Construction of novel subtilisin E with high specificity, activity and productivity through multiple amino acid substitutions

Through three cumulative amino acid substitutions, we constructed novel mutant subtilisins E of Bacillus subtilis, all with high specificity, activity and productivity. The substitution of conserved Gly127, constituting P1 substrate-binding pocket, with Ala and Val showed a marked preference for the small P1 substrate. Leu was then substituted for Ile31 next to the catalytic Asp32 to enhance the catalytic activity. Both double mutants (I31L/G127A and I31L/G127V) showed a 3-5- fold increase in catalytic efficiency due to a large kcat, without any change in the specificity of the mutants at position 127. Molecular modeling suggests that large P1 residues were unable to access the pocket because of steric hindrance. A third mutation was introduced by replacing Tyr(-1) with Ala in the propeptide essential for autoprocessing to active mature subtilisin in vivo. A prominent 7-20- fold increase in active enzyme production occurred in the triple mutants (Y-1A/I31L/G127A and Y-1A/I31L/G127V).      

Engineering subtilisin YaB: restriction of substrate specificity by the substitution of Gly124 and Gly151 with Ala

The 3-D structure of subtilisin YaB was computer modelled using the structures of subtilisin BPN', subtilisin Carlsberg and thermitase as templates. Gly124 and Gly151 located on both sides of the waist of the S1 pocket were selected for site-directed mutagenesis based on the modelled structure. The mutated ale genes coding for the mutant subtilisin YaB were expressed in Bacillus subtilis DB104. All of the G124 and G151 series of mutants exhibited an increase of relative catalytic activity for elastin-orcein against casein and myofibrillar proteins. The S1 substrate specificity of G124A, G124V and G151A mutants were assessed using various carbobenzoxy-amino acid-nitrophenyl esters and succinyl-Ala-Ala-(Pro or Val)-(Ala, Phe or Leu)-p- nitroanilide [AA(P/V) (A/F/L)]. While G124A and G124V mutants hydrolyzed only Ala and Gly esters, G151A mutant hydrolyzed Ala, Leu and Gly esters. The G124A and G124V mutants did not hydrolyze AAPF and AAPL. However, these two mutants hydrolyzed AAPA and AAVA with kcat/Km values approximately 3-10-fold higher than those of the wild-type enzyme. The G151A mutant did not hydrolyze AAPF, but hydrolyzed AAPL, AAPA and AAVA with kcat/Km values approximately 1-4-fold higher than those of the wild-type enzyme. These results clearly indicate that the S1 substrate specificity of G124A and G124V mutants was restricted to Ala and Gly, and G151A mutant to Ala, Gly and Leu.      

Engineering the Catalytic Properties of Two-Domain Laccase from Streptomyces griseoflavus Ac-993

Laccases catalyze the oxidation of substrates with the concomitant reduction of oxygen to water. Recently, we found that polar residues located in tunnels leading to Cu2 and Cu3 ions control oxygen entrance (His 165) and proton transport (Arg 240) of two-domain laccase (2D) from Streptomyces griseoflavus (SgfSL). In this work, we have focused on optimizing the substrate-binding pocket (SBP) of SgfSL while simultaneously adjusting the oxygen reduction process. SgfSL variants with three single (Met199Ala, Met199Gly, and Tyr230Ala) and three double amino acid residues substitutions (Met199Gly/His165Ala, His165Ala/Arg240His, Met199Gly/Arg240His) were constructed, purified, and investigated. Combination of substitutions in the SBP and in the tunnel leading to Cu2 ion (Met199Gly/Arg240His) increased SgfSL catalytic activity towards ABTS by 5-fold, and towards 2.6-DMP by 16-fold. The high activity of the Met199Gly/Arg240His variant can be explained by the combined effect of the SBP geometry optimization (Met199Gly) and increased proton flux via the tunnel leading to Cu2 ion (Arg240His). Moreover, the variant with Met199Gly and His165Ala mutations did not significantly increase SgfSL’s activity, but led to a drastic shift in the optimal pH of 2.6-DMP oxidation. These results indicate that His 165 not only regulates oxygen access, but it also participates in proton transport in 2D laccases.     

Mutants of 4-oxalocrotonate tautomerase catalyze the decarboxylation of oxaloacetate through an imine mechanism

A designed single amino acid substitution can alter the catalytic activity and mechanism of 4-oxalocrotonate tautomerase (4-OT). While the wild-type enzyme catalyzes only the tautomerization of oxalocrotonate, the Pro1Ala mutant (P1A) catalyzes two reactions--the original tautomerization reaction and the decarboxylation of oxaloacetate. Although the N-terminal amine group of P1A is involved in both reactions, our results support a nucleophilic mechanism for the decarboxylase activity, in contrast to the general acid/base mechanism that has been previously established for the tautomerase activity. These findings demonstrate that a single catalytic group in a 4-OT mutant can catalyze two reactions by two different mechanisms.     

