Evaluation of the role of His447 in the reaction catalyzed by cholesterol oxidase

Cholesterol oxidase catalyzes the oxidation and isomerization of cholesterol to cholest-4-en-3-one via cholest-5-en-3-one. It has been proposed that His447 acts as the general base catalyst for oxidation, and that the resulting imidazolium ion formed acts as an electrophile for isomerization. In this work, we undertook an assessment of the proposed dual roles of His447 in the oxidation and isomerization reactions. To test its role, we constructed five mutants, H447Q, H447N, H447E, H447D, and H447K, that introduce hydrogen bond donors and acceptors and carboxylate bases at this position, and a sixth mutant, E361Q, to test the interplay between His447 and Glu361. These mutants were characterized using steady-state kinetics and deuterium substrate and solvent isotope effects. For those mutants that catalyze either oxidation of cholesterol or isomerization of cholest-5-en-3-one, the Km's vary no more than 3-fold relative to wild type. H447K is inactive in both oxidation (> 100,000-fold reduced) and isomerization assays (> 10,000-fold reduced). H447E and H447D do not catalyze oxidation (> 100,000-fold reduced), but do catalyze isomerization, 10(4) times slower than wild type. The k(cat) for H447Q is 120-fold lower than wild type for oxidation, and the same as wild type for isomerization. The k(cat) for H447N is 4400-fold lower than wild type for oxidation, and is 30-fold lower than wild type for isomerization. E361Q does not catalyze isomerization (> 10,000-fold reduced), and the k(cat) for oxidation is 30-fold lower than wild type. The substrate deuterium kinetic isotope effects for the wild-type and mutant-catalyzed oxidation reactions suggest that mutation of His447 to an amide results in a change of the rate-determining step from hydride transfer to hydroxyl deprotonation. The deuterium solvent and substrate kinetic isotope effects for isomerization indicate that an amide at position 447 is an effective electrophile to catalyze formation of a dienolic intermediate. Moreover, consideration of kinetic and structural results together suggests that a hydrogen bonding network involving His447, Glu361 and Asn485, Wat541, and substrate serves to position the substrate and coordinate general base and electrophilic catalysis. That is, in addition to its previously demonstrated role as base for deprotonation of carbon-4 during isomerization, Glu361 has a structural role and may act as a general base during oxidation. The His447, Asn485, Glu361, and Wat541 residues are conserved in other GMC oxidoreductases. Observation of this catalytic tetrad in flavoproteins of unknown function may be diagnostic for an ability to oxidize unactivated alcohols.     

Investigation of membrane disruption in the reaction catalyzed by cholesterol oxidase

Dye leakage experiments were undertaken to investigate the membrane disruption properties of cholesterol oxidase. Inspection of the X-ray crystal structures of cholesterol oxidase suggested that an active-site "lid" opens in order to bind substrate [Li, J., Vrielink, A., Brick, P., & Blow, D. M. (1993) Biochemistry 32, 11507-11515]. We tested whether the interaction of the putative active-site lid with the membrane was sufficiently disruptive of the membrane structure to cause leakage or lysis of the cell membrane. Vesicles (100 nm) composed of egg phosphatidylcholine, 2-palmitoyl-3-oleoyl-1-sn-phosphatidylethanolamine, and 2-palmitoyl-3-oleoyl-1-sn-phosphatidylcholine were used in this study to mimic biomembranes. To separate the effects of membrane binding from conversion of cholesterol to cholest-4-en-3-one, the active-site mutant E361Q was utilized. In the reaction catalyzed by E361Q, isomerization of the cholest-5-en-3-one intermediate is suppressed and cholest-5-en-3-one is the major product isolated. Furthermore, E361Q produces cholest-5-en-3-one 20-fold more slowly than wild type produces cholest-4-en-3-one from cholesterol. Wild-type and E361Q cholesterol oxidases bind to vesicles with an apparent K(D) of approximately 25 microM, as measured by quenching of intrinsic tryptophan fluorescence, irrespective of headgroup size and cholesterol content. Membrane disruption was measured by leakage of the encapsulated marker carboxyfluorescein. Leakage was observed with cholesterol-containing vesicles and wild-type enzyme only; the rate of leakage was dependent on the rate of cholest-4-en-3-one production. E361Q did not induce membrane disruption, regardless of vesicle type tested. Thus, binding of cholesterol oxidase to the membrane and partitioning of cholesterol into the active site does not sufficiently perturb the bilayer to cause leakage of vesicle contents. Formation of the product cholest-4-en-3-one, however, does increase membrane permeability. Expansion of the lipid bilayer upon conversion of cholesterol to cholest-4-en-3-one is the likely cause of this increased permeability.     

