Alanine-scanning mutagenesis of plasmatocyte spreading peptide identifies critical residues for biological activity

Plasmatocyte spreading peptide (PSP) is a 23-amino acid cytokine that induces a class of insect immune cells called plasmatocytes to spread on foreign surfaces. The structure of PSP consists of a disordered N terminus (residues 1-6) and a well-defined core (residues 7-23) stabilized by a disulfide bridge between Cys(7) and Cys(19), hydrophobic interactions, and a short beta-hairpin. Structural comparisons also indicate that the core region of PSP adopts an epidermal growth factor (EGF)-like fold very similar to the C-terminal subdomain of EGF-like module 5 of thrombomodulin. To identify residues important for plasmatocyte spreading activity, we bioassayed PSP mutants in which amino acids were either replaced with alanine or deleted. Within the well-defined core of PSP, alanine replacement of Cys(7) and Cys(19) (C7.19A) eliminated all activity. Alanine replacement of Arg(13) reduced activity approximately 1000-fold in comparison to wild-type PSP, whereas replacement of the other charged residues (Asp(16), Arg(18), Lys(20)) surrounding Cys(19) diminished activity to a lesser degree. The point mutants Y11A, T14A, T22A, and F23A had activity identical or only slightly reduced to that of wild-type PSP. The mutant PSP-(7-23) lacked the entire unstructured domain of PSP and was found to have no plasmatocyte spreading activity. Surprisingly, E1A and N2A had higher activity than wild-type PSP, but F3A had almost no activity. We thus concluded that the lack of activity for PSP-(7-23) was largely due to the critical importance of Phe(3). To determine whether reductions in activity correlated with alterations in tertiary structure, we compared the C7.19A, R13A, R18A, and F3A mutants to wild-type PSP by NMR spectroscopy. As expected, the simultaneous replacement of Cys(7) and Cys(19) profoundly affected tertiary structure, but the R13A, R18A, and F3A mutants did not differ from wild-type PSP. Collectively, these results indicate that residues in both the unstructured and structured domains of PSP are required for plasmatocyte-spreading activity.     

Modification of the response to opioid and nonopioid drugs by chronic opioid antagonist treatment

Chronic exposure to opioid antagonists increases the analgesic actions of opioids such as morphine. In the present studies, morphine's analgesic potency was increased (supersensitivity) following an 8 day subcutaneous naltrexone implant in mice, but not following a 1 day implant. Supersensitivity was maximal 24hr following the 8 day implant and declined linearly and had returned to control levels by 120hr. Implantation of naltrexone pellets for 8 days was found to increase the relative analgesic potency of methadone by 120%, while the lethal potency of cocaine was slightly (19%), but significantly, decreased. In contrast, identical treatment did not alter the potency of the benzodiazepine alprazolam to induce ataxia.     

Estrogenic activity of two standardized red clover extracts (Menoflavon) intended for large scale use in hormone replacement therapy

Isoflavones are the most potent estrogenic compounds in red clover extracts. Standardized extracts have been discussed as an alternative for hormone replacement therapy. Variation due to extraction procedure and natural seasonal variation and variations originating from agricultural conditions have prevented the large scale use of such phytochemicals. An improved extraction procedure and careful analysis of the raw material yielded in a highly standardized preparation (Menoflavon) with an average isoflavone content of approximately 9% (dry weight) determined by HPLC. The estrogenic activity has been further evaluated by a yeast two plasmid system using estrogen receptor alpha (ER alpha) and estrogen receptor beta (ER beta). An estrogenic activity corresponding to a transactivational capacity of ca. 18 microg 17 beta-estradiol per g red clover extract for ER alpha and ca. 78 microg 17 beta-estradiol per g red clover for ER beta was obtained. The difference is explained by the higher affinity of ER beta to isoflavones than that observed for ER alpha. Calculation of potency from isoflavone content measured by HPLC yielded a comparable potency to that experimentally determined by the bioassay. The high content of isoflavones as well as the higher transactivational potency for ER beta than ER alpha make these extracts interesting candidates for HRT.     

Phosphorylation of synthetic peptide analogs of rabbit cardiac troponin inhibitory subunit by the cyclic AMP-dependent protein kinase.

