Biotechnological Production of Caffeic Acid by Bacterial Cytochrome P450 CYP199A2

Caffeic acid is a biologically active molecule that has various beneficial properties, including antioxidant, anticancer, and anti-inflammatory activities. In this study, we explored the catalytic potential of a bacterial cytochrome P450, CYP199A2, for the biotechnological production of caffeic acid. When the CYP199A2 enzyme was reacted with p-coumaric acid, it stoichiometrically produced caffeic acid. The crystal structure of CYP199A2 shows that Phe at position 185 is situated directly above, and only 6.35 Å from, the heme iron. This F185 residue was replaced with hydrophobic or hydroxylated amino acids using site-directed mutagenesis to create mutants with novel and improved catalytic properties. In whole-cell assays with the known substrate of CYP199A2, 2-naphthoic acid, only the wild-type enzyme hydroxylated 2-naphthoic acid at the C-7 and C-8 positions, whereas all of the active F185 mutants exhibited a preference for C-5 hydroxylation. Interestingly, several F185 mutants (F185V, F185L, F185I, F185G, and F185A mutants) also acquired the ability to hydroxylate cinnamic acid, which was not hydroxylated by the wild-type enzyme. These results demonstrate that F185 is an important residue that controls the regioselectivity and the substrate specificity of CYP199A2. Furthermore, Escherichia coli cells expressing the F185L mutant exhibited 5.5 times higher hydroxylation activity for p-coumaric acid than those expressing the wild-type enzyme. By using the F185L whole-cell catalyst, the production of caffeic acid reached 15 mM (2.8 g/liter), which is the highest level so far attained in biotechnological production of this compound.     

Rational evolution of a medium chain-specific cytochrome P-450 BM-3 variant

The single mutant F87A of cytochrome P-450 BM-3 from Bacillus megaterium was engineered by rational evolution to achieve improved hydroxylation activity for medium chain length substrates (C8-C10). Rational evolution combines rational design and directed evolution to overcome the drawbacks of these methods when applied individually. Based on the X-ray structure of the enzyme, eight mutation sites (P25, V26, R47, Y51, S72, A74, L188, and M354) were identified by modeling. Sublibraries created by site-specific randomization mutagenesis of each single site were screened using a spectroscopic assay based on omega-p-nitrophenoxycarboxylic acids (pNCA). The mutants showing activity for shorter chain length substrates were combined, and these combi-libraries were screened again for mutants with even better catalytic properties. Using this approach, a P-450 BM-3 variant with five mutations (V26T, R47F, A74G, L188K, and F87A) that efficiently hydrolyzes 8-pNCA was obtained. The catalytic efficiency of this mutant towards omega-p-nitrophenoxydecanoic acid (10-pNCA) and omega-p-nitrophenoxydodecanoic acid (12-pNCA) is comparable to that of the wild-type P-450 BM-3.     

Modulation of activity and substrate specificity by modifying the backbone length of the distant interdomain loop of D-amino acid aminotransferase.

The activity and substrate specificity of D-amino acid aminotransferase (D-AAT) (EC 2.6.1.21) can be rationally modulated by replacing the loop core (P119-R120-P121) with glycine chains of different lengths: 1, 3, or 5 glycines. The mutant enzymes were much more active than the wild-type enzyme in the overall reactions between various amino acids and pyruvate. The presteady-state kinetic analyses of half-reactions revealed that the 5-glycine mutant has the highest affinity (Kd) among all mutant enzymes and the wild-type enzyme towards various amino acids except D-aspartate. The 5-glycine mutant was much more efficient as a catalyst than the wild-type enzyme because the mutant enzyme showed the highest value of specificity constant (kmax/Kd) for all amino acids except D-aspartate and D-glutamate. The kmax/Kd values of the three mutants decreased with decrease in glycine chain length for each amino acid examined. Our findings may provide a new approach to rational modulation of enzymes.     

