Functional roles of the N-terminal amino acid residues in the Mn(II)-L- malate binding and subunit interactions of pigeon liver malic enzyme

Pigeon liver malic enzyme has an N-terminal amino acid sequence of Met- Lys-Lys-Gly-Tyr-Glu-. In this work, various mutants of the enzyme with individual or combinational deletion (delta) or substitution at these amino acids were constructed and functionally expressed in Escherichia coli cells. A major protein band corresponding to an Mr of approximately 65000 was observed for all recombinant enzymes in sodium dodecyl sulfate polyacrylamide gel electrophoresis. However, when examining by polyacrylamide gel electrophoresis under native conditions, the recombinant enzymes were found to possess a tetrameric structure with Mr approximately 260000 or a mixture of tetramers and dimers with the exception of delta(K2K3G4) and delta(1-16) mutants, which existed exclusively as dimers at the protein concentration we employed. K3A and K3E also dissociated substantially. K(2,3)A was a tetramer but K(2,3)E essentially existed as dimers. All tetramers and dimers were enzymatically active in the gels. All mutants displayed a similar apparent Km value for NADP+. The apparent Km for L-malate and Mn(II), on the other hand, was increased by 4-27-fold for the delta(K2/K3) and the delta(1-16) mutants. The small binding affinity of delta(K2/K3) with Mn(II)-L-malate was specific. With additional deletion at positions 3 and/or 4, the delta(K2K3), delta(K2G4/K3G4) or delta(K2K3G4) mutants exhibited similar kinetic properties for the wild type. The lysine residues at the positions 2 or 3 seem to be crucial for the correct active site conformation. The results indicate that the N-terminus of malic enzyme is located at the Mn(II)-L-malate binding domain of the active center and is also near the subunit's interface. These results were interpreted with our asymmetric double-dimer model for the enzyme in which the N-terminus was involved in the head-to-tail monomer-monomer interactions but not the dimer-dimer interactions.      

Crystal structures of L201A mutant of D-amino acid aminotransferase at 2.0 A resolution: implication of the structural role of Leu201 in transamination

The leucine-to-alanine mutation at residue 201 of D-amino acid aminotransferase provides a unique enzyme which gradually loses its activity while catalyzing the normal transamination; the co-enzyme form is converted from pyridoxal 5'-phosphate to pyridoxamine 5'-phosphate upon the inactivation [Kishimoto,K., Yoshimura,T., Esaki,N., Sugio,S., Manning,J.M. and Soda,K. (1995) J. Biochem., 117, 691-696]. Crystal structures of both co-enzyme forms of the mutant enzyme have been determined at 2.0 A resolution: they are virtually identical, and are quite similar to that of the wild-type enzyme. Significant differences in both forms of the mutant are localized only on the bound co-enzyme, the side chains of Lys145 and Tyr31, and a water molecule sitting on the putative substrate binding site. Detailed comparisons of the structures of the mutant, together with that of the pyridoxamine-5'- phosphate form of the wild-type enzyme, imply that Leu201 would play a crucial role in the transamination reaction by keeping the pyridoxyl ring in the proper location without disturbing its oscillating motion, although the residue seems to not be especially important for the structural integrity of the enzyme.      

Stability of aspartate aminotransferase from Sulfolobus solfataricus

Aspartate aminotransferase from Sulfolobus solfataricus (SsAspAT) is an extremely thermophilic and thermostable dimeric enzyme which retains its structure and reaches maximal activity at 100 degrees C. The structural stability of this protein was investigated by coupling isothermally and thermally induced denaturation studies to molecular modeling. Gel filtration analysis indicated that SsAspAT unfolds with an N2 reversible 2D mechanism. In the molecular model, a cluster of hydrophobic residues was shown at the interface between the subunits of SsAspAT and suggested this cluster as a structural feature stabilizing the enzyme quaternary structure. At 25 degrees C, SsAspAT is less resistant to guanidinium chloride-induced denaturation than the cytosolic aspartate aminotransferase from pig heart (cpAspAT), which was chosen as a mesophilic counterpart in the thermodynamic analysis since it shares with SsAspAT the two-state unfolding mechanism. Therefore, in the case of aspartate aminotransferases, thermal stability does not correlate with the stability against chemical denaturants. Isothermal denaturation curves at 25 degrees C and melting profiles recorded in the presence of guanidinium chloride showed that the delta G degrees (H2O) at 25 degrees C of SsAspAT exceeds that of cpAspAT by roughly 15 kJ/mol; the parameter delta n, related to the number of binding sites for the denaturant differentially exposed in unfolded and folded states, is higher for SsAspAT than for cpAspAT; and delta Cp is lower for the thermophilic enzyme than for the mesophilic one by 8 kJ/K.mol. These results are indicative of a less hydrophobic core for SsAspAT than cpAspAT. In agreement with this, the molecular model predicts that some charged side chains are buried in SsAspAT and interact to form an H-bond/ion-pair network.      

