FMO3 allelic variants in Sicilian and Sardinian populations: Trimethylaminuria and absence of fish-like body odor

The N-oxygenation of amines by the human flavin-containing monooxygenase (form 3) (FMO3) represents an important means for the conversion of lipophilic nucleophilic heteroatom-containing compounds into more polar and readily excreted products. In healthy individuals, virtually all Trimethylamine (TMA) are metabolized to Trimethylamine N-oxide (TMAO). Several single nucleotide polymorphisms (SNPs) of the FMO3 gene have been described and result in an enzyme with decreased or abolished functional activity for TMA N-oxygenation thus leading to TMAU, or fish-like odor syndrome. Three coding region variants, c. G472A (p.E158K) in exon 4, c. G769A (p.V257M) in exon 6, and c.A923G (p.E308G) in exon 7, are common polymorphisms identified in all population examined so far and are associated with normal or slightly reduced TMA N-oxygenation activity. However, simultaneous occurrence of 158K and 308G variants results in a more pronounced decrease in FMO3 activity. A fourth polymorphism, c. G1424A (p.G475D) in exon 9, less common in the general population, was observed in individuals suffering severe or moderate trimethylaminuria.     

Mutation, polymorphism and perspectives for the future of human flavin-containing monooxygenase 3

Flavin-containing monooxygenases (FMOs) catalyze NADPH-dependent monooxygenation of soft-nucleophilic nitrogen, sulfur, and phosphorous atoms contained within various drugs, pesticides, and xenobiotics. Flavin-containing monooxygenase 3 (FMO3) is responsible for the majority of FMO-mediated xenobiotic metabolism in the adult human liver. Mutations in the FMO3 gene can result in defective trimethylamine (TMA) N-oxygenation, which gives rise to the disorder known as trimethylaminuria (TMAU) or "fish-odour syndrome". To date 18 mutations of FMO3 gene have been reported that cause TMAU, and polymorphic variants of the gene have also been identified. Interindividual variability in the expression of FMO3 may affect drug and foreign chemical metabolism in the liver and other tissues. It is important therefore to study how base sequence variation of the FMO3 gene might affect the ability of individuals and different ethnic population groups to deal with the variety of environmental chemicals and pharmaceutical products that are substrates for FMO3.     

Allele and genotype frequencies of polymorphic FMO3 gene in two genetically distinct populations

The aims of this study were to analyze flavin-containing monooxygenase 3 (FMO3) polymorphisms and allele and genotype frequencies in 256 Han Chinese and 50 African-American individuals, to compare the allele and genotype frequencies of these populations with those of other world populations. For Han Chinese, genotyping of three common single nucleotide polymorphisms, E158K, V257M and E308G was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). For African-Americans, genotyping of all coding exons was performed by modified PCR-single strand conformational polymorphism (SSCP). Evolutionary rates of FMO3 were estimated computationally. We found that there were significant differences in allele and genotype frequencies among Han Chinese, African-Americans and other world populations. In Han Chinese, the minor allele frequencies (MAFs) were 0.229 (E158K), 0.203 (V257M) and 0.148 (E308G), respectively. In African-Americans, MAFs were 0.48 (E158K), 0.05 (V257M) and 0 (E308G), respectively. There was rapid evolution during the divergence of primate FMO3. This is the first report comparing FMO alleles and genotypes between Han Chinese and African-Americans. A Han Chinese population database has been established for three gene polymorphisms. The data presented here justify further pharmacogenetic studies for potentially optimizing recommended drug dosages and evaluating relationships with disease processes.     