Metal-catalyzed oxidation and mutagenesis studies on the iron(II) binding site of 1-aminocyclopropane-1-carboxylate oxidase

The final step in the biosynthesis of the plant signaling molecule ethylene is catalyzed by 1-aminocyclopropane-1-carboxylate (ACC) oxidase, a member of the non-heme iron(II) dependent family of oxygenases and oxidases, which has a requirement for ascorbate as a co-substrate and carbon dioxide as an activator. ACC oxidase (tomato) has a particularly short half-life under catalytic conditions undergoing metal-catalyzed oxidative (MCO) fragmentation. Sequence comparisons of ACC oxidases with isopenicillin N synthase (IPNS) and members of the 2-oxoglutarate Fe(II) dependent dioxygenases show an aspartate and two of six ACC oxidase conserved histidine residues are completely conserved throughout this subfamily of Fe(II) dependent oxygenases/oxidases. Previous mutagenesis, spectroscopic, and crystallographic studies on IPNS indicate that the two completely conserved histidine and aspartate residues act as Fe(II) ligands. To investigate the role of the conserved aspartate and histidine residues in ACC oxidase (tomato fruit), they were substituted via site-directed mutagenesis. Modified ACC oxidases produced were H39Q, H56Q, H94Q, H177Q, H177D, H177E, D179E, D179N, H177D&D179E, H211Q, H234Q, H234D, and H234E. Among those histidine mutants replaced by glutamine, H39Q, H56Q, H94Q, and H211Q were catalytically active, indicating these histidines are not essential for catalysis. Mutant enzymes H177D, H177Q, D179N, H177D&D179E, H234Q, H234D, and H234E were catalytically inactive consistent with the assignment of H177, D179, and H234 as iron ligands. Replacement of H177 with glutamate or D179 with glutamate resulted in modified ACC oxidases which still effected the conversion of ACC to ethylene, albeit at a very low level of activity, which was stimulated by bicarbonate. The H177D (inactive), H177E (low activity), D179E (low activity), and H234Q (inactive) modified ACC oxidases all underwent MCO fragmentation, indicating that they can bind iron, dioxygen, ACC, and ascorbate. The results suggest that MCO cleavage results from active site-mediated reactions and imply that, while H177, D179, and H234 are all involved in metal ligation during catalysis, ligation to H234 is not required for fragmentation. It is possible that MCO fragmentation results from reaction of incorrectly folded or "primed" ACC oxidase.     

The active site of hamster 3-hydroxy-3-methylglutaryl-CoA reductase resides at the subunit interface and incorporates catalytically essential acidic residues from separate polypeptides

We employed site-directed mutagenesis based on sequence comparisons and characterization of purified mutant enzymes to identify Glu558 and Asp766 of Syrian hamster 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) as essential for catalysis. Mutant enzymes E558D, E558Q, and D766N had wild-type Km values for (S)-HMG-CoA and NADPH, but exhibited less than 0.5% of the wild-type catalytic activity. The inactive mutant polypeptides E558Q and D766N nevertheless can associate to generate an active enzyme. In vitro, 6% of the wild-type activity was observed when mutant polypeptides E558D and D766N were mixed in the absence of chaotropic agents. When mutant polypeptides E558Q and D766N were co-expressed in Escherichia coli, the resulting purified enzyme had 25% of wild-type activity. Hamster HMG-CoA reductase thus is a two-site, dimeric enzyme whose subunits associate to form an active site in which each monomer contributes at least one residue (e.g. Glu558 from one monomer and Asp766 from the other). The wild-type enzyme behaves as a dimer during size exclusion chromatography and has one HMG-CoA binding site per monomer. Syrian hamster HMG-CoA reductase thus appears to be a homodimer with two active sites which are located at the subunit interface.     