Improvement in the alkaline stability of subtilisin using an efficient random mutagenesis and screening procedure

An efficient random mutagenesis procedure coupled to a replicaplate screen facilitated the isolation of mutant subtilisinsfrom Bacillus amyloliquefaciens that had altered autolytic stabilityunder alkaline conditions. Out of about 4000 clones screened,approximately 70 produced subtilisins with reduced stability(negatives). Two dones produced a more stable subtilisin (positives)and were identified as having a single mutation, either IIe107Valor Lys2l3Arg (the wild-type amino acid is followed by the codonposition and the mutant amino acid). One of the negative mutants,Met50Val, was at a site where other homologous subtilisins containeda Phe. When the Met50Phe mutation was introduced into the B.amyloliquefaciens gene, the mutant subtilisin was more alkalinestable. The double mutant IIe107Val/Lys2l3Arg) was more stablethan the isolated single mutant parents. The triple mutant (Met50Phe/IIel07Val/Lys2l3Arg)was even more stable than IIe107Val/Lys2l3Arg (up to two timesthe autolytic half-time of wild-type at pH 12). These studiesdemonstrate the feasibility for improving the alkaline stabilityof proteins by random mutagenesis and identifying potentialsites where substitutions from homologous proteins can improvealkaline stability.     

Subunit interface mutation disrupting an aromatic cluster in Plasmodium falciparum triosephosphate isomerase: effect on dimer stability

A mutation at the dimer interface of Plasmodium falciparum triosephosphateisomerase (PfTIM) was created by mutating a tyrosine residueat position 74, at the subunit interface, to glycine. Tyr74is a critical residue, forming a part of an aromatic clusterat the interface. The resultant mutant, Y74G, was found to haveconsiderably reduced stability compared with the wild-type protein(TIMWT). The mutant was found to be much less stable to denaturingagents such as urea and guanidinium chloride. Fluorescence andcircular dichroism studies revealed that the Y74G mutant andTIMWT have similar spectroscopic properties, suggestive of similarfolded structures. Further, the Y74G mutant also exhibited aconcentration-dependent loss of enzymatic activity over therange 0.1–10 µM. In contrast, the wild-type enzymedid not show a concentration dependence of activity in thisrange. Fluorescence quenching of intrinsic tryptophan emissionwas much more efficient in case of Y74G than TIMWT, suggestiveof greater exposure of Trp11, which lies adjacent to the dimerinterface. Analytical gel filtration studies revealed that inY74G, monomeric and dimeric species are in dynamic equilibrium,with the former predominating at low protein concentration.Spectroscopic studies established that the monomeric form ofthe mutant is largely folded. Low concentrations of urea alsodrive the equilibrium towards the monomeric form. These studiessuggest that the replacement of tyrosine with a small residueat the interface of triosephosphate isomerase weakens the subunit–subunitinteractions, giving rise to structured, but enzymatically inactive,monomers at low protein concentration.     