Isolation of BamHI variants with reduced cleavage activities

Derivation of the bamhIR sequence (Brooks, J. E., Nathan, P.D., Landry, D., Sznyter, L.A., Waite-Rees, P., Ives, C. C., Mazzola, L. M., Slatko, B. E., and Benner, J. S. (1991) Nucleic Acids Res., in press), the gene coding for BamHI endonuclease, has facilitated construction of an Escherichia coli strain that overproduces BamHI endonuclease (W. E. Jack, L. Greenough, L. F. Dorner, S. Y. Xu, T. Strezelecka, A. K. Aggarwal, and I. Schildkraut, submitted for publication). As expected, low-level constitutive expression of the bamhIR gene in E. coli from the Ptac promotor construct is lethal to the host unless the bamHIM gene, which encodes the BamHI methylase, is also expressed within the cell. We identified four classes of BamHI endonuclease variants deficient in catalysis by selecting for survival of a host deficient for bamHIM gene, transformed with mutagenized copies of the bamhIR gene, and then screening the surviving cell extracts for DNA cleavage and binding activities. Class I variants (G56S, G91S/T153I, T114I, G130R, E135K, T153I, T157I, G194D) displayed 0.1-1% of the wild-type cleavage activity; class II variant (D94N) lacked cleavage activity but retained wild-type DNA binding specificity; class III variants (E77K, E113K) lacked cleavage activity but bound DNA more tightly; class IV variants (G56D, G90D, G91S, R122H, R155H) lacked both binding and cleavage activities. Variants with residual cleavage activities induced the E. coli SOS response and thus are presumed to cleave chromosomal DNA in vivo. We conclude that Glu77, Asp94, and Glu113 residues are essential for BamHI catalytic function.     

Sequencing, expression, and characterization of cDNA expressed flavin-containing monooxygenase 2 from mouse

The cDNA clone of mouse flavin-containing monooxygenase 2 (FMO2) was obtained as an expressed sequence tag (EST) isolated from a female mouse kidney cDNA library from the I.M.A.G.E. consortium (I.M.A.G.E. CloneID 1432164). Complete sequencing of the EST derived a nucleotide sequence for mouse FMO2, which contains 112 bases of 5' flanking region, 1607 bases of coding region, and 309 bases of 3' flanking region. This FMO2 sequence encodes a protein of 535 amino acids including two putative pyrophosphate binding sequences (GxGxxG/A) beginning at positions 9 and 191. Additionally, this mouse FMO protein sequence shows 87 and 86% homology to rabbit and human FMO2 respectively. The mouse FMO2 sequence was subcloned into the expression vector pJL-2, a derivative of pKK233-2 and used to transform XL1-Blue Escherichia coli. FMO activity in particulate fractions isolated from isopropyl-beta-D-thiogalactopyanoside (IPTG) induced cells was heat stable (45 degrees C for 5 min) and demonstrated optimal activity at a relatively high pH of 10.5. The expressed FMO2 enzyme showed catalytic activity towards the FMO substrate methimazole and further analysis of E. coli fractions utilizing NADPH oxidation demonstrated that the mouse FMO2 enzyme also exhibits catalytic activity towards thiourea, trimethylamine, and the insecticide phorate.     

Electrochemical sensing of trimethylamine based on polypyrrole-flavin-containing monooxygenase (FMO3) and ferrocene as redox probe for evaluation of fish freshness

Amperometric and impedimetric biosensor for detecting trimethylamine (TMA) which represents good parameters for estimating fish freshness has been developed. The biosensor is based on a conducting polypyrrole substituted with ferrocenyl, where flavin-containing monooxygenase 3 (FMO3) enzyme was immobilised by covalent bonding. FMO3 catalyzes the monooxygenation TMA to trimethylamine N-oxide (TMO). For catalysis FMO require flavin adenine (FAD) as a prosthetic group, NADPH as a cofactor and molecular oxygen as cosubstrate. Ferrocenyl group substituted on the polypyrrole matrix will serve as redox probe for monitoring the response of the biosensor to TMA. The construction of the biosensor was characterized by FT-IR, cyclic voltammetry and impedance measurements. Detection is done through the analysis of the current of oxidation signal of the ferrocenyl groups and compared to the measurement of impedance related to the electrical properties of the layers. Amperometric and impedimetric response were measured as a function of TMA concentration in range of 0.4 μgm L(-1)-80 μgm L(-1) (6.5 μmol L(-1)-1.5 mmol L(-1)). Amperometric measurements show a decrease in current response which is in correlation with the increase of the charge transfer resistance demonstrated by impedance. Calibration curve obtained by impedance spectroscopy shows a high sensitivity with a dynamic range from (0.4 μgm L(-1) to 80 μgm L(-1)). We demonstrated, using ferrocene as redox probe for catalytic reaction of FMO3, that high sensitivity and dynamic range was obtained. The biosensor was stable during 16 days. The biosensor shows high selectivity and its sensitivity to TMA in real samples was evaluated using fish extract after deterioration during storage.     

