Intramolecular electron transfer in cytochrome o of Escherichia coli: events following the photolysis of fully and partially reduced CO-bound forms of the bo3 and oo3 enzymes

The events which follow photolysis of CO-inhibited fully reduced and CO-bound mixed-valence cytochrome o have been studied in two variants of the enzyme, one of which contains heme B at the low-spin site (bo3) and the other of which contains heme O (oo3). For this, isolated enzyme was prepared from three different strains of Escherichia coli which produce these two variants in different relative amounts [Puustinen, A., Morgan, J. E., Verkhovsky, M., Thomas, J. W., Gennis, R. B., & Wikstr?m, M. (1992) Biochemistry 31, 10363-10369]. In both types of enzyme microsecond electron redistribution was observed from the oxygen-binding heme to the low-spin heme. In the bo3 enzyme, the rate was similar to that in the bovine enzyme (3 microseconds), but in the oo3 enzyme, it was several times slower. However, in both types of cytochrome o, the same electron redistribution process was also apparently observed on other time scales, some faster and some slower. The rate of CO rebinding in the mixed-valence enzyme was found to be slower than in the fully reduced enzyme, apparently because of the subpopulation of oxidized oxygen-binding heme produced by the electron redistribution. The extent of this electron redistribution, and thus the inter-heme delta Em, can be calculated from this change in rate. The heme B and heme O containing low-spin sites have Em values about 20 and 50 mV lower, respectively, than the oxygen-binding heme.     

Axial coordination and reduction potentials of the sixteen hemes in high-molecular-mass cytochrome c from Desulfovibrio vulgaris (Hildenborough)

A spectroelectrochemical study is described of the sixteen hemes in the high-molecular-mass, monomeric cytochrome c (Hmc) from the periplasmic space of Desulfovibrio vulgaris, strain Hildenborough. One of the hemes has special properties. In the oxidized state at pH 7 it is predominantly high-spin, S = 5/2, with a g perpendicular value of less than 6 indicative of quantum-mechanical mixing with a low-lying (800 cm-1) S = 3/2 state; the balance is probably a low-spin derivative. The high-spin heme has an Em.7.5 value of +61 mV. The fifteen other hemes are low-spin bis-histidine coordinated with Em.7.5 values of approximately -0.20 V. Two of these hemes exhibit very anisotropic EPR spectra with a g1 value of 3.65 characteristic for strained bis-histidine coordination. A previous proposal, namely that methionine is coordinated to one of the hemes [Pollock, W.B.R., Loufti, M. Bruschi, M. Rapp-Giles, B.J., Wall, J. & Voordouw, G. (1991) J. Bacteriol. 173, 220] is disproved using spectroscopic evidence. Contrasting electrochemical data sets from two previous studies [Tan, J. & Cowan, J.A. (1990) Biochemistry 29, 4886; Bruschi, M., Bertrand, P., More, C., Leroy, G., Bonicel, J., Haladjian, J., Chottard, G., Pollock, W.B.R. & Voordouw, G. (1992) Biochemistry 31, 3281] are not consistent with our EPR titration results and are not reproducible. Hmc can be reduced by D. vulgaris Fe-hydrogenase in the presence of molecular hydrogen.     

Resonance Raman spectroscopic identification of a histidine ligand of b595 and the nature of the ligation of chlorin d in the fully reduced Escherichia coli cytochrome bd oxidase