Thermostable variants of bovine {beta}-lactoglobulin

The thermal stability of bovine ß-lactoglobulin (BLG)has been enhanced by the introduction of an additional disulfidebond. Wild-type BLG has two disulfide bonds, C106–C119and C66–C160, with a free cysteine at position 121. Wehave designed, with the aid of molecular modeling calculations,two mutants of a recombinant BLG (rBLG), L104C and A132C. Moleculardynamics simulations were performed at 300K to study the effectof these alterations on the conformation of the protein. Thesemutants were then created by site-directed mutagenesis and purifiedfrom Escherichia coli carrying a tac expression vector usinga two-step renaturation method. Formation of disulfide linkagesin the correct arrangement, as designed, was confirmed by peptidemapping. In contrast to wild-type rBLG, which polymerizes attemperatures >65°C, neither of the mutant proteins polymerized.The conformational stability of the L104C and A132C mutant proteinsagainst thermal denaturation has been substantially increased(8- 10°C) as compared with wild-type rBLG. Furthermore,the A132C rBLG exhibits an enhanced stability against denaturationby guanidine hydrocnloride as compared with the wild-type orL104C rBLG     

Probing the role of threonine and serine residues of E.coli asparaginase II by site-specific mutagenesis

Site-specific mutagenesis has been used to probe amino acidresidues proposed to be critical in catalysis by Escherichiacoli asparaginase II. Thr12 is conserved in all known asparaginases.The catalytic constant of a T12A mutant towards L-aspartk acidß-hydroxamate was reduced to 0.04% of wild type activity,while its An, and stability against urea denaturation were unchanged.The mutant enzyme T12S exhibited almost normal activity butaltered substrate specificity. Replacement of Thr119 with Alaled to a 90% decrease of activity without markedly affectingsubstrate binding. The mutant enzyme S122A showed normal catalyticfunction but impaired stability in urea solutions. These dataindicate that the hydroxyl group of Thr12 is directly involvedin catalysis, probably by favorably interacting with a transitionstate or intermediate. By contrast, Thr119 and Ser122, bothputative target sites of the inactivator DONV, are functionallyless important.     

Thermal and mechanical properties of plastics molded from urea-modified soy protein isolates

Soy protein has been considered as a potential alternative of some petroleum polymers in the manufacture of plastics. The purpose of this investigation was to characterize the thermal and mechanical properties of plastics made from urea-modified soy protein. Soy protein isolate was separated from the defatted soy flour, modified with various urea concentrations, and compression-molded into plastics. Differential scanning calorimetry showed that the temperatures of denaturation and the enthalpies of denaturation of the modified soy protein decreased as urea concentrations increased above 1 M. At the same urea concentration, molded plastics made from the modified soy proteins showed a similar temperature of denaturation as the modified soy protein, but a lower enthalpy of denaturation. Tensile strength and Young's modulus of the molded plastics from the modified soy proteins increased as urea concentration increased and reached their maximum values at 8 M urea modification. Both storage modulus and glass transition temperature of the plastics from the modified soy proteins increased as urea concentration increased. The plastics made from the 2 M urea-modified soy proteins showed improvements in elongation, tough fracture behavior, and water resistance. The urea may function as a denaturant, a plasticizer, and a filler.     

Thermal stabilization of penicillolysin, a thermolabile 19 kDa Zn2+-protease, obtained by site-directed mutagenesis

Penicillolysin is a member of the clan MX and the family of M35 proteases. The enzyme is a thermolabile Zn(2+)- protease from Penicillium citrinum with a unique substrate profile. We expressed recombinant penicillolysin in Aspergillus oryzae and generated several site-directed mutants, R33E/E60R, A167E and T81P, with the intention of exploring thermal stabilization of this protein. We based our choice of mutations on the structures of homologous thermally stable enzymes, deuterolysin (EC 3.4.24.39) from A.oryzae and a peptidyl-Lys metallopeptidase (GfMEP) from the edible mushroom Grifora frondsa. The resulting mutant proteins exhibited comparable catalytic efficiency to the wild-type enzyme and some showed a higher tolerance to temperature.     