A series of synthetic peptide analogs of the cardiac troponin inhibitory subunit (TN-1) phosphorylation site sequence, Arg12-Pro-Ala-Pro-Ala-Val-Arg18-Arg19-Ser20-Asp21-Arg22-Ala, have been tested as substrates for the catalytic subunit of the cyclic AMP-dependent protein kinase (EC 2.7.1.37, ATP:protein phosphotransferase). As substrates, these peptides were generally inferior to the pyruvate kinase analog peptide Leu-Arg-Arg-Ala-Ser-Leu-Gly or its COOH-terminal amide analog. Replacing Arg-19 with alanine had only a minor effect on the kinetics of phosphorylation of the TN-1 peptide analog. In contrast, replacement of Arg-22 and Arg-18 with alanine resulted in marked enhancement and reduction of the Vmax, respectively. The results of this study have demonstrated that synthetic peptide analogs of the local phosphorylation site sequences of natural substrates may differ widely in their capacity to act as substrates for this protein kinase. In the case of the TN-1 peptide analogs, the contribution of the 4 arginine residues can be distinguished in terms of their influence on the kinetics of phosphorylation.     

Novel glycosylated [Lys(7)]-dermorphin analogues: synthesis, biological activity and conformational investigations.

Syntheses of the [Lys(7)]- and [Hyp(6),Lys(7)]-dermorphin analogues in which either Tyr(5) or Hyp(6) are O-glucosylated are described. For comparison, the carbohydrate-free peptides have also been prepared. Structural investigations by FT-IR and CD measurements were carried out on the synthetic analogues and some preliminary pharmacological experiments were also performed.The biological potency of the glucosylated analogues was compared with that of the micro-opioid receptor agonist dermorphin in GPI preparations. Glucosylation of either Tyr(5) or Hyp(6) reduces the potency of both [Lys(7)]-dermorphin and [Hyp(6),Lys(7)]-dermorphin. The effect induced by the Tyr(5) glucosylation is quite strong and the potency of both peptides is reduced by about 150 times. A similar but less dramatic effect is induced by the glucosylation of the Hyp(6) residue, and the potency of the parent peptide is reduced by about 15 times. The presence of acetyl groups on the sugar hydroxyl functions further reduces the agonistic potency of the glucosylated analogues. The analgesic potency of [Hyp(6),Lys(7)]-, [Hyp(betaGlc)(6),Lys(7)]- and [Tyr(betaGlc)(5),Lys(7)]-dermorphin were also tested in vivo by the tail-flick test. The glucosylated hydroxyproline-containing analogue is 8-10 times less active than the parent peptide, but its analgesic effect lasts significantly longer.     

Human beta-endorphin: synthesis and biological activity of analogs with substitutions in positions 2, 9, 18, 27 and 31

Four analogs of the opioid peptide human beta-endorphin (Bh-EP) have been synthesized: [D-Lys9, Phe27, Gly31]-beta h-EP, [D-PHe18,Phe27,Gly31]-beta h-EP, [D-Thr2,D-Lys9,Phe27,Gly31]-beta h-EP, and [D-Thr2,D-Phe18,Phe27,Gly31]-beta h-EP. All are practically indistinguishable from beta h-EP in the guinea pig ileum assay. All show diminished analgesic potency in the mouse tail-flick assay.     

ACUTE ETHANOL EXPOSURE DECREASES THE ANALGESIC POTENCY OF MORPHINE IN MICE

Chronic (7 days), forced ethanol drinking can decrease the analgesic potency of opioid agonists in mice. In the present study, the effect of short-term ethanol treatment was examined using forced ethanol access and ethanol injection protocols. Mice were given forced access to 1, 3 or 7% (v/v) ethanol for 24 hr and then tested for s.c. morphine analgesia using the tailflick assay. Controls had access to water. Another group of mice was injected i.p. with 2.5 g/kg ethanol or water 4 times over a 21 hr period and tested 3 hr after the final injection for morphine analgesia. Other mice were injected once i.p. with 1, 2 or 3 g/kg ethanol or water and tested 24 hr later using the tailflick. In the forced access study, ethanol dose-dependently decreased morphine's analgesic potency with the highest dose (7%) producing a 1.6-fold shift in the ED₅₀. This decrease in morphine potency was similar to that found in a related study using 7% ethanol for 7 days (1.8-fold shift). Repeated ethanol injections significantly reduced the analgesic potency of morphine (1.9-fold shift), whereas, a single injection of 1, 2 or 3 g/kg ethanol did not alter the potency of morphine. Control studies indicated that neither 24 hr water nor food deprivation affected morphine potency. Overall, these data show that sustained exposure to ethanol over a 24 hr period will dose-dependently decrease morphine's analgesic potency. © 1998 Elsevier Science Inc.     