Isolation and characterization of the heat-responsive genes in Escherichia coli

Isocitrate lyase from Escherichia coli has been expressed in transformed E. coli JE10 cells lacking the isocitrate lyase (icl) gene. After directed mutagenesis of icl by the restriction-site elimination method, partially purified isocitrate lyase mutants in which His 356 has been converted to Lys, Arg, Gln, Asp, or Leu have been characterized after induction of transformed, induced JE10 cells. Values of kcat compared to those for wild-type (wt) enzyme (100) at 37 degrees C, pH 7.3, are 18, 1, <1, 0, and 0 for H356K, H356R, H356E, H356Q, and H356L mutant enzymes, respectively. Km values for the 1:1 Mg-isocitrate complex (in millimolar units) are: 0.13, wt; 0.11, H356K; and 0.63, H356R. Further chromatographic purification of isocitrate lyase yields highly purified wt, H356K, and H356R enzymes. The pH profile of the stability of isocitrate lyase, which has never been reported, showed that the H356R enzyme was unstable in the pH range investigated; the wt and H356R variant differed but each was sufficiently stable to study the pH dependence of catalysis. The log kcat/pH profiles for highly purified wt and H356K enzymes are roughly bell-shaped and have pKa and pKb values for dissociation of an ionizable group on the enzyme-substrate complex of <6.3 and 8.4 for wt and 5.9 and 7.9 for H356K enzymes. Plots of pKm vs pH were different for the wt and H356K variant. Values of pKa and pKb (derived from log kcat/Km plots vs pH) for the dissociation of an activity-related ionizable group on the variant were 5.3 and 7.6, whereas the analogous pKb value for the wt enzyme was 8.4. The data suggest that His 356 is an important functional residue in isocitrate lyase, perhaps in deprotonating isocitrate during catalytic cleavage.     

Mechanism of phosphatidylinositol-specific phospholipase C: a unified view of the mechanism of catalysis

The mechanism of phosphatidylinositol-specific phospholipase C (PI-PLC) has been suggested to resemble that of ribonuclease A. The goal of this work is to rigorously evaluate the mechanism of PI-PLC from Bacillus thuringiensis by examining the functional and structural roles of His-32 and His-82, along with the two nearby residues Asp-274 and Asp-33 (which form a hydrogen bond with His-32 and His-82, respectively), using site-directed mutagenesis. In all, twelve mutants were constructed, which, except D274E, showed little structural perturbation on the basis of 1D NMR and 2D NOESY analyses. The H32A, H32N, H32Q, H82A, H82N, H82Q, H82D, and D274A mutants showed a 10(4)-10(5)-fold decrease in specific activity toward phosphatidylinositol; the D274N, D33A, and D33N mutants retained 0. 1-1% activity, whereas the D274E mutant retained 13% activity. Steady-state kinetic analysis of mutants using (2R)-1, 2-dipalmitoyloxypropane-3-(thiophospho-1d-myo-inositol) (DPsPI) as a substrate generally agreed well with the specific activity toward phosphatidylinositol. The results suggest a mechanism in which His-32 functions as a general base to abstract the proton from 2-OH and facilitates the attack of the deprotonated 2-oxygen on the phosphorus atom. This general base function is augmented by the carboxylate group of Asp-274 which forms a diad with His-32. The H82A and D33A mutants showed an unusually high activity with substrates featuring low pKa leaving groups, such as DPsPI and p-nitrophenyl inositol phosphate (NPIPs). These results suggest that His-82 functions as the general acid with assistance from Asp-33, facilitating the departure of the leaving group by protonation of the glycerol O3 oxygen. The Bronsted coefficients obtained for the WT and the D33N mutant indicate a high degree of proton transfer to the leaving group and further underscore the "helper" function of Asp-33. The complete mechanism also includes activation of the phosphate group toward nucleophilic attack by a hydrogen bond between Arg-69 and a nonbridging oxygen atom. The overall mechanism can be described as "complex" general acid-general base since three elements are required for efficient catalysis.     

Involvement of glutamate 399 and lysine 192 in the mechanism of human liver mitochondrial aldehyde dehydrogenase

Mutation to the conserved Glu399 or Lys192 caused the rate-limiting step of human liver mitochondrial aldehyde dehydrogenase (ALDH2) to change from deacylation to hydride transfer (Sheikh, S., Ni, L., Hurley, T. D., and Weiner, H. (1997) J. Biol. Chem. 272, 18817-18822). Here we further investigated the role of these two NAD+-ribose-binding residues. The E399Q/K/H/D and K192Q mutants had lower dehydrogenase activity when compared with the native enzyme. No pre-steady state burst of NADH formation was found with the E399Q/K and K192Q enzymes when propionaldehyde was used as the substrate; furthermore, each mutant oxidized chloroacetaldehyde slower than propionaldehyde, and a primary isotope effect was observed for each mutant when [2H]acetaldehyde was used as a substrate. However, no isotope effect was observed for each mutant when alpha-[2H]benzaldehyde was the substrate. A pre-steady state burst of NADH formation was observed for the E399Q/K and K192Q mutants with benzaldehyde, and p-nitrobenzaldehyde was oxidized faster than benzaldehyde. Hence, when aromatic aldehydes were used as substrates, the rate-limiting step remained deacylation for all these mutants. The rate-limiting step remained deacylation for the E399H/D mutants when either aliphatic or aromatic aldehydes were used as substrates. The K192Q mutant displayed a change in substrate specificity, with aromatic aldehydes becoming better substrates than aliphatic aldehydes.     