Haplotype frequency distribution and linkage disequilibrium analysis of single nucleotide polymorphisms at the human FMO3 gene locus

We analyzed flavin-containing monooxygenase 3 (FMO3) polymorphisms, haplotype structure, and linkage disequilibrium (LD) in 256 Han Chinese and 50 African-American individuals to compare their haplotype frequencies and LD with other world populations. For the Han Chinese, genotyping of three haplotype tag single nucleotide polymorphisms (E158K, V257M, and E308G) was performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism. For the African-Americans, genotyping of all coding exons was performed by modified PCR-single strand conformational polymorphism. Haplotype frequencies, LD, and evolutionary rates were inferred and estimated computationally. There were significant differences in haplotype frequency distribution and LD pattern among Han Chinese, African-Americans, and other world populations. Four major haplotypes of Han Chinese were EVE, KVE, EME, and EVG. Two major haplotypes of African-Americans were EVE and KVE. We found that sites 158 and 257 are in significant LD in both populations. This is the first report comparing FMO haplotypes and LD of Han Chinese with African-Americans. The data presented here justify further pharmacogenetic studies for potentially optimizing recommended drug dosages and evaluating relationships with disease processes.     

Fishy taint in chicken eggs is associated with a substitution within a conserved motif of the FMO3 gene

Fishy odor of urine and other secretions is a characteristic of trimethylaminuria in humans, resulting from loss-of-function mutations in the flavin-containing mono-oxygenase isoform FMO3. A similar phenotype exists in cattle, in which a nonsense mutation in the bovine orthologue causes fishy off-flavor in cow's milk. The fishy odor is caused by an elevated level of excreted odorous trimethylamine (TMA), due to deficient oxidation of TMA. We report the mapping of a similar disorder (fishy taint of eggs) and the chicken FMO3 gene to chicken chromosome 8. The only nonsynonymous mutation identified in the chicken FMO3 gene (T329S) changes an evolutionarily highly conserved amino acid and is associated with elevated levels of TMA and fishy taint in the egg yolk in several chicken lines. No differences in the expression of FMO3 were found among individuals with different associated genotypes, indicating that the trait is not caused by a linked polymorphism causing altered expression of the gene. The results support the importance and function of the evolutionarily conserved motif FATGY, which has been speculated to be a substrate recognition pocket of N-hydroxylating siderophore enzymes and flavin-containing mono-oxygenases.     

Characterization and suppression of dysfunctional human alpha1-antitrypsin variants

Human alpha1-antitrypsin-deficient variants may aggregate in the liver, with subsequent deficiency in the plasma, which can lead to emphysema. The structural and functional characteristics of 10 dysfunctional alpha1-antitrypsin variants (R39C, S53F, V55P, I92N, G115S, N158K, E264V, A336T, P369S, and P369L) were analyzed in detail. Most of them were unstable, as compared to the wild-type molecule, and many of the variants folded into an intermediate form. When five thermostable mutations (T68A, A70G, M374I, S381A, and K387R) were introduced into dysfunctional alpha1-antitrypsin variants, the stabilities and inhibitory activities of most of the variants were restored to levels comparable to those of the wild-type molecule. However, the extremely unstable S53F variant was not stabilized sufficiently by these mutations so as to exhibit function. N158K variant, which carries a mutation in the region critical for the reactive site loop insertion into beta-sheet A, exhibited a reduced level of inhibitory activity, despite conformational stabilization. These results show that aberrant folding caused by conformational destabilization due to mutations can be compensated for by increasing the overall stability of the alpha1-antitrypsin molecule, with exception of a mutation in the highly localized region critical for functional execution.     

Flavin containing monooxygenase 3 genetic polymorphisms Glu158Lys and Glu308Gly and their relation to ischemic stroke