Critical role of the residue size at position 87 in H2O2- dependent substrate hydroxylation activity and H2O2 inactivation of cytochrome P450BM-3

Replacement of phenylalanine 87 with alanine or glycine (mutant F87A or F87G) greatly increased the H2O2-supported substrate hydroxylation activity of cytochrome P450BM-3, whose original H2O2-supported activity is hardly detectable. On the other hand, replacement of phenylalanine 87 with valine (mutant F87V) did not. In the oxidation of p-nitrophenoxydodecanoic acid (12-pNCA), the turnover numbers of the mutant F87A in the presence of NADPH and O2, or H2O2 were 493 and 162 nmol/min/nmol, respectively. The H2O2-supported F87A hydroxylation activity was further confirmed with free fatty acids as substrates. Moreover, the stability of F87A in H2O2 solutions also largely increased. The order of the stability of the wild type (WT), F87A, and their substrate (12-pNCA)-binding complexes in H2O2 solutions listed from high to low was F87A, WT, F87A substrate-binding complex, and WT substrate-binding complex. We propose that the free space size in the vicinity of the heme iron significantly influences P450BM-3 H2O2-supported activity and H2O2 inactivation.     

Biotransformation of β-ionone by engineered cytochrome P450 BM-3

Wild-type cytochrome P450 monooxygenase from Bacillus megaterium (P450 BM-3) has a low hydroxylation activity for β-ionone (<1 min⁻¹). Substitution of phenylalanine by valine at position 87 led to a more than 100-fold increase in β-ionone hydroxylation activity (115 min⁻¹). Enzyme activity could be further increased by both site-directed and random mutagenesis. The mutant R47L Y51F F87V, designed by site-directed mutagenesis, and the mutant A74E F87V P386S, obtained after two rounds of error-prone polymerase chain reaction, exhibited an increase in activity of up to 300-fold compared to the wild-type enzyme. The triple mutant R47 LY51F F87V exhibited moderate enantioselectivity, forming (R)-4-hydroxy-β-ionone with an optical purity of 39%. All mutants regioselectively converted β-ionone into 4-hydroxy-β-ionone. The regioselectivity is determined amongst others by the absolute configuration of the substrate.     

Site-directed Mutagenesis Identifies Amino Acid Residues Associated with the Dehydrogenase and Isomerase Activities of Human Type I (Placental) 3β-Hydroxysteroid Dehydrogenase/Isomerase

3β-Hydroxysteroid dehydrogenase/steroid Δ^{5 → 4}-isomerase (3β-HSD/isomerase) was expressed by baculovirus in Spodoptera fungiperda (Sf9) insect cells from cDNA sequences encoding human wild-type I (placental) and the human type I mutants - H261R, Y253F and Y253,254F. Western blots of SDS-polyacrylamide gels showed that the baculovirus-infected Sf9 cells expressed the immunoreactive wild-type, H261R, Y253F or Y253,254F protein that co-migrated with purified placental 3β-HSD/isomerase (monomeric M_r=42,000 Da). The wild-type, H261R and Y253F enzymes were each purified as a single, homogeneous protein from a suspension of the Sf9 cells (5.01). In kinetic studies with purified enzyme, the H261R mutant enzyme had no 3β-HSD activity, whereas the K_m and V_max values of the isomerase substrate were similar to the values obtained with the wild-type and native enzymes. The V_max (88 nmol/min/mg) for the conversion of 5-androstene-3,17-dione to androstenedione by the Y253F isomerase activity was 7.0-fold less than the mean V_max (620 nmol/min/mg) measured for the isomerase activity of the wild-type and native placental enzymes. In microsomal preparations, isomerase activity was completely abolished in the Y253,254F mutant enzyme, but Y253,254F had 45% of the 3β-HSD activity of the wild-type enzyme. In contrast, the purified Y253F, wild-type and native enzymes had similar V_max values for substrate oxidation by the 3β-HSD activity. The 3β-HSD activities of the Y253F, Y253,254F and wild-type enzymes reduced NAD⁺ with similar kinetic values. Although NADH activated the isomerase activities of the H261R and wild-type enzymes with similar kinetics, the activation of the isomerase activity of H261R by NAD⁺ was dramatically decreased. Based on these kinetic measurements, His²⁶¹ appears to be a critical amino acid residue for the 3β-HSD activity, and Tyr²⁵³ or Tyr²⁵⁴ participates in the isomerase activity of human type I (placental) enzyme.     