Identification of N,N'-dicyclohexylcarbodiimide-reactive glutamic and aspartic acid residues in Escherichia coli transhydrogenase and the exchange of these by site-specific mutagenesis

Pyridine nucleotide transhydrogenase (EC 1.6.1.1) from Escherichia coli was investigated with respect to the role of glutamic and aspartic acid residues reactive to N,N'-dicyclohexylcarbodiimide (DCCD) and potentially involved in the proton-pumping mechanism of the enzyme. The E. coli transhydrogenase consists of an alpha (510 residues) and a beta (462 residues) subunit. DCCD reacts with the enzyme to inhibit catalytic activity and proton pumping. This reagent modifies Asp alpha 232, Glu alpha 238, and Glu alpha 240 as well as amino acid residue(s) in the beta subunit. Using the cloned and overexpressed E. coli transhydrogenase genes (Clarke, D. M., and Bragg, P. D. (1985) J. Bacteriol. 162, 367-373), Asp alpha 232 and Glu alpha 238 were replaced independently by site-specific mutagenesis. In addition, Asp alpha 232, Glu alpha 238, and Glu alpha 240 were replaced to generate triple mutants. The specific catalytic activities of the mutant transhydrogenases alpha D232N, alpha D232E, alpha D232K, alpha D232H, alpha E238K, and alpha E238Q as well as of the triple mutants alpha D232N, alpha E238Q, alpha E240Q and alpha D232H, alpha E238Q, alpha E240Q were in the range of 40-90% of the wild-type activity. Proton-pumping activity was present in all mutants. Examination of the extent of subunit modification by [14C]DCCD revealed that the label was still incorporated into both alpha and beta subunits in the Asp alpha 232 mutants, but that the alpha subunit was not labeled in the triple mutants. Catalytic and proton-pumping activities were nearly insensitive to DCCD in the triple mutants. This suggests that loss of catalytic and proton-pumping activities is associated with modification of the aspartic and glutamic acid residues of the alpha subunit. In the presence of the substrate NADPH, the rate of modification of the beta subunit by [14C]DCCD was increased, and there was a greater extent of enzyme inactivation. By contrast, NADH and 3-acetylpyridine-NAD+ protected the catalytic activity of the transhydrogenase from inhibition by DCCD. The protection was particularly marked in the E238Q and E238K mutants. It is concluded that the Asp alpha 232, Glu alpha 238, and Glu alpha 240 residues are not essential for catalytic activity or proton pumping. The inactivation by DCCD is likely due to the introduction of a sterically hindering group that reacts with the identified acidic residues close to the NAD(H)-binding site.     

Mutational analysis of the role of the distal histidine and glutamine residues of prostaglandin-endoperoxide synthase-2 in peroxidase catalysis, hydroperoxide reduction, and cyclooxygenase activation

Site-directed mutants of prostaglandin-endoperoxide synthase-2 (PGHS-2) with changes in the peroxidase active site were prepared by mutagenesis, expressed in Sf-9 cells, and purified to homogeneity. The distal histidine, His193, was mutated to alanine and the distal glutamine, Gln189, was changed to asparagine, valine, and arginine. The guaiacol peroxidase activities of H193A, Q189V, and Q189R were drastically reduced to levels observed in the absence of protein; only Q189N retained wild-type PGHS-2 (wtPGHS-2) activity. The mechanism of hydroperoxide reduction by the PGHS-2 mutants was investigated using 15-hydroperoxyeicosatetraenoic acid (15-HPETE), a diagnostic probe of hydroperoxide reduction pathways. The hydroperoxide reduction activity of Q189V and Q189R was reduced to that of free Fe(III) protoporphyrin IX levels, whereas Q189N catalyzed more reduction events than wtPGHS-2. The percentage of two-electron reduction events was identical for wtPGHS-2 and Q189N. The number of hydroperoxide reductions catalyzed by H193A was reduced to approximately 60% of wtPGHS-2 activity, but the majority of products were the one-electron reduction products, 15-KETE and epoxyalcohols. Thus, mutation of the distal histidine to alanine leads to a change in the mechanism of hydroperoxide reduction. Reaction of wtPGHS-2, Q189N, and H193A with varying concentrations of 15-HPETE revealed a change in product profile that suggests that 15-HPETE can compete with the reducing substrate for oxidation by the peroxidase higher oxidation state, compound I. The ability of the PGHS-2 proteins to catalyze two-electron hydroperoxide reduction correlated with the activation of cyclooxygenase activity. The reduced ability of H193A to catalyze two-electron hydroperoxide reduction resulted in a substantial lag phase in the cyclooxygenase assay. The addition of 2-methylimidazole chemically reconstituted the two-electron hydroperoxide reduction activity of H193A and abolished the cyclooxygenase lag phase. These observations are consistent with the involvement of the two-electron oxidized peroxidase intermediate, compound I, as the mediator of the activation of the cyclooxygenase of PGHS.     