The biological potency of a series of analogues of human calcitonin correlates with their interactions with phospholipids

The conformational and lipid binding properties of several calcitonin analogs were compared. These analogs were designed to have the central amphipathic helical region of salmon calcitonin and N- and C-terminal segments similar to human calcitonin. The various analogs differed from one another either by removal of Leu19 from this hybrid analog, replacement of Leu19 with Gly19 or having a carboxyl terminus more closely related to salmon calcitonin. It had been found that replacement of Leu19 with Gly19 caused a marked reduction in the hypocalemic activity of the analog. The ability of the analogs to form helical structures in the presence of dimyristoylphosphatidylglycerol as well as their ability to lower the enthalpy of the calorimetric phase transition of this phospholipid correlates well with the hypocalcemic potency of the peptide.     

The insulin receptor juxtamembrane region contains two independent tyrosine/beta-turn internalization signals

We have investigated the role of tyrosine residues in the insulin receptor cytoplasmic juxtamembrane region (Tyr953 and Tyr960) during endocytosis. Analysis of the secondary structure of the juxtamembrane region by the Chou-Fasman algorithms predicts that both the sequences GPLY953 and NPEY960 form tyrosine-containing beta-turns. Similarly, analysis of model peptides by 1-D and 2-D NMR show that these sequences form beta-turns in solution, whereas replacement of the tyrosine residues with alanine destabilizes the beta-turn. CHO cell lines were prepared expressing mutant receptors in which each tyrosine was mutated to phenylalanine or alanine, and an additional mutant contained alanine at both positions. These mutations had no effect on insulin binding or receptor autophosphorylation. Replacements with phenylalanine had no effect on the rate of [125I]insulin endocytosis, whereas single substitutions with alanine reduced [125I]insulin endocytosis by 40-50%. Replacement of both tyrosines with alanine reduced internalization by 70%. These data suggest that the insulin receptor contains two tyrosine/beta-turns which contribute independently and additively to insulin-stimulated endocytosis.     

Implications of invariant residue LeuB6 in insulin-receptor interactions

By the chemical synthesis of modified insulin B chains and the combination of the synthetic B chains with natural insulin A chains, we have prepared insulin analogs with natural and unnatural amino acid replacements of invariant residue LeuB6. Analogs have been investigated by reference to their potencies for interaction with the insulin receptor (as assessed by competition for 125I-labeled binding to isolated canine hepatocytes) and to their abilities to undergo the structural transitions that are characteristic of insulin self-aggregation (as assessed by the spectroscopic analysis of analog complexes with cobalt). Our results identify that (a) replacement of LeuB6 by glycine has nearly the equivalent effect as deletion of residues B1-B6 in decreasing receptor binding potency of the analog to only about 0.05% of that of insulin; (b) relative to the GlyB6 derivative, replacements that increase the relative hydrophobicity of the residue B6 side chain also increase the relative receptor binding potencies of the resulting analogs; (c) negative steric effects resulting from substitutions by valine, phenylalanine, and gamma-ethylnorleucine limit the potential for enhancing potency as the result of increased hydrophobicity; and (d) two analogs with disparate potency for receptor interaction (those with alanine and gamma-ethylnorleucine at position B6, analogs exhibiting about 1 and 48% of the potency of insulin, respectively) undergo the T6----R6 structural transition in the presence of Co2+ and phenol which is typical of insulin but result in hexameric complexes with greatly reduced stability. We conclude that leucine provides a closely determined best fit at insulin position B6, and we discuss our findings in terms of insulin conformations that may apply to the receptor-bound state of the hormone.     