Rational stabilization of the C-LytA affinity tag by protein engineering

The C-LytA protein constitutes the choline-binding module of the LytA amidase from Streptococcus pneumoniae. Owing to its affinity for choline and analogs, it is regularly used as an affinity tag for the purification of proteins in a single chromatographic step. In an attempt to build a robust variant against thermal denaturation, we have engineered several salt bridges on the protein surface. All the stabilizing mutations were pooled in a single variant, C-LytAm7, which contained seven changes: Y25K, F27K, M33E, N51K, S52K, T85K and T108K. The mutant displays a 7 degrees C thermal stabilization compared with the wild-type form, together with a complete reversibility upon heating and a higher kinetic stability. Moreover, the accumulation of intermediates in the unfolding of C-LytA is virtually abolished for C-LytAm7. The differences in stability become more evident when the proteins are bound to a DEAE-cellulose affinity column, as most of wild-type C-LytA is denatured at approximately 65 degrees C, whereas C-LytAm7 may stand temperatures up to 90 degrees C. Finally, the change in the isoelectric point of C-LytAm7 enhances its solubility at acidic pHs. Therefore, C-LytAm7 behaves as an improved affinity tag and supports the engineering of surface salt bridges as an effective approach for protein stabilization.     

A lysine to arginine substitution at position 146 of rabbit aldolase A changes the rate-determining step to Schiff base formation

Lys146 of rabbit aldolase A [D-fructose-1,6-bis(phosphate):D-glyceraldehyde-3-phosphate lyase, EC 4.1.2.13 [EC] ] was changedto arginine by site-directed mutagenesis. The k_cat of the resultingmutant protein, K146R, was 500 times slower than wild-type insteady-state kinetic assays for both cleavage and condensationof fructose-1,6-bis(phosphate), while the Km for this substratewas unchanged. Analysis of the rate of formation of catalyticintermediates showed K146R was significantly different fromthe wild-type enzyme and other enzymes mutated at this site.Single-turnover experiments using acid precipitation to trapthe Schiff base intermediate on the wild-type enzyme failedto show a build-up of this intermediate on K146R. However, K146Rretained the ability to form the Schiff base intermediate asshown by the significant amounts of Schiff base intermediatetrapped with NaBH₄. In the single-turnover experiments it appearedthat the Schiff base intermediate was converted to productsmore rapidly than it was produced. This suggested a maximalrate of Schiff base formation of 0.022 s^–1, which wasclose to the value of k_cat for this enzyme. This observationis strikingly different from the wild-type enzyme in which Schiffbase formation is >100 times faster than k_cat. For K146Rit appears that steps up to and including Schiff base formationare rate limiting for the catalytic reaction. The carbanionintermediate derived from either substrate or product, and theequilibrium concentrations of covalent enzyme-substrate intermediates,were much lower on K146R than on the wild-type enzyme. The greaterbulk of the guanidino moiety may destabilize the covalent enzyme-substrateintermediates, thereby slowing the rate of Schiff base formationsuch that it becomes rate limiting. The K146R mutant enzymeis significantly more active than other enzymes mutated at thissite, perhaps because it maintains a positively charged groupat an essential position in the active site or perhaps the Argfunctionally substitutes as a general acid/base catalyst inboth Schiff base formation and in subsequent abstraction ofthe C4-hydroxyl proton.     

Possible role of two phenylalanine residues in the active site of human cytidine deaminase

Site-directed mutagenesis on human cytidine deaminase (CDA)was employed to mutate specifically two highly conserved phenylalanineresidues, F36 and F137, to tryptophan; at the same time, theunique tryptophan residue present in the sequence at position113 was mutated to phenylalanine. These double mutations wereperformed in order to have for each protein a single tryptophansignal for fluorescence studies relative to position 36 or 137.The mutant enzymes thus obtained, W113F, F36W/W113F and F137W/W113F,showed by circular dicroism and thermal stability an overallstructure not greatly affected by the mutations. The titrationof Trp residues by N-bromosuccinimide (NBS) suggested that residueW113 of the wild-type CDA and W36 of mutant F36W/W113F are buriedin the tertiary structure of the enzyme, whereas the residueW137 of mutant F137W/W113F is located near the surface of themolecule. Kinetic experiments and equilibrium experiments withFZEB showed that the residue W113 seems not to be part of theactive site of the enzyme whereas the Phe/Trp substitution inF36W/W113F and F137W/W113F mutant enzymes had a negative effecton substrate binding and catalysis, suggesting that F137 andF36 of the wild-type CDA are involved in a stabilizing interactionbetween ligand and enzyme.     