Spectroscopic evidence for interaction between transmembrane helices 3 and 5 in rhodopsin

Recent molecular models of rhodopsin (Rho) propose a specific interaction between transmembrane (TM) helices 3 and 5, which appears to be mediated by amino acid residues Glu122 and His211 on TM helices 3 and 5, respectively. To test this proposed interaction, four single-site histidine replacement mutants (H100N, H152N, H211N, and H211F), two single-site glutamic acid replacement mutants (E122Q and E122A), and three double-site replacement mutants (E122Q/H211F, E122Q/H211N, and E122A/H211F) of Rho were prepared. The expressed mutant pigments reconstituted into membranes were studied by FTIR difference spectroscopy addressing especially the transition to metarhodopsin I (MI). It is shown that the lipid environment influences bands typical of the MI state. Spectra of mutants with substituted Glu122 allowed assignments of the C=O stretch of protonated Glu122 in the dark state and in MI of Rho. Mutation of His211, but not of other histidine residues, affects these vibrational modes assigned to Glu122. In addition, replacements of His211 affect protein modes that are proposed to arise from a third, hydroxyl-bearing group, which also interacts with Glu122. These modes are influenced as well when Glu122 is replaced by Ala in mutant E122A but not when it is replaced by Gln in mutant E122Q. These results provide direct experimental evidence for an interaction between TM helices 3 and 5 in Rho, which is mediated by Glu122 and His211.     

Histidine77, glutamic acid81, glutamic acid123, threonine126, asparagine194, and tryptophan197 of the human emopamil binding protein are required for in vivo sterol delta 8-delta 7 isomerization

The human emopamil binding protein (hEBP) exhibits sterol Delta8-Delta7 isomerase activity (EC 5.3.3.5) upon heterologous expression in a sterol Delta8-Delta7 isomerization-deficient erg2-3 yeast strain. Ala scanning mutagenesis was used to identify residues in the four putative transmembrane alpha-helices of hEBP that are required for catalytic activity. Isomerization was assayed in vivo by spectrophotometric quantification of Delta5,7-sterols. Out of 64 Ala mutants of hEBP only H77A-, E81A-, E123A-, T126A-, N194A-, and W197A-expressing yeast strains contained 10% or less of wild-type (wt) Delta5,7-sterols. All substitutions of these six residues with functionally or structurally similar amino acid residues failed to fully restore catalytic activity. Mutants E81D, T126S, N194Q, and W197F, but not H77N and E123D, still bound the enzyme inhibitor 3H-ifenprodil. Changed equilibrium and kinetic binding properties of the mutant enzymes confirmed our previous suggestion that residues required for catalytic activity are also involved in inhibitor binding [Moebius et al. (1996) Biochemistry 35, 16871-16878]. His77, Glu81, Glu123, Thr126, Asn194, and Trp197 are localized in the cytoplasmic halves of the transmembrane segments 2-4 and are proposed to line the catalytic cleft. Ala mutants of Trp102, Tyr105, Asp109, Arg111, and Tyr112 in a conserved cytoplasmic domain (WKEYXKGDSRY) between transmembrane segments 2 and 3 contained less than 10% of wt Delta5,7-sterols, implying that this region also could be functionally important. The in vivo complementation of enzyme-deficient yeast strains with mutated cDNAs is a simple and sensitive method to rapidly analyze the functional consequences of mutations in sterol modifying enzymes.     