Ischemic stroke is a multifactorial disease leading to severe long-term disability and it is the third leading cause of death in developed countries. Although many studies have been reported to elucidate etiological and pathological mechanisms of stroke, the genetic and molecular basis of disease remains poorly understood. Recent studies have shown that reactive oxygen species causing oxidative stress play a pivotal role in the pathogenesis of atherosclerosis that is the main cause of a group of cardiovascular diseases including ischemic stroke. In this study, we aimed to investigate the relationship between FMO3 Glu158Lys and Glu308Gly variants, and the risk of incidence of ischemic stroke in Turkish population. Two single nucleotide polymorphisms (SNPs) within the FMO3 gene were genotyped by using PCR-RFLP technique in a sample set of 245 cases and 145 controls. In the case-control analysis, no significant difference was observed between stroke patients and controls with respect to FMO3 Glu158Lys and Glu308Gly polymorphisms' genotype and allele frequency distribution. However, heterozygote 158Glu/Lys (OR = 6.110, P < 0.001) and 308Glu/Gly (OR = 6.000, P = 0.006) genotypes increase the risk of stroke 6 times in hypertensive subjects. On the other hand, the wild type genotypes 158Glu/Glu and 308Glu/Glu had 6.2-fold and 4.8-fold higher risk of ischemic stroke in obese subgroup, respectively. Our results clearly showed that the risk of hypertension-related ischemic stroke was higher in the heterozygote genotype carriers. This is the first study conducted regarding the association of FMO3 Glu158Lys and Glu308Gly genetic polymorphisms and ischemic stroke risk in Turkish population.     

Cytochrome b5 reductase: role of the si-face residues, proline 92 and tyrosine 93, in structure and catalysis

The conserved sequence motif "RxY(T)(S)xx(S)(N)" coordinates flavin binding in NADH:cytochrome b(5) reductase (cb(5)r) and other members of the flavin transhydrogenase superfamily of oxidoreductases. To investigate the roles of Y93, the third and only aromatic residue of the "RxY(T)(S)xx(S)(N)" motif, that stacks against the si-face of the flavin isoalloxazine ring, and P92, the second residue in the motif that is also in close proximity to the FAD moiety, a series of rat cb(5)r variants were produced with substitutions at either P92 or Y93, respectively. The proline mutants P92A, G, and S together with the tyrosine mutants Y93A, D, F, H, S, and W were recombinantly expressed in E. coli and purified to homogeneity. Each mutant protein was found to bind FAD in a 1:1 cofactor:protein stoichiometry while UV CD spectra suggested similar secondary structure organization among all nine variants. The tyrosine variants Y93A, D, F, H, and S exhibited varying degrees of blue-shift in the flavin visible absorption maxima while visible CD spectra of the Y93A, D, H, S, and W mutants exhibited similar blue-shifted maxima together with changes in absorption intensity. Intrinsic flavin fluorescence was quenched in the wild type, P92S and A, and Y93H and W mutants while Y93A, D, F, and S mutants exhibited increased fluorescence when compared to free FAD. The tyrosine variants Y93A, D, F, and S also exhibited greater thermolability of FAD binding. The specificity constant (k(cat)/K(m)(NADH)) for NADH:FR activity decreased in the order wild type > P92S > P92A > P92G > Y93F > Y93S > Y93A > Y93D > Y93H > Y93W with the Y93W variant retaining only 0.5% of wild-type efficiency. Both K(s)(H4NAD) and K(s)(NAD+) values suggested that Y93A, F, and W mutants had compromised NADH and NAD(+) binding. Thermodynamic measurements of the midpoint potential (E degrees ', n = 2) of the FAD/FADH(2) redox couple revealed that the potentials of the Y93A and S variants were approximately 30 mV more positive than that of wild-type cb(5)r (E degrees ' = -268 mV) while that of Y93H was approximately 30 mV more negative. These results indicate that neither P92 nor Y93 are critical for flavin incorporation in cb(5)r and that an aromatic side chain is not essential at position 93, but they demonstrate that Y93 forms contacts with the FAD that effectively modulate the spectroscopic, catalytic, and thermodynamic properties of the bound cofactor.     

alpha Arg-237 in Methylophilus methylotrophus (sp. W3A1) electron-transferring flavoprotein affords approximately 200-millivolt stabilization of the FAD anionic semiquinone and a kinetic block on full reduction to the dihydroquinone