Altering the regioselectivity of the subterminal fatty acid hydroxylase P450 BM-3 towards gamma- and delta-positions

Cytochrome P450 BM-3 monooxygenase from Bacillus megaterium (CYP102A1) catalyzes the subterminal hydroxylation of fatty acids with a chain length of 12-22 carbons. Wild-type P450 BM-3 oxidizes saturated fatty acids at subterminal positions producing a mixture of omega-1, omega-2 and omega-3 hydroxylated products. Using a rational site-directed mutagenesis approach, three new elements have been introduced into the substrate binding pocket of the monooxygenase, which greatly changed the product pattern of lauric acid hydroxylation. Particularly, substitutions at positions S72, V78 and I263 had an effect on the enzyme regioselectivity. The P450 BM-3 mutants V78A F87A I263G and S72Y V78A F87A were able to oxidize lauric acid not only at delta-position (14% and 16%, respectively), but also produced gamma- and beta-hydroxylated products. delta-Hydroxy lauric and gamma-hydroxy lauric acid are important synthons for the production of the corresponding lactones.     

A continuous spectrophotometric assay for P450 BM-3, a fatty acid hydroxylating enzyme, and its mutant F87A

Cytochrome P450 BM-3 from Bacillus megaterium catalyzes the subterminal hydroxylation of medium- and long-chain fatty acids at the positions omega-1, omega-2, and omega-3. A rapid and continuous spectrophotometric activity assay for cytochrome P450 BM-3 based on the conversion of p-nitrophenoxycarboxylic acids (pNCA) to omega-oxycarboxylic acids and the chromophore p-nitrophenolate was developed. In contrast to the commonly used activity assays for this enzyme, relying on the consumption of oxygen or NADPH or the use of 14C-labeled carboxylic acids, the pNCA assay can even be used with crude extracts of the recombinant enzyme from lysed Escherichia coli cells. The kinetics of p-nitrophenolate formation are directly measured at a wavelength of 410 nm using a spectrophotometer or microtiter plate reader. Sensitivity of the assay is greatly enhanced if p-nitrophenoxydodecanoic or p-nitrophenoxypentadecanoic acid are used with the F87A mutant instead of the wild-type P450 BM-3 enzyme.     

A P450 BM-3 mutant hydroxylates alkanes, cycloalkanes, arenes and heteroarenes

P450 monooxygenases from microorganisms, similar to those of eukaryotic mitochondria, display a rather narrow substrate specificity. For native P450 BM-3, no other substrates than fatty acids or an indolyl-fatty acid derivative have been reported (Li, Q.S., Schwaneberg, U., Fischer, P., Schmid, R.D., 2000. Directed evolution of the fatty-acid hydroxylase P450BM-3 into an indole-hydroxylating catalyst. Chem. Eur. J. 6 (9), 1531-1536). Engineering the substrate specificity of Bacillus megaterium cytochrome P-450 BM3: hydroxylation of alkyl trimethylammonium compounds. Biochem. J. 327, 537-544). We thus were quite surprised to observe, in the course of our investigations on the rational evolution of this enzyme towards mutants, capable of hydroxylating shorter-chain fatty acids, that a triple mutant P450 BM-3 (Phe87Val, Leu188-Gln, Ala74Gly, BM-3 mutant) could efficiently hydroxylate indole, leading to the formation of indigo and indirubin (Li, Q.S., Schwaneberg, U., Fischer, P., Schmid, R.D., 2000. Directed evolution of the fatty-acid hydroxylase P450BM-3 into an indole-hydroxylating catalyst. Chem. Eur. J. 6 (9), 1531-1536). Indole is not oxidized by the wild-type enzyme; it lacks the carboxylate group by which the proper fatty acid substrates are supposed to be bound at the active site of the native enzyme, via hydrogen bonds to the charged amino acid residues Arg47 and Tyr51. Our attempts to predict the putative binding mode of indole to P450 BM-3 or the triple mutant by molecular dynamics simulations did not provide any useful clue. Encouraged by the unexpected activity of the triple mutant towards indole, we investigated in a preliminary, but systematic manner several alkanes, alicyclic, aromatic, and heterocyclic compounds, all of which are unaffected by the native enzyme, for their potential as substrates. We here report that this triple mutant indeed is capable to hydroxylate a respectable range of other substrates, all of which bear little or no resemblance to the fatty acid substrates of the native enzyme.     