Stabilization of Aspergillus awamori glucoamylase by proline substitution and combining stabilizing mutations

To stabilize Aspergillus awamori glucoamylase (GA), three proline substitution mutations were constructed. When expressed in Saccharomyces cerevisiae, Ser30-->Pro (S30P) stabilized the enzyme without decreased activity, whereas Asp345-->Pro (D345P) did not significantly alter and Glu408-->Pro (E408P) greatly decreased enzyme thermostability. The S30P mutation was combined with two previously identified stabilizing mutations: Gly137-->Ala, and Asn20-->Cys/Ala27-- >Cys (which creates a disulfide bond between positions 20 and 27). The combined mutants demonstrated cumulative stabilization as shown by decreased irreversible thermoinactivation rates between 65 and 80 degrees C. Additionally, two of the combined mutants outperformed wild- type GA in high-temperature (65 degrees C) saccharifications of DE 10 maltodextrin and were more active than the wild-type enzyme when assayed using maltose as substrate.      

Elucidation of the role of Ser329 and the C-terminal region in the catalytic activity of Pseudomonas stutzeri L-rhamnose isomerase

Pseudomonas stutzeri l-rhamnose isomerase (l-RhI) is capable of catalyzing the isomerization between various aldoses and ketoses, showing high catalytic activity with broad substrate-specificity compared with Escherichia coli l-RhI. In a previous study, the crystal structure of P. stutzeri l-RhI revealed an active site comparable with that of E. coli l-RhI and d-xylose isomerases (d-XIs) with structurally conserved amino acids, but also with a different residue seemingly responsible for the specificity of P. stutzeri l-RhI, though the residue itself does not interact with the bound substrate. This residue, Ser329, corresponds to Phe336 in E. coli l-RhI and Lys294 in Actinoplanes missouriensis d-XI. To elucidate the role of Ser329 in P. stutzeri l-RhI, we constructed mutants, S329F (E. coli l-RhI type), S329K (A. missouriensis d-XI type), S329L and S329A. Analyses of the catalytic activity and crystal structure of the mutants revealed a hydroxyl group of Ser329 to be crucial for catalytic activity via interaction with a water molecule. In addition, in complexes with substrate, the mutants S329F and S329L exhibited significant electron density in the C-terminal region not observed in the wild-type P. stutzeri l-RhI. The C-terminal region of P. stutzeri l-RhI has flexibility and shows a flip-flop movement at the inter-molecular surface of the dimeric form.     

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.     

Computational design of the lasso peptide antibiotic microcin J25

Microcin J25 (MccJ25) is a 21 amino acid (aa) ribosomally synthesized antimicrobial peptide with an unusual structure in which the eight N-terminal residues form a covalently cyclized macrolactam ring through which the remaining 13 aa tail is fed. An open question is the extent of sequence space that can occupy such an extraordinary, highly constrained peptide fold. To begin answering this question, here we have undertaken a computational redesign of the MccJ25 peptide using a two-stage sequence selection procedure based on both energy minimization and fold specificity. Eight of the most highly ranked sequences from the design algorithm, each of which contained two or three amino acid substitutions, were expressed in Escherichia coli and tested for production and antimicrobial activity. Six of the eight variants were successfully produced by E.coli at production levels comparable with that of the wild-type peptide. Of these six variants, three retain detectable antimicrobial activity, although this activity is reduced relative to wild-type MccJ25. The results here build upon previous findings that even rigid, constrained structures like the lasso architecture are amenable to redesign. Furthermore, this work provides evidence that a large amount of amino acid variation is tolerated by the lasso peptide fold.     

Computational Study on Substrate Specificity of a Novel Cysteine Protease 1 Precursor from Zea mays

Cysteine protease 1 precursor from Zea mays (zmCP1) is classified as a member of the C1A family of peptidases (papain-like cysteine protease) in MEROPS (the Peptidase Database). The 3D structure and substrate specificity of the zmCP1 is still unknown. This study is the first one to build the 3D structure of zmCP1 by computer-assisted homology modeling. In order to determine the substrate specificity of zmCP1, docking study is used for rapid and convenient analysis of large populations of ligand–enzyme complexes. Docking results show that zmCP1 has preference for P1 position and P2 position for Arg and a large hydrophobic residue (such as Phe). Gly147, Gly191, Cys189, and Asp190 are predicted to function as active residues at the S1 subsite, and the S2 subsite contains Leu283, Leu193, Ala259, Met194, and Ala286. SIFt results indicate that Gly144, Arg268, Trp308, and Ser311 play important roles in substrate binding. Then Molecular Mechanics-Poisson-Boltzmann Surface Area (MM-PBSA) method was used to explain the substrate specificity for P1 position of zmCp1. This study provides insights into the molecular basis of zmCP1 activity and substrate specificity.     