Cytochrome bd oxidase is a bacterial terminal oxidase that contains three cofactors: a low-spin heme (b558), a high-spin heme (b595), and a chlorin d. The center of dioxygen reduction has been proposed to be a binuclear b595/d site, whereas b558 is mainly involved in transferring electrons from ubiquinol to the oxidase. Information on the nature of the axial ligands of the three heme centers has come from site-directed mutagenesis and spectroscopy, which have implicated a His/Met coordination for b558 (Spinner, F., Cheesman, M. R., Thomson, A. J., Kaysser, T., Gennis, R. B., Peng, Q., & Peterson, J. (1995) Biochem. J. 308, 641-644; Kaysser, T. M., Ghaim, J. B., Georgiou, C., & Gennis, R. B. (1995) Biochemistry 34, 13491-13501), but the ligands to b595 and d are not known with certainty. In this work, the three heme chromophores of the fully reduced cytochrome bd oxidase are studied individually by selective enhancement of their resonance Raman (rR) spectra at particular excitation wavelengths. The rR spectrum obtained with 413.1-nm excitation is dominated by the bands of the 5cHS b595(2+) cofactor. Excitation close to 560 nm yields a rR spectrum dominated by the 6cLS b558(2+) heme. Wavelengths between these values enhance contributions from both b595(2+) and b558(2+) chromophores. The rR bands of the ferrous chlorin become the major features with red laser excitation (595-650 nm). The rR data indicate that d2+ is a 5cHS system whose axial ligand is either a weakly coordinating protein donor or a water molecule. In the low-frequency region of the 441.6-nm spectrum, we assign a rR band at 225 cm-1 to the (b595)Fe(II)-N(His) stretching vibration, based on its 1.2-cm(-1) upshift in the 54Fe-labeled enzyme. This observation provides the first physical evidence that the proximal ligand of b595 is a histidine. Site-directed mutagenesis had suggested that His 19 is associated with either b595 or d (Fang, H., Lin, R. -J., & Gennis, R. B. (1989) J. Biol. Chem. 264, 8026-8032). On the basis of the present study, we propose that the proximal ligand of b595 is His 19. We have also studied the reaction of cyanide with the fully reduced cytochrome bd oxidase. In approximately 700-fold excess cyanide (approximately 35 mM), the 629-nm UV/vis band of d2+ is blue-shifted to 625 nm and diminished in intensity. However, the rR spectra at each of three different gamma(0) (413.1, 514.5, and 647.1 nm) are identical with or without cyanide, thus indicating that both b595 and d remain as 5cHS species in the presence of CN-. This observation leads to the proposal that a native ligand of ferrous chlorin d is replaced by CN- to form the 5cHS d2+ cyano adduct. These findings corroborate our companion study of the "as-isolated" enzyme in which we proposed a 5cHS d3+ cyano adduct (Sun, J., Osborne, J. P., Kahlow, M. A., Kaysser, T. M., Hill, J. J., Gennis, R. B., & Loehr, T. M. (1995) Biochemistry 34, 12144-12151). To further characterize the unusual and unexpected nature of these proposed high-spin cyanide adducts, we have obtained EPR spectral evidence that binding of cyanide to fully oxidized cytochrome bd oxidase perturbs a spin-state equilibrium in the chlorin d3+ to yield entirely the high-spin form of the cofactor.     

Over-production of stereoselective nitrile hydratase from Pseudomonas putida 5B in Escherichia coli: activity requires a novel downstream protein

The stereoselective nitrile hydratase (NHase) from Pseudomonas putida 5B has been over-produced in Escherichia coli. Maximal enzyme activity requires the co-expression of a novel downstream gene encoding a protein (P14K) of 127 amino acids, which shows no significant homology to any sequences in the protein database. Nitrile hydratase produced in transformed E. coli showed activity as high as 472 units/mg dry cell (sixfold higher than 5B), and retained the stereoselectivity observed in the native organism. Separated from the end of the beta subunit by only 51 bp, P14K appears to be part of an operon that includes the alpha and beta structural genes of nitrile hydratase, and other potential coding sequences.     

Spectroscopic characterization of a binuclear metal site in Bacillus cereus beta-lactamase II

The zinc metalloenzyme beta-lactamase II (betaLII) from Bacillus cereus has been overexpressed in Escherichia coli as a fusion protein with glutathione-S-transferase, and the metal binding properties of recombinant betaLII toward Zn(II) and Co(II) have been studied by fluorescence and activity measurements. The apoenzyme is able to bind two metal ion equivalents, which confer on betaLII its maximum enzymatic efficiency. The enzyme is partially active with one metal ion equivalent. The diCo(II) and a mixed Zn(II)Co(II) derivative of betaLII were obtained and probed by electronic and paramagnetic NMR spectroscopy. In the high-affinity site, the metal is bound to three His residues and a solvent molecule, adopting a tetrahedral geometry. A Cys, a His, and an Asp residue are coordinated to the low-affinity metal site, together with two or three solvent molecules. This coordination polyhedron resembles the binuclear metal site of the Bacteroides fragilis beta-lactamase [Concha, N., Rasmussen, B. A., Bush, K., and Herzberg, O. (1996) Structure 4, 823-836; Carfi, A., Duée, E., Paul-Soto, R., Galleni, M., Frère, J. M., and Dideberg, O. (1998) Acta Crystallogr. D54, 47-57] but differs from that resulting from the X-ray study of betaLII [Carfi, A., Pares, S., Duée, E., Galleni, M., Duez, C., Frère, J. M., and Dideberg, O. (1995) EMBO J. 14, 4914-4921]. These results suggest that this binuclear metal site may be a general feature of metallo-beta-lactamases.     