Filling a hole in cytochrome P450 BM3 improves substrate binding and catalytic efficiency

Cytochrome P450BM3 (CYP102A1) from Bacillus megaterium, a fatty acid hydroxylase, is a member of a very large superfamily of monooxygenase enzymes. The available crystal structures of the enzyme show non-productive binding of substrates with their omega-end distant from the iron in a hydrophobic pocket at one side of the active site. We have constructed and characterised mutants in which this pocket is filled by large hydrophobic side-chains replacing alanine at position 82. The mutants having phenylalanine or tryptophan at this position have very much (approximately 800-fold) greater affinity for substrate, with a greater conversion of the haem iron to the high-spin state, and similarly increased catalytic efficiency. The enzyme as isolated contains bound palmitate, reflecting this much higher affinity. We have determined the crystal structure of the haem domain of the Ala82Phe mutant with bound palmitate; this shows that the substrate is binding differently from the wild-type enzyme but still distant from the haem iron. Detailed analysis of the structure indicates that the tighter binding in the mutant reflects a shift in the conformational equilibrium of the substrate-free enzyme towards the conformation seen in the substrate complex rather than differences in the enzyme-substrate interactions. On this basis, we outline a sequence of events for the initial stages of the catalytic cycle. The Ala82Phe and Ala82Trp mutants are also very much more effective catalysts of indole hydroxylation than the wild-type enzyme, suggesting that they will be valuable starting points for the design of mutants to catalyse synthetically useful hydroxylation reactions.     

A highly active adipyl-cephalosporin acylase obtained via rational randomization

There is strong interest in creating an enzyme that can deacylate natural cephalosporins such as cephalosporin C in order to efficiently acquire the starting compound for the industrial production of semisynthetic cephalosporin antibiotics. In this study, the active site of the glutaryl acylase from Pseudomonas SY-77 was randomized rationally. Several mutations that were found in previous studies to enhance the activity of the enzyme towards adipyl-7-aminodesacetoxycephalosporanic acid (ADCA) and cephalosporin C have now been combined, and libraries have been made in which random amino acid substitutions at these positions are joined. The mutants were expressed in a leucine-deficient Escherichia coli strain and subjected to growth selection with adipyl-leucine or amino-adipyl-leucine as sole leucine source. The mutants growing on these media were selected and purified, and their hydrolysis activities towards adipyl-7-ADCA and cephalosporin C were tested. Several mutants with highly improved activities towards the desired substrates were found in these rationally randomized libraries. The best mutant was selected from a library of totally randomized residues: 178, 266, and 375. This mutant comprises two mutations, Y178F + F375H, which synergistically improve the catalytic efficiency towards adipyl-7-ADCA 36-fold. The activity of this mutant towards adipyl-7-ADCA is 50% of the activity of the wild-type enzyme towards the preferred substrate glutaryl-7-aminocephalosporanic acid, and therefore the characteristics of this mutant approach those needed for industrial application.     