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.     

Directed evolution of (βα)(8)-barrel enzymes: establishing phosphoribosylanthranilate isomerisation activity on the scaffold of the tryptophan synthase α-subunit

Phosphoribosylanthranilate (PRA) isomerase (TrpF) and tryptophan synthase α-subunit (TrpA) are (βα)(8)-barrel enzymes that are involved in the biosynthesis of tryptophan. They contain a conserved phosphate binding site, which indicates a common evolutionary origin. In order to experimentally back this hypothesis, we have established TrpF activity on the scaffold of TrpA from Salmonella typhimurium using protein engineering. Based on the superposition of crystal structures with bound ligands, two residues in the active site of TrpA were replaced with catalytic residues from TrpF using site-directed mutagenesis. This TrpA variant as well as wild-type TrpA were each subjected to random mutagenesis using error-prone PCR. The two resulting trpA gene libraries were used to transform an auxotrophic Escherichia coli trpF deletion strain, and TrpA variants with PRA isomerisation activity were isolated by in vivo complementation. The amino acid substitutions of the selected TrpA variants were recombined by DNA shuffling, again followed by complementation in vivo. Several TrpA variants were produced in E. coli and purified, and their catalytic TrpF activities were determined in vitro by steady-state enzyme kinetics. Our results support that TrpA and TrpF have evolved by gene duplication and diversification from each other or a common predecessor, and provide insights into the minimum requirements for the catalysis of PRA isomerisation.     

Chemical rescue of active site mutants of S. pneumoniae surface endonuclease EndA and other nucleases of the HNH family by imidazole

The His-Asn-His (HNH) motif characterizes the active sites of a large number of different nucleases such as homing endonucleases, restriction endonucleases, structure-specific nucleases and, in particular, nonspecific nucleases. Several biochemical studies have revealed an essential catalytic function for the first amino acid of this motif in HNH nucleases. This histidine residue was identified as the general base that activates a water molecule for a nucleophilic attack on the sugar phosphate backbone of nucleic acids. Replacement of histidine by an amino acid such as glycine or alanine, which lack the catalytically active imidazole side chain, leads to decreases of several orders of magnitude in the nucleolytic activities of members of this nuclease family. We were able, however, to restore the activity of HNH nuclease variants (i.e., EndA (Streptococcus pneumoniae), SmaNuc (Serratia marcescens) and NucA (Anabaena sp.)) that had been inactivated by His→Gly or His→Ala substitution by adding excess imidazole to the inactive enzymes in vitro. Imidazole clearly replaces the missing histidine side chain and thereby restores nucleolytic activity. Significantly, this chemical rescue could also be observed in vivo (Escherichia coli). The in vivo assay might be a promising starting point for the development of a high-throughput screening system for functional EndA inhibitors because, unlike the wild-type enzyme, the H160G and H160A variants of EndA can easily be produced in E. coli. A simple viability assay would allow inhibitors of EndA to be identified because these would counteract the toxicities of the chemically rescued EndA variants. Such inhibitors could be used to block the nucleolytic activity of EndA, which as a surface-exposed enzyme in its natural host destroys the DNA scaffolds of neutrophil extracellular traps (NETs) and thereby allows S. pneumoniae to escape the innate immune response.     

Mutations to alter Aspergillus awamori glucoamylase selectivity. II. Mutation of residues 119 and 121

Mutations Ser119-->Glu, Ser119-->Gly, Ser119-->Trp, Gly121-->Ala and Gly121-->Ala/Ser411-->Gly were constructed in glucoamylase to change substrate specificity. Mutation Ser411-->Gly was already known to decrease glucoamylase selectivity toward isomaltose formation and to increase peak glucose yield. All mutated glucoamylases had slightly lower specific activities on maltose than on wild-type glucoamylase. Ser119-->Glu, Ser119-->Gly and Ser119-->Trp glucoamylases were about as active on isomaltose and DP 4-7 maltooligosaccharides as wild-type glucoamylase. Gly121-->Ala and Gly121-->Ala/Ser411-->Gly glucoamylases were less active. At 55 degrees C Ser119-->Glu, wild-type, Ser119-- >Trp, Ser119-->Gly, Gly121-->Ala and Gly121-->Ala/Ser411-->Gly glucoamylases had progressively higher peak glucose yields, generally in the opposite order to their activities. There was also an inverse correlation between peak glucose yield and ratio of initial rate of isomaltose production from glucose condensation to that of glucose production from maltodextrin hydrolysis. The effect of mutations Gly121- ->Ala and Ser411-->Gly was not additive in predicting the effect of the double mutation on the ratio or on peak glucose yield.      