Characterization of the structure and self-recognition of the human centrosomal protein NA14: implications for stability and function

The protein NA14 is a key adaptor protein mediating the intermolecular interactions of microtubules and Spastin. To gain insight into its structure and function, we have expressed, purified and characterized human NA14 and some variants. NA14 is rather insoluble and tends to oligomerize and form fibrils. Successive mutation of the three Cys and two potentially exposed Leu residues (83 and 93) yielded a water-soluble quintuple variant, named 3CS-2LR. NA14 and its variants have a high helical content as determined by circular dichroism (CD). Based on nuclear magnetic resonance data of the quintuple mutant and the wild-type (wt) protein in the presence of dodecylphosphocholine micelles, the N-(M1-N13) and C-termini (K105-S119) were found to lack preferred structure. The remaining residues (14-104) participate in NA14 self-association, probably by forming a parallel coiled-coil structure. We hypothesize that Leu 83 and Leu 93 mediate interactions among NA14, Spastin and microtubules. We have also examined urea and thermal denaturation of the quintuple and other NA14 variants at different pH values by CD. The pH dependence of the conformational stability and the elevated native-state pK(a) determined for the two conserved Tyr allow us to propose that the NA14 structure may be stabilized by two Glu-COO(-) ||| HO-Tyr H-bonds, highly conserved in NA14-like proteins in other species.     

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.     

Stabilization of apoflavodoxin by replacing hydrogen-bonded charged Asp or Glu residues by the neutral isosteric Asn or Gln

Knowledge of protein stability principles provides a means toincrease protein stability in a rational way. Here we explorethe feasibility of stabilizing proteins by replacing solvent-exposedhydrogen-bonded charged Asp or Glu residues by the neutral isostericAsn or Gln. The rationale behind this is a previous observationthat, in some cases, neutral hydrogen bonds may be more stablethat charged ones. We identified, in the apoflavodoxin fromAnabaena PCC 7119, three surface-exposed aspartate or glutamateresidues involved in hydrogen bonding with a single partnerand we mutated them to asparagine or glutamine, respectively.The effect of the mutations on apoflavodoxin stability was measuredby both urea and temperature denaturation. We observed thatthe three mutant proteins are more stable than wild-type (onaverage 0.43 kcal/mol from urea denaturation and 2.8°C froma two-state analysis of fluorescence thermal unfolding data).At high ionic strength, where potential electrostatic repulsionsin the acidic apoflavodoxin should be masked, the three mutantsare similarly more stable (on average 0.46 kcal/mol). To ruleout further that the stabilization observed is due to removalof electrostatic repulsions in apoflavodoxin upon mutation,we analysed three control mutants and showed that, when thecharged residue mutated to a neutral one is not hydrogen bonded,there is no general stabilizing effect. Replacing hydrogen-bondedcharged Asp or Glu residues by Asn or Gln, respectively, couldbe a straightforward strategy to increase protein stability.     

Specificity studies of bacillus 1,3-1,4-beta-glucanases and application to glycosynthase-catalyzed transglycosylation

Bacillus 1,3-1,4-beta-glucanases hydrolyze 1,3-1,4-beta-gluco-oligosaccharides with a retaining mechanism. The binding-site cleft of these endoglycosidases is composed of six subsites (-4 to +2) of which subsite -3 makes the largest contribution to transition state stabilization. The specificity of this subsite is here analyzed for both glycosidase and glycosynthase activities in the wild-type and the nucleophile-less E134A mutant Bacillus licheniformis enzymes. A D-galactosyl residue on the nonreducing end of a trisaccharide substrate is accepted by the enzyme and binds at subsite -3 in the productive enzyme-substrate complex. The wild-type enzyme catalyzes the hydrolysis of the substrate Glcbeta4Glcbeta3GlcbetaMU (Glc=glucosyl, MU=4-methylumbelliferyl) with a k(cat)/K(M) value only 1.3-fold higher than for the Galbeta4Glcbeta3GlcbetaMU (Gal=galactosyl) substrate. The corresponding alpha-fluorides act as good donors for the glycosynthase condensation reaction with mono- and disaccharide acceptors catalyzed by the E134A mutant. Whereas self-condensation and elongation products are also obtained as minor compounds with the Glcbeta4Glcbeta3GlcalphaF donor, nearly quantitative yields of single condensation products are obtained with the Galbeta4Glcbeta3GlcalphaF donor, in which the axial configuration of the 4-OH group on the nonreducing end prevents self-condensation and elongation reactions.     