How enzymes control the reactivity of adenosylcobalamin: effect on coenzyme binding and catalysis of mutations in the conserved histidine-aspartate pair of glutamate mutase

Glutamate mutase is one of a group of adenosylcobalamin-dependent enzymes that catalyze unusual isomerizations that proceed through the formation of radical intermediates. It shares a structurally similar cobalamin-binding domain with methylcobalamin-dependent methionine synthase. In particular, both proteins contain the "DXHXXG" cobalamin-binding motif, in which the histidine provides the axial ligand to cobalt. The effects of mutating the conserved histidine and aspartate residues in methionine synthase have recently been described [Jarrett, J. T., Amaratunga, M., Drennan, C. L., Scholten, J. D., Sands, R. H., Ludwig, M. L., & Matthews, R. G. (1996) Biochemistry 35, 2464-2475]. Here, we describe how similar mutations in the "DXHXXG" motif of glutamate mutase affect coenzyme binding and catalysis in an adenosylcobalamin-dependent reaction. The mutations made in the MutS subunit of glutamate mutase were His16Gly, His16Gln, Asp14Asn, Asp14Glu, and Asp14Ala. All the mutations affect, in varying degrees, the rate of catalysis, the affinity of the protein for the coenzyme, and the coordination of cobalt. Mutations of either Asp14 or His16 decrease k(cat) by 1000-fold, and whereas cob(II)alamin accumulates as an intermediate in the wild-type enzyme, it does not accumulate in the mutants, suggesting the rate-determining step is altered. The apparent Kd for adenosylcobalamin is raised by about 50-fold when His16 is mutated and by 5-10-fold when Asp16 is mutated. There are extensive differences between the UV-visible spectra of wild-type and mutant holoenzymes, indicating that the mutant enzymes coordinate cobalt less well. Overall, the properties of these mutants differ quite markedly from those observed when similar mutations were introduced into methionine synthase.     

Purification, regulation, and molecular and biochemical characterization of pyruvate carboxylase from Methanobacterium thermoautotrophicum strain deltaH

We discovered that Methanobacterium thermoautotrophicum strain DeltaH possessed pyruvate carboxylase (PYC), and this biotin prototroph required exogenously supplied biotin to exhibit detectable amounts of PYC activity. The enzyme was highly labile and was stabilized by 10% inositol in buffers to an extent that allowed purification to homogeneity and characterization. The purified enzyme was absolutely dependent on ATP, Mg2+ (or Mn2+ or Co2+), pyruvate, and bicarbonate for activity; phosphoenolpyruvate could not replace pyruvate, and acetyl-CoA was not required. The enzyme was inhibited by ADP and alpha-ketoglutarate but not by aspartate or glutamate. ATP was inhibitory at high concentrations. The enzyme, unlike other PYCs, exhibited nonlinear kinetics with respect to bicarbonate and was inhibited by excess Mg2+, Mn2+, or Co2+. The 540-kDa enzyme of A4B4 composition contained a non-biotinylated 52-kDa subunit (PYCA) and a 75-kDa biotinylated subunit (PYCB). The pycB gene was probably monocistronic and followed by a putative gene of a DNA-binding protein on the opposite strand. The pycA was about 727 kilobase pairs away from pycB on the chromosome and was probably co-transcribed with the biotin ligase gene (birA). PYCA and PYCB showed substantial sequence identities (33-62%) to, respectively, the biotin carboxylase and biotin carboxyl carrier + carboxyltransferase domains or subunits of known biotin-dependent carboxylases/decarboxylases. We discovered that PYCB and probably the equivalent domains or subunits of all biotin-dependent carboxylases harbored the serine/threonine dehydratase types of pyridoxal-phosphate attachment site. Our results and the existence of an alternative oxaloacetate synthesizing enzyme phosphoenolpyruvate carboxylase in M. thermoautotrophicum strain DeltaH (Kenealy, W. R., and Zeikus, J. G. (1982) FEMS Microbiol. Lett. 14, 7-10) raise several questions for future investigations.     