The midpoint reduction potentials of the FAD cofactor in wild-type Methylophilus methylotrophus (sp. W3A1) electron-transferring flavoprotein (ETF) and the alphaR237A mutant were determined by anaerobic redox titration. The FAD reduction potential of the oxidized-semiquinone couple in wild-type ETF (E'(1)) is +153 +/- 2 mV, indicating exceptional stabilization of the flavin anionic semiquinone species. Conversion to the dihydroquinone is incomplete (E'(2) < -250 mV), because of the presence of both kinetic and thermodynamic blocks on full reduction of the FAD. A structural model of ETF (Chohan, K. K., Scrutton, N. S., and Sutcliffe, M. J. (1998) Protein Pept. Lett. 5, 231-236) suggests that the guanidinium group of Arg-237, which is located over the si face of the flavin isoalloxazine ring, plays a key role in the exceptional stabilization of the anionic semiquinone in wild-type ETF. The major effect of exchanging alphaArg-237 for Ala in M. methylotrophus ETF is to engineer a remarkable approximately 200-mV destabilization of the flavin anionic semiquinone (E'(2) = -31 +/- 2 mV, and E'(1) = -43 +/- 2 mV). In addition, reduction to the FAD dihydroquinone in alphaR237A ETF is relatively facile, indicating that the kinetic block seen in wild-type ETF is substantially removed in the alphaR237A ETF. Thus, kinetic (as well as thermodynamic) considerations are important in populating the redox forms of the protein-bound flavin. Additionally, we show that electron transfer from trimethylamine dehydrogenase to alphaR237A ETF is severely compromised, because of impaired assembly of the electron transfer complex.     

Diminished FAD binding in the Y459H and V492E Antley-Bixler syndrome mutants of human cytochrome P450 reductase

Numerous mutations/polymorphisms of the POR gene, encoding NADPH:cytochrome P450 oxidoreductase (CYPOR), have been described in patients with Antley-Bixler syndrome (ABS), presenting with craniofacial dysmorphogenesis, and/or disordered steroidogenesis, exhibiting ambiguous genitalia. CYPOR is the obligate electron donor to 51 microsomal cytochromes P450 that catalyze critical steroidogenic and xenobiotic reactions, and to two heme oxygenase isoforms, among other redox partners. To address the molecular basis of CYPOR dysfunction in ABS patients, the soluble catalytic domain of human CYPOR was bacterially expressed. WT enzyme was green, due to air-stable FMN semiquinone (blue) and oxidized FAD (yellow). The ABS mutant V492E was blue-gray. Flavin analysis indicated that WT had a protein:FAD:FMN ratio of approximately 1:1:1, whereas approximately 1:0.1:0.9 was observed for V492E, which retained 9% of the WT k(cat)/K(m) in NADPH:cytochrome c reductase assays. V492E was reconstituted upon addition of FAD, post-purification, as shown by flavin analysis, activity assay, and near UV-visible CD. Both Y459H and V492E were expressed as membrane anchor-containing proteins, which also exhibited FAD deficiency. CYP4A4-catalyzed omega-hydroxylation of prostaglandin E1 was supported by WT CYPOR but not by either of the ABS mutants. Hydroxylation activity was rescued for both Y459H and V492E upon addition of FAD to the reaction. Based on these findings, decreased FAD-binding affinity is proposed as the basis of the observed loss of CYPOR function in the Y459H and V492E POR mutations in ABS.     

A peptide from the eel pancreas with structural similarity to human pancreatic secretory trypsin inhibitor

The primary structure of a 61-amino-acid residue peptide from the pancreas of the European eel (Anguilla anguilla) has been established as E E K S G(5)L Y R K P(10)S C G E M(15)S A M H A(20)C P M N F(25)A P V C G(30)T D G N T(35)Y P N E C(40)S L C F Q(45)R Q N T K(50)T D I L I(55)T K D D R(60)C. There was no indication of microheterogeneity. This peptide shows structural similarity to pancreatic secretory trypsin inhibitors from several mammalian species and to a cholecystokinin-releasing peptide isolated from rat pancreatic juice. A comparison of the amino acid sequences of the peptides has identified a domain in the central region of the molecules that has been strongly conserved during evolution. In contrast, the amino acid sequence in the region corresponding to the reactive centre of the mammalian trypsin inhibitors is very poorly conserved in the eel peptide. The P1-P1' reactive site lysine-isoleucine (or arginine-isoleucine) bond in the mammalian trypsin inhibitors is replaced by a methionine-asparagine bond. This region does, however, show limited homology to the reactive centre of human alpha 1-protease inhibitor suggesting that the eel peptide may function as an inhibitor of other proteolytic enzymes in the pancreas.     