Role of Asn-16 and Ser-19 in anthopleurin B binding. Implications for the electrostatic nature of Na(V) site 3

Anthopleurin B (ApB) is a type 1 sea anemone toxin, which binds to voltage-sensitive sodium channels (Na(V)'s), thereby delaying channel inactivation. Previous work from our laboratories has demonstrated that the structurally unconstrained region involving residues 8-17 of this polypeptide, designated the Arg-14 loop, is important for full toxin affinity (Seibert et al., (2003) Biochemistry 42, 14515). Within this region, important contributions are made by residues Arg-12 and Leu-18 (Gallagher and Blumenthal, (1994) J. Biol. Chem. 269, 254; Dias-Kadambi et al., (1996) J. Biol. Chem. 271, 23828). Moreover, replacement of glycine residues found at positions 10 or 15 of the loop by alanine has been shown to have profound, isoform-selective effects on toxin-binding kinetics (Seibert et al., (2003)Biochemistry 42, 14515). To thoroughly understand the importance of this entire region, the work described here investigates the contribution of ApB residues Asn-16, Thr-17, and Ser-19 to toxin affinity and isoform selectivity. Our results demonstrate that residues within and proximal to the C terminus of the Arg-14 loop are important modulators of ApB affinity for Na(V) channels, indicating that the loop and channel site 3 are likely in close contact. A comparison of the effects of multiple replacements at each position reveals that Asn-16 and Ser-19 are involved in binding, whereas Thr-17 is not. The fact that anionic replacements for Asn-16 or Ser-19 are highly deleterious for toxin binding strongly suggests that site 3 contains either formal anionic residues or regions of high electron density, which could be formed by aromatic clusters. These data represent the first indication of the presence of such residues or regions within Na(V) site 3.     

Primary structural determinants essential for potent inhibition of cAMP-dependent protein kinase by inhibitory peptides corresponding to the active portion of the heat-stable inhibitor protein

PKI-(5-24)-amide is a 20-residue peptide with the sequence, Thr5-Thr-Tyr-Ala-Asp-Phe-Ile-Ala-Ser-Gly-Arg-Thr-Gly-Arg-Arg-Asn-A la-Ile-His- Asp24-NH2, that corresponds to the active portion of the heat-stable inhibitor protein of cAMP-dependent protein kinase (Cheng, H.-C., Kemp, B. E., Pearson, R. B., Smith, A. J., Misconi, L., Van Patten, S. M., and Walsh, D. A. (1986) J. Biol. Chem. 261, 989-992). Amino acid residues in PKI-(5-24)-amide responsible for the potent inhibition (Ki = 2.3 nM) of the catalytic subunit of protein kinase were further investigated using deletion and substitution analogs of the synthetic peptide. Residues 5, 23, and 24 were not required for activity since the 17-residue PKI-(6-22)-amide retained full potency. Sequential removal of the first seven amino acids from the NH2 terminus of PKI-(5-24)-amide caused a progressive 50-fold loss of inhibitory potency. In contrast, substitution of either Thr6, Asp9, or Ile11 with alanine, or Ala8 by leucine, in PKI-(5-22)-amide produced less than 3-fold decreases in potency. Of the 2 aromatic residues in PKI-(5-22)-amide, the individual substitution of Phe10 and Tyr7 by alanine caused, respectively, 90- and 5-fold decreases in inhibitory potency, demonstrating important roles for each. This NH2-terminal portion of the peptide is believed to contain a significant portion of alpha-helix. Many recognition or structural determinants are also essential in the COOH-terminal portion of PKI-(5-22)-amide. In addition to the basic subsite provided by the three arginines, several other of the residues are critical for full inhibitory potency. Substitution of Ile22 by glycine in either PKI-(5-22)-amide or PKI-(14-22)-amide lowered the inhibitory potency by 150- and 50-fold, respectively. Separate replacement of Gly17 or Asn20, in either PKI-(5-22)-amide or PKI-(14-22)-amide, caused 7-15-fold decreases in potency. Substitution of both Gly17 and Asn20 together (in PKI-(14-22)-amide) produced a synergistic loss of inhibitory activity. [Leu13,Ile14]PKI-(5-22)-amide, a doubly substituted analog exhibited a 42-fold increase in Ki value. We conclude that Ser13 and/or Gly14, Gly17, Asn20, and Ile22 each contribute important features to the binding of these inhibitory peptides to the protein kinase, either by providing recognition determinants, inducing structure, and/or allowing essential peptide backbone flexibility.(ABSTRACT TRUNCATED AT 400 WORDS)     

Analysis of a nuclear localization signal of simian virus 40 major capsid protein Vp1.