Enzymatic synthesis of structured phenolic lipids by incorporation of selected phenolic acids into triolein

The synthesis of structured phenolic lipids by lipase-catalyzed transesterification of selected phenolic acids, including p-hydroxyphenyl acetic, p-coumaric, sinapic, ferulic and 3,4-dihydroxybenzoic acids, with triolein was investigated. The highest enzymatic activity (248 nmol esterified phenolic acid/g solid enzyme/min) and bioconversion (62%) was obtained for the transesterification of p-hydroxyphenyl acetic acid with triolein. In addition, the transesterification of p-coumaric with triolein resulted in a higher enzymatic activity (87 nmol esterified phenolic acid/g solid enzyme/min) and bioconversion (46%) than those obtained for the transesterfication of ferulic and sinapic acids. The results also showed that using p-hydroxyphenyl acetic, p-coumaric and ferulic acids as substrate, the maximum bioconversion of phenolic monoacylglycerols was close to that of phenolic diacylglycerols. Although p-coumaric acid had very low radical scavenging activity (2%) compared to that of ferulic acid (62%), the p-coumaroylated lipids demonstrated a higher scavenging potency (16%) than that of the feruloylated one (10%).     

Blocking the tunnel: engineering of Candida rugosa lipase mutants with short chain length specificity

The molecular basis of chain length specificity of Candida rugosa lipase 1 was investigated by molecular modeling and site-directed mutagenesis. The synthetic lip1 gene and the lipase mutants were expressed in Pichia pastoris and assayed for their chain length specificity in single substrate assays using triglycerides as well as in a competitive substrate assay using a randomized oil. Mutation of amino acids at different locations inside the tunnel (P246F, L413F, L410W, L410F/S300E, L410F/S365L) resulted in mutants with a different chain length specificity. Mutants P246F and L413F have a strong preference for short chain lengths whereas substrates longer than C10 are hardly hydrolyzed. Increasing the bulkiness of the amino acid at position 410 led to mutants that show a strong discrimination of chain lengths longer than C14. The results obtained can be explained by a simple mechanical model: the activity for a fatty acid sharply decreases as it becomes long enough to reach the mutated site. In contrast, a mutation at the entrance of the tunnel (L304F) has a strong impact on C4 and C6 substrates. This mutant is nevertheless capable of hydrolyzing chain lengths longer than C8.     

Biochemical and ultrastructural studies on the interaction of basic proteins with mitochondria: a primary effect on membrane configuration

The cytochrome P-450_K containing monooxygenase system of rat kidney cortex microsomes catalyzes the hydroxylation of various saturated fatty acids of medium chain length to the corresponding ω- and (ω-1)-hydroxy derivatives. The hydroxylation activity, as well as the ratio between the two hydroxylated products, vary with the carbon chain length of the fatty acid. Optimal hydroxylation activity is observed with myristic acid which yields the 13- and 14-hydroxylated products at a ratio of about 1. The ω/(ω-1)-hydroxylation ratio decreases with increasing carbon chain length of the fatty acid. On the other hand, with lauric acid as a substrate the ratio between ω- and (ω-1)-hydroxylation does not change significantly with varying time of incubation or substrate concentration, or incubation in a medium containing D₂O or after induction of enhanced hydroxylation activity by starvation of the animals. Furthermore, 12-hydroxylauric acid and capric acid—which is almost exclusively ω-hydroxylated by rat kidney cortex microsomes—inhibit both 11- and 12-hydroxylation of lauric acid to a similar extent whereas 11-hydroxylauric acid does not seem to inhibit either 11- or 12-hydroxylation.C₁₀-C₁₆ fatty acids produce the type I spectral change upon addition to rat kidney cortex microsomes and seem to interact with similar amounts of the cytochrome P-450_K present in these particles. In agreement with the metabolic studies, 12-hydroxylauric acid interacts with cytochrome P-450_K giving rise to a reverse type I spectral change, whereas 11-hydroxylauric acid does not produce an observable spectral change. Finally, results of binding experiments with a series of derivatives of dodecane suggest that type I binding to cytochrome P-450_K requires, besides a proper chain length, the presence of a carbonyl group together with an electron pair on a neighboring atom at the end of the carbon chain. A chain length of 14 carbon atoms seems to be optimal and it is suggested that this chain length may correspond to the distance between a possible binding site and the catalytic site of cytochrome P-450_K     