Toward identification of acid/base catalysts in the active site of enolase: comparison of the properties of K345A, E168Q, and E211Q variants

High-resolution crystallographic data show that Glu 168 and Glu 211 lie on opposite surfaces of the active site from Lys 345. Two different proposals for general base catalysis have emerged from these structural studies. In one scheme, the carboxylate side chains of Glu 168 and Glu 211 are proposed to ionize a trapped water molecule and the OH- serves as the base [Lebioda, L., & Stec, B. (1991) Biochemistry 30, 2817-2822]. In the other proposal, the epsilon-amino group of Lys 345 functions in general base catalysis [Wedekind, J. E., Poyner, R. R., Reed, G. H., & Rayment, I. (1994) Biochemistry 33, 9333-9342]. Genes encoding site specific mutations of these active site residues of yeast enolase, K345A, E168Q, and E211Q, have been prepared. The respective protein products of the wild type and mutant genes were expressed in Escherichia coli and isolated in homogeneous form. All three mutant proteins possess severely depressed activities in the overall reaction- < 1 part in 10(5) of wild type activity. Properties of the three mutant proteins in partial reactions were examined to define more clearly the roles of these residues in the catalytic cycle. The K345A variant fails to catalyze the exchange of the C-2 proton of 2-phospho-D-glycerate with deuterium in D2O, whereas both the E211Q and E168Q mutant proteins are functional in this partial reaction. For E211Q and E168Q enolases, exchange is essentially complete prior to appearance of product, and this observation provides further support for an intermediate in the normal reaction. K345A enolase is inactive in the ionization of tartronate semialdehyde phosphate (TSP), whereas both E168Q and E211Q proteins alter the tautomeric state or catalyze ionization of bound TSP. Wild type enolase catalyzes hydrolysis of (Z)-3-chloro-2-phosphoenolpyruvate by addition of OH- and elimination of Cl- at C-3. This reaction mimics the addition of OH- to C-3 of phosphoenolpyruvate in the reverse reaction with the normal product. All three mutant proteins are depressed in their abilities to carry out this reaction. In single-turnover assays, the activities vary in the order K345A > E168Q > E211Q. These results suggest that Lys 345 functions as the base in the ionization of 2-PGA and that Glu 211 participates in the second step of the reaction.     

The importance of GLU361 position in the reaction catalyzed by cholesterol oxidase

Cholesterol oxidase stereospecifically isomerizes cholest-5-en-3-one to cholest-4-en-3-one. When the base catalyst for isomerization, Glu361, is mutated to Asp, the rate of deprotonation of cholest-5-en-3-one is not affected, but protonation of the dienolic intermediate becomes rate-limiting. This may be a consequence of the large distance between the catalytic base and carbon-6 of the intermediate in the mutant enzyme.     

Neonatal intrahepatic cholestasis with hepatic siderosis and steatosis

Conversion of cholesterol into cholic acid in mammalian liver requires a 12alpha-hydroxylation step. Results have been presented suggesting that two different enzymes are involved in this hydroxylation with different activities towards the two steroids believed to be the physiological substrates for the enzyme, 7alpha-hydroxy-4-cholesten-3-one and 5beta-cholestane-3alpha,7alpha-diol. It is shown here that rabbit liver microsomes and partly purified sterol 12alpha-hydroxylase as well as COS cells transfected with a cDNA coding for this enzyme are able to catalyze 12alpha-hydroxylation of the two substrates at similar relative rates. Also 7alpha-hydroxycholesterol and 3alpha,7alpha-dihydroxy-5beta-cholestanoic acid are 12alpha-hydroxylated by the three systems. It is concluded that rabbit liver contains one major sterol 12alpha-hydroxylase with a broad substrate specificity.     

Clinico-statistical analysis of the approach to retained foreign bodies in the extremities

Secondary structures, functionally important residues, antigenic sites, membrane spanning segments and hydropathicity of light harvesting chlorophyll a/b binding polypeptides (LHC) are predicted by theoretical methods from the amino acid sequence of the polypeptides. The reported structural features of the Pea LHC (Lhcb 1 gene product) from electron crystallographic studies have been compared by alignment with other types of chlorophyll a/b binding polypeptides for structural prediction. Fifteen conserved residues D85, D89, E113, H116, E/Q133, E/Q181, E189, D/N233, E252, N/H255, Q/E269, E/D/Q280, N281, H285, D288 (number indicates position in the aligned sequence), are identified which are potential ligands to Mg2+ of chlorophylls. Three amino acid residues D89, E/Q131 and D/N 233 are proposed as ligands to chlorophylls b2, a7 and b2 respectively, for which ligands are not identified in electron crystallographic study.     