Increased proteolytic resistance of ribonuclease A by protein engineering

Although highly stable toward unfolding, native ribonucleaseA is known to be cleaved by unspecific proteases in the flexibleloop region near Ala20. With the aim to create a protease-resistantribonuclease A, Ala20 was substituted for Pro by site-directedmutagenesis. The resulting mutant enzyme was nearly identicalto the wild-type enzyme in the near-UV and far-UV circular dichroismspectra, in its activity to 2',3'-cCMP and in its thermodynamicstability. However, the proteolytic resistance to proteinaseK and subtilisin Carlsberg was extremely increased. Pseudo-first-orderrate constants of proteolysis, determined by densitometric analysisof the bands of intact protein in SDS–PAGE, decreasedby two orders of magnitude. In contrast, the rate constant ofproteolysis with elastase was similar to that of the wild-typeenzyme. These differences can be explained by the analysis ofthe fragments occurring in proteolysis with elastase. Ser21–Ser22was identified as the main primary cleavage site in the degradationof the mutant enzyme by elastase. Obviously, this bond is notcleavable by proteinase K or subtilisin Carlsberg. The resultsdemonstrate the high potential of a single mutation in proteinstabilization to proteolytic degradation.     

Improvement of cyclodextrin glucanotransferase as an antistaling enzyme by error-prone PCR

In an effort to improve the properties of cyclodextrin glucanotransferase (CGTase) as an antistaling enzyme, error-prone PCR was used to introduce random mutations into a CGTase cloned from alkalophilic Bacillus sp. I-5 (CGTase I-5). A mutant CGTase[3-18] with the three mutations M234T, F259I and V591A was selected by agar plate assay. Sequence alignment of various CGTases indicated that M234 and F259 are located in the vicinity of the catalytic sites of the enzyme and V591 in the starch binding domain E. The cyclization activity of CGTase[3-18] was dramatically decreased by 10-fold, while the hydrolyzing activity was increased by up to 15-fold. These mutations near subsite +1 (M234T) and at subsite +2 (F259I) are likely to alter the enzyme activity in a concerted manner, promoting hydrolysis of substrate while retarding cyclization. The addition of CGTase[3-18] reduced the retrogradation rate of bread by as much as did the commercial antistaling enzyme Novamyl during 7-day storage at 4 degrees C. No cyclodextrin (CD) was detected in bread treated with CGTase[3-18], whereas 21 mg of CD per 10 g of bread was produced in bread treated with wild-type CGTase.     

Selective hydroxylation of highly branched fatty acids and their derivatives by CYP102A1 from Bacillus megaterium

Highly branched fatty acids, the main components of the preen-gland waxes of the domestic goose and the Muscovy duck, and their derivatives are promising chiral precursors for the synthesis of macrolide antibiotics. The key step in the utilisation of these compounds is their regioselective hydroxylation, which cannot be achieved in a classical chemical approach. Three P450 monooxygenases, CYP102A1, CYP102A2 and CYP102A3, demonstrating high turnover numbers in the hydroxylation of iso and anteiso fatty acids (>400 min(-1)), were tested for their activity towards these substrates. CYP102A1 from Bacillus megaterium and its A74G F87V L188Q triple mutant hydroxylate a variety of these substrates with high activity and regioselectivity. In all cases, the triple mutant showed much higher activities than the wild-type enzyme. The binding constants, determined for wild-type CYP102A1 and the triple mutant with tetramethylnonanol as substrate, were >200 microM and approximately 23 microM, respectively. Data derived from binding analysis support the differences in activity found for the wild-type CYP102A1 and the triple mutant. Surprisingly, CYP102A2 and CYP102A3 from Bacillus subtilis did not show any activity. Substrate binding spectra, recorded to investigate substrate accessibility to the enzyme's active sites, revealed that the substrates either could not access the active site of the Bacillus subtilis monooxygenases, or did not come into proximity with the heme.