Engineering Cytochrome P450 BM-3 for Oxidation of Polycyclic Aromatic Hydrocarbons

Cytochrome P450 BM-3, a self-sufficient P450 enzyme from Bacillus megaterium that catalyzes the subterminal hydroxylation of long-chain fatty acids, has been engineered into a catalyst for the oxidation of polycyclic aromatic hydrocarbons. The activities of a triplet mutant (A74G/F87V/L188Q) towards naphthalene, fluorene, acenaphthene, acenaphthylene, and 9-methylanthracene were 160, 53, 109, 287, and 22/min, respectively. Compared with the activities of the wild type towards these polycyclic aromatic hydrocarbons, those of the mutant were improved by up to 4 orders of magnitude. The coupling efficiencies of the mutant towards naphthalene, fluorene, acenaphthene, acenaphthylene, and 9-methylanthracene were 11, 26, 5.4, 15, and 3.2%, respectively, which were also improved several to hundreds fold. The high activities of the mutant towards polycyclic aromatic hydrocarbons indicate the potential of engineering P450 BM-3 for the biodegradation of these compounds in the environment.     

Engineering cytochrome P450 BM-3 for oxidation of polycyclic aromatic hydrocarbons.

Cytochrome P450 BM-3, a self-sufficient P450 enzyme from Bacillus megaterium that catalyzes the subterminal hydroxylation of long-chain fatty acids, has been engineered into a catalyst for the oxidation of polycyclic aromatic hydrocarbons. The activities of a triplet mutant (A74G/F87V/L188Q) towards naphthalene, fluorene, acenaphthene, acenaphthylene, and 9-methylanthracene were 160, 53, 109, 287, and 22/min, respectively. Compared with the activities of the wild type towards these polycyclic aromatic hydrocarbons, those of the mutant were improved by up to 4 orders of magnitude. The coupling efficiencies of the mutant towards naphthalene, fluorene, acenaphthene, acenaphthylene, and 9-methylanthracene were 11, 26, 5.4, 15, and 3.2%, respectively, which were also improved several to hundreds fold. The high activities of the mutant towards polycyclic aromatic hydrocarbons indicate the potential of engineering P450 BM-3 for the biodegradation of these compounds in the environment.     

Directed evolution of the alpha-L-fucosidase from Thermotoga maritima into an alpha-L-transfucosidase

The alpha-L-fucosidase from Thermotoga maritima (Tm alpha fuc) was converted into alpha-L-transfucosidase variants by directed evolution. The wild-type enzyme catalyzes oligosaccharide synthesis by transfer of a fucosyl residue from a pNP-fucoside donor to pNP-fucoside (self-condensation) with alpha-(1-->3) regioselectivity or pNP-galactoside (transglycosylation) with alpha-(1-->2) regioselectivity at low yields (7%). The wild-type enzyme was submitted to one cycle of mutagenesis, followed by rational recombination of the selected mutations, which allowed identification of variants with improved transferase activity. The transferase and hydrolytic kinetics of all the mutants were assessed by NMR methods and capillary electrophoresis. It was shown that the best mutant exhibited a dramatic 32-fold increase in the transferase/hydrolytic kinetic ratio, while keeping 60% of the overall wild-type enzyme activity. Accordingly, the maximum yield of a specific transglycosylation product [pNP-Gal-alpha-(1-->2)-Fuc] reached more than 60% compared to 7% with WT enzyme at equimolar and low concentrations of donor and acceptor (10 mM). Such an improvement was obtained with only three mutations (T264A, Y267F, L322P), which were all located in the second amino acid shell of the fucosidase active site. Molecular modeling suggested that some of these mutations (T264A, Y267F) cause a reorientation of the amino acids that are in direct contact with the substrates, resulting in a better docking energy. Such mutants with high transglycosidase activity may constitute novel enzymatic tools for the synthesis of fucooligosaccharides.     