Effects of Specific Amino Acid Substitutions on Activities of Dinitrogenase Reductase-Activating Glycohydrolase from Rhodospirillum rubrum

Site-directed mutagenesis of the draG gene was used to generate altered forms of dinitrogenase reductase-activating glycohydrolase (DRAG) with D123A, H142L, H158N, D243G, and E279R substitutions. The amino acid residues H142 and E279 are not required either for the coordination to the metal center or for catalysis since the variants H142L and E279R retained both catalytic and electron paramagnetic resonance spectral properties similar to those of the wild-type enzyme. Since DRAG-H158N and DRAG-D243G variants lost their ability to bind Mn(II) and to catalyze the hydrolysis of the substrate, H158 and D243 residues could be involved in the coordination of the binuclear Mn(II) center in DRAG.     

Haplotype Frequency Distribution and Linkage Disequilibrium Analysis of Single Nucleotide Polymorphisms at the Human FMO3 Gene Locus

We analyzed flavin-containing monooxygenase 3 (FMO3) polymorphisms, haplotype structure, and linkage disequilibrium (LD) in 256 Han Chinese and 50 African-American individuals to compare their haplotype frequencies and LD with other world populations. For the Han Chinese, genotyping of three haplotype tag single nucleotide polymorphisms (E158K, V257M, and E308G) was performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism. For the African-Americans, genotyping of all coding exons was performed by modified PCR-single strand conformational polymorphism. Haplotype frequencies, LD, and evolutionary rates were inferred and estimated computationally. There were significant differences in haplotype frequency distribution and LD pattern among Han Chinese, African-Americans, and other world populations. Four major haplotypes of Han Chinese were EVE, KVE, EME, and EVG. Two major haplotypes of African-Americans were EVE and KVE. We found that sites 158 and 257 are in significant LD in both populations. This is the first report comparing FMO haplotypes and LD of Han Chinese with African-Americans. The data presented here justify further pharmacogenetic studies for potentially optimizing recommended drug dosages and evaluating relationships with disease processes.     

Molecular cloning, sequence characterization, SNP detection, and tissue expression analysis of duck FMO3 gene

Flavin-containing monooxygenase 3 (FMO3) is an important monooxygenase for catalytic oxygenation of many harmful xenobiotics. Mutations in the FMO3 gene have been identified as causing trimethylaminuria in human and fishy off-flavor in cow milk and chicken eggs. In this study, the full-length cDNA sequence of Pekin duck FMO3 gene was cloned, sequenced, and characterized. The full-length cDNA sequence consisted of 1,846 bp and contained a 1,599 bp open-reading frame encoding 532 amino acids. Duck FMO3 gene shared a similar nine exon–eight intron structure with chicken and human. The duck FMO3 putative protein sequence showed high identity with that of chicken (82 %), and relative low identity with those of mammals (61–66 %). We also found that the duck FMO3 gene was dramatically expressed in liver, lung, and kidney compared to that in other tissues in the ducks, indicating the possible roles the FMO3 gene could play in the three tissues. By bidirectional sequencing, we also found one nonsense mutation, 5 nonsynonymous, and 21 synonymous mutations in the coding region of the FMO3 gene in 11 duck breeds and some of them were predicted to be potentially associated with the activities of FMO3 protein.     

Differential contributions of NADPH-cytochrome P450 oxidoreductase FAD binding site residues to flavin binding and catalysis

Transfer of reducing equivalents from NADPH to the cytochromes P450 is mediated by NADPH-cytochrome P450 oxidoreductase, which contains stoichiometric amounts of tightly bound FMN and FAD. Hydrogen bonding and van der Waals interactions between FAD and amino acid residues in the FAD binding site of the reductase serve to regulate both flavin binding and reactivity. The precise orientation of key residues (Arg(454), Tyr(456), Cys(472), Gly(488), Thr(491), and Trp(677)) has been defined by x-ray crystallography (Wang, M., Roberts, D. L., Paschke, R., Shea, T. M., Masters, B. S., Kim, J.-J. P. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 8411-8416). The current study examines the relative contributions of these residues to FAD binding and catalysis by site-directed mutagenesis and kinetic analysis. Mutation of either Tyr(456), which makes van der Waals contact with the FAD isoalloxazine ring and also hydrogen-bonds to the ribityl 4'-hydroxyl, or Arg(454), which bonds to the FAD pyrophosphate, decreases the affinity for FAD 8000- and 25,000-fold, respectively, with corresponding decreases in cytochrome c reductase activity. In contrast, substitution of Thr(491), which also interacts with the pyrophosphate grouping, had a relatively modest effect on both FAD binding (100-fold decrease) and catalytic activity (2-fold decrease), while the G488L mutant exhibited, respectively, 800- and 50-fold decreases in FAD binding and catalytic activity. Enzymic activity of each of these mutants could be restored by addition of FAD. Kinetic properties and the FMN content of these mutants were not affected by these substitutions, with the exception of a 3-fold increase in Y456S K(m)(cyt )(c) and a 70% decrease in R454E FMN content, suggesting that the FMN- and FAD-binding domains are largely, but not completely, independent. Even though Trp(677) is stacked against the re-face of FAD, suggesting an important role in FAD binding, deletion of both Trp(677) and the carboxyl-terminal Ser(678) decreased catalytic activity 50-fold without affecting FAD content.     