Effects of site-directed mutagenesis on the serine residues of human lecithin: Cholesterol acyltransferase

Lecithin:cholesterol acyltransferase (LCAT) is a serine protease-type enzyme that esterifies cholesterol in human plasma and is activated by apolipoprotein A-I in high-density lipoproteins. LCAT contains 22 serine residues, including Ser 181, which is thought to be part of the catalytic site. In order to determine the importance of these serine residues in LCAT, we prepared six LCAT mutants: LCAT (Ser19→Ala), LCAT (Ser181→Gly), LCAT (Ser208→Ala), LCAT (Ser216→Ala), LCAT (Ser225→Ala) and LCAT (Ser383→Ala). We also replaced the adjacent asparagine residues in two additional mutants, LCAT (Ser19→Ala, Asn20→Thr) and LCAT (Ser383→Ala, Asn384→Thr), in order to ascertain the effect of the serines onN-glycosylation. The mutant complementary DNA (cDNA) were subcloned into a eukaryotic expression vector (pSG5) and expressed in COS-6 cells. By polymerase chain reaction analysis, LCAT-specific messenger RNA (mRNA) was found in all mutant and wild-type transfectants. Western blot analysis revealed LCAT-specific bands in media and lysates of the transfected cells. With two exceptions, the amounts of LCAT mass secreted by the transfectants were similar to that of the wild type (mean, 90% mass of wild type; range, 34–138%). Except for LCAT (Ser181→Gly), which was inactive, the specific activities of the remainder of the mutant enzymes were also similar (mean, 95% activity of wild type; range, 65–169%). These results indicate that Ser181 is part of the catalytic site and that stereoconservative substitutions for serines have minor effects on the synthesis, secretion and specific activities of human LCAT.     

Computationally designed variants of Escherichia coli chorismate mutase show altered catalytic activity

Computational protein design methods were used to predict five variants of monofunctional Escherichia coli chorismate mutase expected to maintain catalytic activity. The variants were tested experimentally and three active site mutants exhibited catalytic activity similar to or greater than the wild-type enzyme. One mutant, Ala32Ser, showed increased catalytic efficiency.     

Cumulative stabilizing effects of glycine to alanine substitutions in Bacillus subtilis neutral protease

Oligonucleotide-directed mutagenesis has been used to replaceglycine residues by alanine in neutral protease from Bacillussubtilis. One Gly to Ala substitution (G147A) was located ina helical region of the protein, while the other (G189A) wasin a loop. The effects of mutational substitutions on the functional,conformational and stability properties of the enzyme have beeninvestigated using enzymatic assays and spectroscopic measurements.Single substitutions of both G1y147 and Gly189 with Ala residuesaffect the enzyme kinetic properties using synthetic peptidesas substrates. When Gly replacements were concurrently introducedat both positions, the kinetic characteristics of the doublemutant were roughly intermediate between those of the two singlemutants, and similar to those of the wild-type protease. Bothmutants G147A and G189A were found to be more stable towardsirreversible thermal inactivation/unfolding than the wild-typespecies. Moreover, the stabilizing effect of the Gly to Alasubstitution was roughly additive in the double mutant G147A/G189A,which shows a 3.2°C increase in T_m with respect to the wild-typeprotein. These findings indicate that the Gly to Ala substitutioncan be used as a strategy to stabilize globular proteins. Thepossible mechanisms of protein stabilization are also discussed.     

Model polypeptide of mussel adhesive protein. I. Synthesis and adhesive studies of sequential polypeptides (X‐Tyr‐Lys)n and (Y‐Lys)n

