts in the chemical preparation of enantiomerically pure-hydroxyacid synthons. For such applications in synthetic organicchemistry it would be desirable to have enzymes which tolerateelevated temperatures for prolonged reaction times, to increaseproductivity and to extend then applicability to poor substrates.Here, two examples are reported of significant thermostabilizations,induced by sitedirected mutagenesis, of an already thermostableprotein, the L-lactate dehydrogenase (EC 1.1.1.27 [EC] , 35 kDa permonomer subunit) from Bacillus stearothermophilus. Thermal inactivationof this enzyme is accompanied by irreversible unfolding of thenative protein structure. The replacement of Argl71 by Tyr stabilizesthe enzyme against thermal inactivation and unfolding. Thisstabilizing effect appears to be based on improved interactionsbetween the subunits in the core of the active dimeric or tetramericforms of the enzyme. The thermal stability of L-lactate dehydrogenasevariants with an active site Arg residue, either in the 171(wild-type) or in the 102 position, is further increased bysulfate ions. The two stabilizing effects are additive, as foundfor the Argl71Tyr/ Gln1O2Arg double mutant, for which the stabilityof the protein in 100 mM sulfate solution reaches that of L-lactatedehydrogenases from extreme thermophiles. All mutant proteinsretain significant catalytic activity, both in the presenceand absence of stnhilfoing salts, and are viable catalysts inpreparative scale reactions.     

Directed evolution of subtilisin E in Bacillus subtilis to enhance total activity in aqueous dimethylformamide

Sequential rounds of error-prone PCR to introduce random mutationsand screenrng of the resultant mutant libraries have been usedto enhance the total catalytic activity of subtilisin E significantlyin a non-natural environment, aqueous dimethylformamide (DMF).Seven DNA substitutions coding for three new amino acid substitutionswere identified in a mutant isolated after two additional generationsof directed evolution carried out on 10M subtilisin E, previously‘evolved’ to increase its specific activity in DMF.A Bacillus subtilis-Escherichia coli shuttle vector was developedin order to increase the size of the mutant library that couldbe established in B.subtilis and the stringency of the screeningprocess was increased to reflect total as well as specific activity.This directed evolution approach has been extremely effectivefor improving enzyme activity in a non-natural environment:the resulting-evolved 13M subtilisin exhibits specific catalyticefficiency towards the hydrolysis of a peptide substrate succlnyl-Ala-Ala-Pro-Phe-p-nitroanilidein 60% DMF solution that is three times that of the parent 10Mand 471 times that of wild type subtilisin E. The total activityof the 13M culture supernatant is enhanced 16-fold over thatof the parent 10M.     

Directed evolution of PDZ variants to generate high-affinity detection reagents

High-throughput protease assays are used to identify new protease inhibitors which have the potential to become valuable therapeutic products. Antibodies are of great utility as affinity reagents to detect proteolysis products in protease assays, but isolating and producing such antibodies is unreliable, slow and costly. It has been shown previously that PDZ domains can also be used to detect proteolysis products in high-throughput homogeneous assays but their limited natural repertoire restricts their use to only a few peptides. Here we show that directed evolution is an efficient way to create new PDZ domains for detection of protease activity. We report the first use of phage display to alter the specificity of a PDZ domain, yielding three variants with up to 25-fold increased affinity for a peptide cleavage product of HIV protease. Three distinct roles are assigned to the amino acid substitutions found in the selected variants of the NHERF PDZ domain: specific 'beta1-beta3' interaction with ligand residue -1, interactions with ligand residues -4 to -7 and improvement in phage display efficiency. The variants, having affinities as high as 620 nM, display improvements in assay sensitivity of over 5-fold while requiring smaller amounts of reagents. The approach demonstrated here leads the way to highly sensitive reagents for drug discovery that can be isolated more reliably and produced less expensively.     

Changes in the specificity of antibodies by site-specific mutagenesis followed by random mutagenesis

The specificity for 11-deoxycortisol (11-DOC) of a monoclonalantibody (mAb), designated SCET, was changed to specificityfor cortisol (CS) by site-specific mutagenesis followed by randommutagenesis. The Fab form of SCET was expressed on the surfaceof a phage. During the first step, mutations were introducedat 14 amino acid positions in three complementarity-determiningregions (CDRs) of the V_H domain that seemed likely to form thesteroid-binding pocket. A clone, DcC16, was isolated from theresultant library with multiple mutations and this clone wasshown to have CS-binding activity but also to retain high 11-DOC-bindingactivity. During the second step, mutations were introducedrandomly into the entire V_H-coding region of the DcC16 cloneby an error-prone polymerase chain reaction, and CS-specificmutant antibodies were selected in the presence of 11-DOC asa competitor. Three representative clones were analyzed withthe BIAcore instrument, and each revealed a large increase inthe binding constant for CS and a decrease in that for 11-DOC.Structural models, constructed by computer simulation, indicatedthe probable molecular basis for these changes in specificity.     