Dimer formation of octaprenyl-diphosphate synthase (IspB) is essential for chain length determination of ubiquinone

Ubiquinone (Q), composed of a quinone core and an isoprenoid side chain, is a key component of the respiratory chain and is an important antioxidant. In Escherichia coli, the side chain of Q-8 is synthesized by octaprenyl-diphosphate synthase, which is encoded by an essential gene, ispB. To determine how IspB regulates the length of the isoprenoid, we constructed 15 ispB mutants and expressed them in E. coli and Saccharomyces cerevisiae. The Y38A and R321V mutants produced Q-6 and Q-7, and the Y38A/R321V double mutant produced Q-5 and Q-6, indicating that these residues are involved in the determination of chain length. E. coli cells (ispB::cat) harboring an Arg-321 mutant were temperature-sensitive for growth, which indicates that Arg-321 is important for thermostability of IspB. Intriguingly, E. coli cells harboring wild-type ispB and the A79Y mutant produced mainly Q-6, although the activity of the enzyme with the A79Y mutation was completely abolished. When a heterodimer of His-tagged wild-type IspB and glutathione S-transferase-tagged IspB(A79Y) was formed, the enzyme produced a shorter length isoprenoid. These results indicate that although the A79Y mutant is functionally inactive, it can regulate activity upon forming a heterodimer with wild-type IspB, and this dimer formation is important for the determination of the isoprenoid chain length.     

Active site mutations change the cleavage specificity of neprilysin

Neprilysin (NEP), a member of the M13 subgroup of the zinc-dependent endopeptidase family is a membrane bound peptidase capable of cleaving a variety of physiological peptides. We have generated a series of neprilysin variants containing mutations at either one of two active site residues, Phe⁵⁶³ and Ser⁵⁴⁶. Among the mutants studied in detail we observed changes in their activity towards leucine⁵-enkephalin, insulin B chain, and amyloid β_1–40. For example, NEP^F563I displayed an increase in preference towards cleaving leucine⁵-enkephalin relative to insulin B chain, while mutant NEP^S546E was less discriminating than neprilysin. Mutants NEP^F563L and NEP^S546E exhibit different cleavage site preferences than neprilysin with insulin B chain and amyloid ß_1–40 as substrates. These data indicate that it is possible to alter the cleavage site specificity of neprilysin opening the way for the development of substrate specific or substrate exclusive forms of the enzyme with enhanced therapeutic potential.     

Indole hydroxylation by bacterial cytochrome P450 BM-3 and modulation of activity by cumene hydroperoxide

Cytochrome P450 BM-3 from Bacillus megaterium catalyzed NADPH-supported indole hydroxylation under alkaline conditions with homotropic cooperativity toward indole. The activity was also found with the support of H2O2, tert-butyl hydroperoxide (tBuOOH), or cumene hydroperoxide (CuOOH). Enhanced activity and heterotropic cooperativity were observed in CuOOH-supported hydroxylation, and both the Hill coefficient and substrate concentration required for half-maximal activity in the CuOOH-supported reaction were much lower than those in the H2O2-, tBuOOH-, or NADPH-supported reactions. CuOOH greatly enhanced NADPH consumption and indole hydroxylation in the NADPH-supported reaction. However, when CuOOH was replaced by tBuOOH or H2O2, heterotropic cooperativity was not observed. Spectral studies also confirmed that CuOOH stimulated indole binding to P450 BM-3. Interestingly, a mutant enzyme with enhanced indole-hydroxylation activity, F87V (Phe87 was replaced by Val), lost homotropic cooperativity towards indole and heterotropic cooperativity towards CuOOH, indicating that the active-site structure affects the cooperativities.