Structural basis of thermostability. Analysis of stabilizing mutations in subtilisin BPN'

The crystal structures of two thermally stabilized subtilisin BPN' variants, S63 and S88, are reported here at 1.8 and 1.9 A resolution, respectively. The micromolar affinity calcium binding site (site A) has been deleted (Delta75-83) in these variants, enabling the activity and thermostability measurements in chelating conditions. Each of the variants includes mutations known previously to increase the thermostability of calcium-independent subtilisin in addition to new stabilizing mutations. S63 has eight amino acid replacements: D41A, M50F, A73L, Q206W, Y217K, N218S, S221C, and Q271E. S63 has 75-fold greater stability than wild type subtilisin in chelating conditions (10 mm EDTA). The other variant, S88, has ten site-specific changes: Q2K, S3C, P5S, K43N, M50F, A73L, Q206C, Y217K, N218S, and Q271E. The two new cysteines form a disulfide bond, and S88 has 1000 times greater stability than wild type subtilisin in chelating conditions. Comparisons of the two new crystal structures (S63 in space group P2(1) with A cell constants 41.2, 78.1, 36.7, and beta = 114.6 degrees and S88 in space group P2(1)2(1)2(1) with cell constants 54.2, 60.4, and 82.7) with previous structures of subtilisin BPN' reveal that the principal changes are in the N-terminal region. The structural bases of the stabilization effects of the new mutations Q2K, S3C, P5S, D41A, Q206C, and Q206W are generally apparent. The effects are attributed to the new disulfide cross-link and to improved hydrophobic packing, new hydrogen bonds, and other rearrangements in the N-terminal region.     

New insights into the role of the N terminus in conformational transitions of the Na,K-ATPase

The deletion of 32 residues from the N terminus of the alpha1 catalytic subunit of the rat Na,K-ATPase (mutant alpha1M32) shifts the E(1)/E(2) conformational equilibrium toward E(1), and the combination of this deletion with mutation E233K in the M2-M3 loop acts synergistically to shift the conformation further toward E(1) (Boxenbaum, N., Daly, S. E., Javaid, Z. Z., Lane, L. K., and Blostein, R. (1998) J. Biol. Chem. 273, 23086-23092). To delimit the region of the cytoplasmic N terminus involved in these interactions, the consequences of a series of N-terminal deletions of alpha1 beyond Delta32 were evaluated. Criteria to assess shifts in conformational equilibrium were based on effects of perturbation of the entire catalytic cycle ((i) sensitivity to vanadate inhibition, (ii) K(+) sensitivity of Na-ATPase measured at micromolar ATP, (iii) changes in K'(ATP), and (iv) catalytic turnover), as well as estimates of the rates of the conformational transitions of phospho- and dephosphoenzyme (E(1)P --> E(2)P and E(2)(K(+)) --> E(1) + K(+)). The results show that, compared with alpha1M32, the deletion of up to 40 residues (alpha1M40) further shifts the poise toward E(1). Remarkably, further deletions (mutants alpha1M46, alpha1M49, and alpha1M56) reverse the effect, such that these mutants increasingly resemble the wild type alpha1. These results suggest novel intramolecular interactions involving domains within the N terminus that impact the manner in which the N terminus/M2-M3 loop regulatory domain interacts with the M4-M5 catalytic loop to effect E(1) <--> E(2) transitions.