The nuclear localization signal of the major structural protein, Vp1, of simian virus 40 was further defined by mutagenesis. The targeting activity was examined in cells microinjected with SV-Vp1 variant viral DNAs bearing either an initiation codon mutation of the agnoprotein or mutations in the Vp1 coding sequence or microinjected with pSG5-Vp1 and pSG5-Vp1 mutant DNAs in which Vp1 or mutant Vp1 is expressed from simian virus 40 early promoter. The Vp1 nuclear localization signal functioned autonomously without agno-protein once the Vp1 protein was synthesized in the cytoplasm. The targeting activity was localized to the amino-terminal 19 residues. While replacement of cysteine 10 with glycine, alanine, or serine did not affect the activity, replacement of arginine 6 with glycine caused the cytoplasmic phenotype. When multiple mutations were introduced among residue 5, 6, 7, 16, 17, or 19, the targeting activity was found to reside in two clusters of basic residues, a cluster of lysine 5, arginine 6, and lysine 7 and a cluster of lysine 16, lysine 17, and lysine 19. The clusters are independently important for nuclear localization activity.     

Mutations in the heparin-binding domains of human basic fibroblast growth factor alter its biological activity

Eleven structural analogues of human basic fibroblast growth factor (bFGF) have been prepared by site-directed mutagenesis of a synthetic bFGF gene to examine the effect of amino acid substitutions in the three putative heparin-binding domains on FGF's biological activity. After expression in Escherichia coli, the mutant proteins were purified to homogeneity by use of heparin-Sepharose chromatography and analyzed for their ability to stimulate DNA synthesis in human foreskin fibroblasts. Recombinant human bFGF 1-146 and [Ala69,Ser87]bFGF, an analogue where two of the four cysteines had been replaced by alanine and serine, were equipotent to standard bovine basic fibroblast growth factor. Substitution of aspartic acid-19 by arginine in the first heparin-binding domain yielded a molecule that stimulated a higher total mitogenic response in fibroblasts as compared to bFGF. In addition, replacement of either arginine-107 in the second domain or glutamine-123 in the third domain with glutamic acid resulted in compounds that were 2 and 4 times more potent than bFGF. In contrast, substitution of arginine-107 with isoleucine reduced the activity of the molecule by 100-fold. Combination of domain substitutions to generate the [Glu107,123]bFGF and [Arg19,Lys123,126]bFGF mutants did not show any additivity of the mutations on biological activity. Alterations in the biological activity of the analogues was dependent on both the site of and the type of modification. Increased positive charge in the first domain and increased negative charge in the second and third domains enhanced biological potency. The altered activities of the derivatives appear to be due in part to changes in the affinity of the analogues for heparin. We conclude that changes in all three of the putative heparin-binding domains result in altered mitogenic activity and heparin interaction of basic fibroblast growth factor.     

Adrenocorticotropin. 48. Synthesis and biological activity of (15, 16-D-lysine, 17, 18-D-arginine)-adrenocorticotropin-(1-19) and an all-D-retropeptide related to the amino terminal octadecapeptide of adrenocorticotropin.

The peptide [15, 16-D-lysine, 17, 18-D-arginine]-adrenocorticotropin-(1-19) and an all-D-retropeptide related to the amino terminal octadecapeptide of adrenocorticotropin have been synthesized by the solid-phase method. The nonadecapeptide was shown to possess 10-15% of the steroidogenic activity and 3% of the lipolytic activity of adrenocorticotropin-(1-19). The all-D-retropeptide showed no activity and exhibited no inhibitory activity in steroidogenesis and lipolysis.     

Synthesis and biological activity of a new progestogen, 16-methylene-17alpha-hydroxy-18-methyl-19-norpregn-4-ene-3, 20-dione acetate

The progestational activity of second- and third-generation progestins in oral contraceptives were markedly increased by addition of an 18-methyl group. A new progestin, the 18-methyl analog of Nestorone, 16-methylene-17alpha-hydroxy-18-methyl-19-norpregn-4-ene-3,2 0-dione acetate (10), was synthesized. The relative binding affinity and biologic activity of 10 was compared with Nestorone, levonorgestrel, and progesterone using a binding assay for rat progesterone receptors, the Clauberg assay in the rabbit, and by assessing pregnancy maintenance in the rat. These studies, as summarized in Table 4, show that 10 is three to ten times more potent than Nestorone. The addition of the 18-methyl group to Nestorone markedly increased its potency as noted above, but is unlikely to change its rate of delivery from sustained release systems. 10 should be ideally suited for administration by implants or small skin patches.     