Engineering a change in metal-ion specificity of the iron-dependent superoxide dismutase from Mycobacterium tuberculosis-- X-ray structure analysis of site-directed mutants

We have refined the X-ray structures of two site-directed mutants of the iron-dependent superoxide dismutase (SOD) from Mycobacterium tuberculosis. These mutations which affect residue 145 in the enzyme (H145Q and H145E) were designed to alter its metal-ion specificity. This residue is either Gln or His in homologous SOD enzymes and has previously been shown to play a role in active-site interactions since its side-chain helps to coordinate the metal ion via a solvent molecule which is thought to be a hydroxide ion. The mutations were based on the observation that in the closely homologous manganese dependent SOD from Mycobacterium leprae, the only significant difference from the M. tuberculosis SOD within 10 A of the metal-binding site is the substitution of Gln for His at position 145. Hence an H145Q mutant of the M. tuberculosis (TB) SOD was engineered to investigate this residue's role in metal ion dependence and an isosteric H145E mutant was also expressed. The X-ray structures of the H145Q and H145E mutants have been solved at resolutions of 4.0 A and 2.5 A, respectively, confirming that neither mutation has any gross effects on the conformation of the enzyme or the structure of the active site. The residue substitutions are accommodated in the enzyme's three-dimensional structure by small local conformational changes. Peroxide inhibition experiments and atomic absorption spectroscopy establish surprisingly the H145E mutant SOD has manganese bound to it whereas the H145Q mutant SOD retains iron as the active-site metal. This alteration in metal specificity may reflect on the preference of manganese ions for anionic ligands.     

How replacements of the 12 conserved histidines of subunit I affect assembly, cofactor binding, and enzymatic activity of the Bradyrhizobium japonicum cbb3-type oxidase

Alignments of the amino acid sequences of subunit I (FixN or CcoN) of the cbb3-type oxidases show 12 conserved histidines. Six of them are diagnostic of heme-copper oxidases and are thought to bind the following cofactors: the low spin heme B and the binuclear high spin heme B-CuB center. The other six are FixN(CcoN)-specific and their function is unknown. To analyze the contribution of these 12 invariant histidines of FixN in cofactor binding and function of the Bradyrhizobium japonicum cbb3-type oxidase, they were substituted by valine or alanine by site-directed mutagenesis. The H131A mutant enzyme had already been reported previously to be defective in oxidase assembly and function (Zufferey, R., Th?ny-Meyer, L., and Hennecke, H. (1996) FEBS Lett. 394, 349-352). Four of the remaining histidines were not essential for activity or assembly (positions 226, 246, 333, and 457); by contrast, histidines 331, 410, and 418 were required both for activity and stability of the enzyme. The last group of mutant enzymes, H420A, H280A, H330A, and H316V, were assembled but not functional. To purify the latter mutant proteins and the wild-type enzyme, a six-histidine tag was added to the C terminus of subunit I. The His6-tagged cbb3-oxidase complexes were purified 20-fold by a three-step purification protocol. With the exception of the H420A mutant oxidase, the mutant enzymes H280A, H316V, and H331A contained normal amounts of copper and heme B, and they displayed similar visible light spectroscopic characteristics like the wild-type His6-tagged enzyme. The His6-tagged H420A mutant oxidase differed from the His6-tagged wild-type protein by showing altered visible light spectroscopic characteristics. No stable mutant oxidase lacking copper or heme B was obtained. This strongly suggests that copper and heme B incorporations in subunit I are prerequisites for assembly of the enzyme.     

Properties of xanthine dehydrogenase variants from rosy mutant strains of Drosophila melanogaster and their relevance to the enzyme's structure and mechanism