Novel features of serine protease active sites and specificity pockets: sequence analysis and modelling studies of glutamate-specific endopeptidases and epidermolytic toxins

With a view to obtaining a better understanding of the structuraldeterminants of P1 glutamate specificity in glutamate-specincendopeptidases (GSEs), the active sites and specificity pocketsof such enzymes from Bacillus ticheniformis (gse-bl), Bacillussubtilis (mpr) and Staphylococcus aureus (v8 protease) weremodelled. This approach was extended to the epidermolytic toxins(ETs), responsible for the staphylococcal scalded skin syndrome.We identify a canonical structure for the S1 subsite, composedof H213 and T190, both of which we predict to interact directlywith the P1 glutamate. The possible importance of R30 (for gse-bland mpr) and of the N-terminus (for gse-bl, mpr and v8 protease)was also examined. In the case of mpr, a G193C substitutionis predicted to participate in a novel disulphide bridge whichstabilizes C193 in such a way as to maintain the oxyanion hole.In v8, the loss or substitution of several important structuralcomponents around D102 of the catalytic triad probably explainsits reduced catalytic efficiency in comparison with other GSEs.In the case of the epidermolytic toxins K216 may be importantfor the previously reported phospholipase C-like activity, sincethe model predicts that it may stabilize the negative chargeon the phosphonyl group.     

Identifying the mechanism of protein loop closure: a molecular dynamics simulation of the Bacillus stearothermophilus LDH loopin solution

The ‘loop’ involving residues 98–110 in Bacillusstearothermophilus lactate dehydrogenase (BSLDH) is of greatinterest as substrate-induced ‘loop’ closure isthought to berate-limiting and essential in catalyzing the reactionand in determining substrate specificity. Consequently, we haveexplored the mechanism underlying ‘loop’ openingin BSLDH through a molecular dynamics simulation at high temperature(1000 K) in the presence of explicit solvent, starting fromthe X-ray structure of BSLDH complexed with the co-enzyme NAD+and oxamate at 2.5 Å. During the simulation, a significantconformational change occurred, as evidenced by sharp dihedralangle transitions, hydrogen bond breaking and formation andlarge root mean square deviations from the starting structure;these changes define the criteria for ‘loop’ opening.The mechanical elements responsible for ‘loop’ opening,i.e. ‘loop’ hinges and flap, are defined througha combination of order parameters, dihedral angle changes andtheir correlations and the dynamical cross-correlation map ofatomic displacements for the ‘loop’ residues. Theresults indicate that the ‘loop’ consists of twoflexible hinge regions on either side of a relatively rigidthree-residue segment that undergoes a significant spatial displacementduring ‘loop’opening. ‘Loop’ openingis made possible through an array of correlated dihedral anglechanges and intra-& ‘loop’ rearrangements ofhydrogen-bond interactions. The presentfindings are comparedto previous work related to ‘loop’ opening and site-directedmutagenesis experiments.     

A theoretical model of restriction endonuclease NlaIV in complex with DNA, predicted by fold recognition and validated by site-directed mutagenesis and circular dichroism spectroscopy

Restriction enzymes (REases) are commercial reagents commonly used in DNA manipulations and mapping. They are regarded as very attractive models for studying protein-DNA interactions and valuable targets for protein engineering. Their amino acid sequences usually show no similarities to other proteins, with rare exceptions of other REases that recognize identical or very similar sequences. Hence, they are extremely hard targets for structure prediction and modeling. NlaIV is a Type II REase, which recognizes the interrupted palindromic sequence GGNNCC (where N indicates any base) and cleaves it in the middle, leaving blunt ends. NlaIV shows no sequence similarity to other proteins and virtually nothing is known about its sequence-structure-function relationships. Using protein fold recognition, we identified a remote relationship between NlaIV and EcoRV, an extensively studied REase, which recognizes the GATATC sequence and whose crystal structure has been determined. Using the 'FRankenstein's monster' approach we constructed a comparative model of NlaIV based on the EcoRV template and used it to predict the catalytic and DNA-binding residues. The model was validated by site-directed mutagenesis and analysis of the activity of the mutants in vivo and in vitro as well as structural characterization of the wild-type enzyme and two mutants by circular dichroism spectroscopy. The structural model of the NlaIV-DNA complex suggests regions of the protein sequence that may interact with the 'non-specific' bases of the target and thus it provides insight into the evolution of sequence specificity in restriction enzymes and may help engineer REases with novel specificities. Before this analysis was carried out, neither the three-dimensional fold of NlaIV, its evolutionary relationships or its catalytic or DNA-binding residues were known. Hence our analysis may be regarded as a paradigm for studies aiming at reducing 'white spaces' on the evolutionary landscape of sequence-function relationships by combining bioinformatics with simple experimental assays.     

A new application of the yeast two-hybrid system in protein engineering

Cytochromes P450 are involved in the biosynthesis of steroid hormones in mitochondria of the adrenal gland. The electrons required for these reactions are provided via a redox chain consisting of adrenodoxin reductase (AdR) and adrenodoxin (Adx). A prerequisite for a fast and efficient electron transfer as well as high catalytic activity is the formation of functional complexes between the different redox partners. To improve the protein-protein interactions by directed evolution, we developed a new in vivo selection system. This high-throughput screening method is based on the yeast two-hybrid system. It enables a background-free screening for increased protein-protein interactions between stable and functional species including cofactor-containing proteins (FAD, [2Fe-2S], heme). The method was successfully applied for the directed evolution of Adx and selected variants were analyzed biochemically and biophysically. All analyzed proteins exhibit typical characteristics of [2Fe-2S]-cluster-type ferredoxins. Adx-dependent substrate conversion assays with different cytochromes demonstrated that the improved ability of the mutants to form complexes results in an enhanced catalytic efficiency of the cytochrome P450 system.