Synthesis and Analgesic Activity of N,N'-Dicarbonyltryptamines

ABSTRACT

5-Methoxytryptamine and L-tryptophan methyl ester were acylated with malonic acid, dimethyl malonate, or succinic anhydride to produce the corresponding N, N'-dicarbonyltryptamine derivatives. The analgesic activity was evaluated by the tail flick test. All of the compounds exhibited desirable analgesic potency. This result is consistent with that of N-(N-acetyl-L-tryptophanyl)-5-methoxytryptamine and confirmed that introducing substituted tryptamine into the amide chain of melatonin does enhance analgesic potency.     

beta-Endorphin: analgesic and receptor binding activity of non-mammalian homologs

Analgesic potencies of turkey, ostrich and des-acetyl salmon beta-endorphins have been measured in the tail-flick test and binding affinities determined by radio-receptor assay. The duration of analgesia and the slope of the dose-response curves generated by these peptides are similar to those elicited by mammalian beta-endorphins. This suggests that they act in vivo and in vitro on the same population of opiate receptors. The ratio of binding to analgesic potencies observed for these peptides varies nearly sixfold. Structure-activity analysis suggests that a basic side-chain at position 9 is required in order to produce a high opiate activity both in vivo and in vitro. A reexamination of the biological activities of camel beta-endorphin shows that the analgesic potency and binding affinity of this peptide are respectively 171 and 2.7 times higher than human beta-endorphin. His-27 and/or Gln-31 may contribute to this increased potency. The dissociation of radioreceptor binding affinity from analgesic potency in these naturally occurring beta-endorphin homologs suggests that either the conditions under which the binding assay is performed mask the true binding potency in the brain or that, once bound to the appropriate receptor, these homologs do not possess equal ability to produce biological effects.     

Brownian dynamics of interactions between aldolase mutants and F-actin

Previous Brownian dynamics (BD) simulations (Ouporov IG, Knull HR and Thomasson KA 1999. Biophys. J. 76: 17-27) of complex formation between rabbit aldolase and F-actin have identified three lysine residues (K288, K293 and K341) on aldolase and acidic residues (DEDE) at the N-terminus of actin as important to binding. BD simulations of computer models of aldolase mutants with any of these lysine residues replaced by alanine show reduced binding energy; the greatest effect of a single substitution is for K341A, and replacement of all three lysines greatly reduces binding. BD simulations of wild-type rabbit aldolase vs altered F-actin show that binding is decreased if any one of the four N-terminal acidic residues is replaced by alanine and binding is greatly reduced if three or more of the N-terminal acidic residues are replaced; none of the four actin residues appear more critical for binding than the others.     

Stereoselective hexobarbital 3'-hydroxylation by CYP2C19 expressed in yeast cells and the roles of amino acid residues at positions 300 and 476

We examined the enzymatic function of recombinant CYP2C19 in enantiomeric hexobarbital (HB) 3'-hydroxylation, and searched the roles of amino acid residues, such as Phe-100, Phe-114, Asp-293, Glu-300, and Phe-476 of CYP2C19 in the stereoselective HB 3'-hydroxylation, using a yeast cell expression system and site-directed mutagenesis method. CYP2C19 wild-type exerted substrate enantioselectivity of (R)-HB>(S)-HB and metabolite diastereoselectivity of 3'(R)<3'(S) in 3'-hydroxylation of HB enantiomers. The substitution of Asp-293 by alanine failed to yield an observable peak at 450 nm in its reduced carbon monoxide-difference spectrum. CYP2C19-E300A and CYP2C19-E300V with alanine and valine, respectively, in place of Glu-300 exerted total HB 3'-hydroxylation activities of 45 and 108%, respectively, that of the wild-type. Interestingly, these two mutants showed substrate enantioselectivity of (R)-HB<(S)-HB, which is opposite to that of the wild-type, while metabolite diasteroselectivity remained unchanged. The replacement of Phe-476 by alanine increased total HB 3'-hydroxylation activity to approximately 3-fold that of the wild-type. Particularly, 3'(S)-OH-(S)-HB-forming activity elevated to 7-fold that of the wild-type, resulting in the reversal of the substrate enantioselectivity. In contrast, the substitution of phenylalanine at positions 100 and 114 by alanine did not produce a remarkable change in the total activity or the substrate enantioselectivity. These results indicate that Glu-300 and Phe-476 are important in stereoselective oxidation of HB enantiomers by CYP2C19.