The sequential polytripeptides and polydipeptides, (X‐Tyr‐Lys)_n, (XGly, Ala, Pro, Ser, Leu, Ile, Phe), (Y‐Lys)_n, (YGly, Tyr), and (Gly‐Tyr)_n, which imitate a mussel adhesive protein, have been synthesized. The molecular weights of the polypeptides were estimated to be 7,200 ∼ 13,400 (19 ∼ 42 repeating units), and the polypeptides were found to have satisfactory amino acid sequences. The polypeptides were crosslinked by tyrosinase, and the optimal pH in the crosslinking reaction was 7.4 in the case of the polytripeptide, (Gly‐Tyr‐Lys)_n. The optimal tyrosinase amount for the adhesive strength of (Gly‐Tyr‐Lys)_n was 0.34 unit/mg (polypeptide) at pH 7.4. The shear adhesive strength of the polytripeptide increased with an increase in the polytripeptide concentration, and was not influenced by the third amino acid, X. The shear adhesive strengths of polytripeptides (X‐Tyr‐Lys)_n were equal to one of the synthetic polydecapeptides, (Ala‐Lys‐Pro‐Ser‐Tyr‐Pro‐Pro‐Thr‐Tyr‐Lys)_n and (Gly‐Pro‐Lys‐Thr‐Tyr‐Pro‐Pro‐Thr‐Tyr‐Lys)_n which were the model polydecapeptides for blue mussel and Californian mussel, respectively. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 76: 929–937, 2000     

无水氨基酸类离子液体-聚乙二醇混合溶剂吸收CO2的动力学

以聚乙二醇400（PEG400）为溶剂,氨基酸类离子液体（AAILs）作为化学吸收剂的混合体系具有蒸汽压极低、热稳定性好、黏度和再生能耗低、CO₂吸收量和选择性高等优点,适用于燃烧前CO₂捕集过程的高温高压吸收条件。本文采用压降法,测定了以四正丁基膦（[P₄₄₄₄]⁺）为阳离子,甘氨酸（Gly）、丙氨酸（Ala）和脯氨酸（Pro）作为阴离子的3种氨基酸类离子液体的混合溶剂体系对CO₂的吸收速率,并建立了该无水体系的CO₂吸收动力学模型。对于反应速率而言,在333.15K时,[P₄₄₄₄][Gly]-PEG400 > [P₄₄₄₄][Pro]-PEG400 > [P₄₄₄₄][Ala]-PEG400,温度升高至373.15K时,[P₄₄₄₄][Pro]-PEG400 > [P4444][Gly]-PEG400 > [P₄₄₄₄][Ala]-PEG400;根据相关吸收动力学参数,推测出CO₂在AAILs-PEG400中的反应均为快反应。通过研究其吸收动力学,获得了关键的吸收动力学数据,为后续的工业开发设计提供基础数据和设计依据。     

Structural Analysis Reveals the Substrate‐Binding Mechanism for the Expanded Substrate Specificity of Mutant meso‐Diaminopimelate Dehydrogenase

A meso‐diaminopimelate dehydrogenase (DAPDH) from Clostridium tetani E88 (CtDAPDH) was found to have low activity toward the D ‐amino acids other than its native substrate. Site‐directed mutagenesis similar to that carried out on the residues mutated by Vedha‐Peters et al. resulted in a mutant enzyme with highly improved catalytic ability for the synthesis of D ‐amino acids. The crystal structures of the CtDAPDH mutant in apo form and in complex with meso‐diaminopimelate (meso‐DAP), D ‐leucine (D ‐leu), and 4‐methyl‐2‐oxopentanoic acid (MOPA) were solved. meso‐DAP was found in an area outside the catalytic cavity; this suggested a possible two‐step substrate‐binding mechanism for meso‐DAP. D ‐leu and MOPA each bound both to Leu154 and to Gly155 in the open form of CtDAPDH, and structural analysis revealed the molecular basis for the expanded substrate specificity of the mutant meso‐diaminopimelate dehydrogenases.     

Novel substrate specificity engineered in the arabinose binding protein

The L-arabinose binding protein (ABP) of Escherichia coli naturallybinds L-arabinose and D-galactose with very high affinity and,with reduced affinity, a variety of other sugars that differonly at the C5 position of the pyranose ring.However, thereare stringent specificity requirements at the 1, 2, 3 and 4positions. Based on the high resolution crystallographic structureof the Ugand-protein complex, remodelling of the binding pocketwas attempted to shift the specificity towards Cl-substitutedgalactosides. To create space in the vicinity of the reducingend of bound galactose, four residues, LyslO, Asp90, Thrl47and Leul45, have been mutated for residues with smaller sidechains. Forty-seven mutants containing different combinationsof these mutations were tested by fluorometry for their abilityto bind methylß-D-galactoside (met-ß-Gal)or iso-propyl-ß-D-thio-galactoside (IPTG). Two double-residuemutants carrying Ser at position 147 and Ala or Gly at position90 appeared of particular interest for being able to bind met-ß-Galor IPTG, respectively, and no longer galactose. Fluorescenceexperiments and molecular modelling indicate that the mode ofbinding of the new substrates to the mutant proteins might besimilar to that of the natural ligands to wild-type ABP.