Xanthine dehydrogenase, a molybdenum, iron-sulfur flavoenzyme encoded in the fruit fly Drosophila melanogaster by the rosy gene, has been characterised both from the wild-type and mutant files. Enzyme assays, using a variety of different oxidising and reducing substrates were supplemented by limited molecular characterisation. Four rosy strains showed no detectable activity in any enzyme assay tried, whereas from four wild-type and three rosy mutant strains, those for the [E89K], [L127F] and [L157P]xanthine dehydrogenases (in all of which the mutation is in the iron-sulfur domain), the enzyme molecules, although present at different levels, had extremely similar or identical properties. This was confirmed by purification of one wild-type and one mutant enzyme. [E89K]xanthine dehydrogenase. These both had ultraviolet-visible absorption spectra similar to milk xanthine oxidase. Both were found to be quite stable molecules, showing very high catalytic-centre activities and with little tendency to become degraded by proteolysis or modified by conversion to oxidase or desulfo forms. In three further rosy strains, giving [G353D]xanthine dehydrogenase and [S357F]xanthine dehydrogenase mutated in the flavin domain, and [G1011E]xanthine dehydrogenase mutated in the molybdenum domain, enzyme activities were selectively diminished in certain assays. For the G353D and S357F mutant enzymes activities to NAD+ as oxidising substrate were diminished, to zero for the latter. In addition for [G353D]xanthine dehydrogenase, there was an increase in apparent Km values both for NAD+ and NADH. These findings indicate involvement of this part of the sequence in the NAD(+)-binding site. The G1011E mutation has a profound effect on the enzyme. As isolated and as present in crude extracts of the files, this xanthine dehydrogenase variant lacks activity to xanthine or pterin as reducing substrate, indicating an impairment of the functioning of its molybdenum centre. However, it retains full activity to NADH with dyes as oxidising substrate. Mild oxidation of the enzyme converts it, apparently irreversibly, to a form showing full activity to xanthine and pterin. The nature of the group that is oxidised is discussed in the light of redox potential data. It is proposed that the process involves oxidation of the pterin of the molybdenum cofactor from the tetrahydro to a dihydro oxidation state. This conclusion is fully consistent with recent information [Rom?o, M. J., Archer, M., Moura, I., Moura. J.J.G., LeGall, J., Engh, R., Schneider, M., Hof, P. & Huber, R. (1995) Science 270. 1170-1176) from X-ray crystallography on the structure of a closely related enzyme from Desulfovibrio gigas. It is proposed, that apparent irreversibility of the oxidative activating process for [G1011E]xanthine dehydrogenase, is due to conversion of its pterin to the tricyclic derivative detected by these workers. The data thus provide the strongest evidence available, that the oxidation state of the pterin can have a controlling influence on the activity of a molybdenum cofactor enzyme. Implications regarding pterin incorporation into xanthine dehydrogenase and in relation to other molybdenum enzymes are discussed.     

Human sulfite oxidase R160Q: identification of the mutation in a sulfite oxidase-deficient patient and expression and characterization of the mutant enzyme

Sulfite oxidase catalyzes the terminal reaction in the degradation of sulfur amino acids. Genetic deficiency of sulfite oxidase results in neurological abnormalities and often leads to death at an early age. The mutation in the sulfite oxidase gene responsible for sulfite oxidase deficiency in a 5-year-old girl was identified by sequence analysis of cDNA obtained from fibroblast mRNA to be a guanine to adenine transition at nucleotide 479 resulting in the amino acid substitution of Arg-160 to Gln. Recombinant protein containing the R160Q mutation was expressed in Escherichia coli, purified, and characterized. The mutant protein contained its full complement of molybdenum and heme, but exhibited 2% of native activity under standard assay conditions. Absorption spectroscopy of the isolated molybdenum domains of native sulfite oxidase and of the R160Q mutant showed significant differences in the 480- and 350-nm absorption bands, suggestive of altered geometry at the molybdenum center. Kinetic analysis of the R160Q protein showed an increase in Km for sulfite combined with a decrease in kcat resulting in a decrease of nearly 1,000-fold in the apparent second-order rate constant kcat/Km. Kinetic parameters for the in vitro generated R160K mutant were found to be intermediate in value between those of the native protein and the R160Q mutant. Native sulfite oxidase was rapidly inactivated by phenylglyoxal, yielding a modified protein with kinetic parameters mimicking those of the R160Q mutant. It is proposed that Arg-160 attracts the anionic substrate sulfite to the binding site near the molybdenum.     

Involvement of cytochrome c oxidase subunit III in energy coupling

The role of the conserved acidic residues of subunit III of cytochrome c oxidase (COIII) in energy transduction was investigated. Using a COIII deletion mutant of Paracoccus denitrificans, complemented with a plasmid expressing either the wild type (wt) COIII gene or site-directed mutants of the COIII gene, we measured cytochrome c oxidase-dependent ATP synthesis, respiration, and membrane potential. Cytochrome c oxidase-dependent ATP synthesis was attenuated in nonacidic mutants of either Glu98 (E98A and E98Q), or Asp259 (D259A) but not in the acidic mutant E98D. The rates of respiration in the energy conversion-defective mutants were as high as or higher than that in the wt. The cytochrome c oxidase-induced increment of membrane potential in the nonacidic mutants was similar to or higher than that in the wt. In contrast, when succinate-driven ATP synthesis was mediated solely by ubiquinol oxidase (e.g., in the presence of myxothiazol), the rates of ATP synthesis in the nonacidic mutants were higher than that in the wt. Moreover, myxothiazol, which inhibited succinate respiration as well as ATP synthesis in wt and E98D, stimulated ATP synthesis, while inhibiting succinate respiration, in the nonacidic mutants. These results indicate that the attenuation of energy conversion in these mutants is limited to cytochrome c oxidase and thus suggest that subunit III plays a role in energy conversion by cytochrome c oxidase.