Metal ion activation of galactosyltransferase

Galactosyltransferase, which functions as the catalytic component of lactose synthase and in the glycosylation of glycoproteins, has been previously reported to have an absolute dependence on Mn2+ for activity, with a Kd for Mn2+ (10(-3) M) 2 to 3 orders of magnitude greater than the physiological range of Mn2+ concentrations (v 10(-6) M). Reinvestigation of the metal ion dependence of this enzyme has shown that Zn2+, Cd2+, Fe2+, Co2+, and Pr3+ also produce activation, although with lower activities at saturation than that attained with Mn2+. Velocity against metal ion concentration curves for all metals, including Mn2+, are sigmoid, suggesting the presence of two or more activating metal binding sites on the enzyme. The presence of two sites is confirmed by studies using both Mn2+ and Ca2+. While galactosyltransferase is inactive in the presence of Ca2+ alone, at low concentrations of Mn2+ (10(-5) M), enzyme activity is stimulated by Ca2+. A more detailed investigation by steady state kinetics has revealed that there is a tight binding site for Mn2+ (site I: Kd of 2 X 10(-6) M) from which Ca2+ is excluded, and a site at which Ca2+ can replace Mn2+ (site II: Kd for Ca2+ of 1.76 X 10(-3) M), to which metal binding has a specific synergistic effect on UDP-galactose binding, possibly as a result of the formation of an enzyme-Ca2+-UDP-galactose bridge complex. The site I Mn2+, site II Ca2+-activated enzyme has a maximum velocity similar to that of the Mn2+-activated enzyme, and is the enzyme form that must act in lactose synthesis in vivo. A trypsin-degraded form of galactose transferase (galactosyltransferase-T) (Powell, J.T., and Brew, K. (1974) Eur. J. Biochem. 48, 217-228) appears to lack site I and is activated by Ca2+ in the absence of Mn2+.     

Evidence that Ser775 in the alpha subunit of the Na,K-ATPase is a residue in the cation binding pocket

Substitution of alanine for Ser775 in a ouabain-resistant alpha1 sheep isoform causes a 30-fold decrease in apparent affinity for K+ as an activator of the Na,K-ATPase, as well as an increase in apparent affinity for ATP (Arguello, J. M., and Lingrel, J. B (1995) J. Biol. Chem. 270, 22764-22771). This study was carried out to determine whether Ser775 is a direct cation-ligating residue or whether the change in apparent affinity for K+ is secondary to a conformational alteration as evidenced in the change in ATP affinity, with the following results. Kinetics of K+(Rb+) influx into intact cells show that the change is due to a change in K+ interaction at the extracellular surface. The K+ dependence of formation of K+-occluded enzyme (E2(K)) and of the rate of formation of deoccluded enzyme from E2(K) indicate that the Ser775 --> Ala mutation results in a marked increase (>/=30-fold) in rate of release of K+ from E2(K). The high affinity Na+-like competitive antagonist 1,3-dibromo2,4,6-tris-(methylisothiouronium)benzene (Br2TITU), which interacts with the E1 conformation and blocks cytoplasmic cation binding (Hoving, S., Bar-Shimon, M., Tijmes, J. J. , Tal, D. M., and Karlish, S. J. D. (1995) J. Biol. Chem. 270, 29788-29793), inhibits Na+-ATPase of the mutant less than the control enzyme. With intact cells, Br2TITU acts as a competitive inhibitor of extracellular K+ activation of both the mutant and control enzymes. In this case, the mutant was more sensitive to inhibition. With vanadate as a probe of conformation, a difference in conformational equilibrium between the mutant and control enzymes could not be detected under turnover conditions (Na+- ATPase) in the absence of K+. These results indicate that the increase in apparent affinity for ATP effected by the Ser775 --> Ala mutation is secondary to a change in intrinsic cation affinity/selectivity. The large change in affinity for extracellular K+ compared with cytoplasmic Na+ and to Br2TITU binding supports the conclusion that the serine hydroxyl is either part of the K+-gate structure or a direct cation-ligating residue that is shared by at least one Na+ ion, albeit with less consequence on rate constants for Na+ binding or release compared with K+.     

Interaction of alcohols and anesthetics with protein kinase Calpha

The key signal transduction enzyme protein kinase C (PKC) contains a hydrophobic binding site for alcohols and anesthetics (Slater, S. J., Cox, K. J. A., Lombardi, J. V., Ho, C., Kelly, M. B., Rubin, E., and Stubbs, C. D. (1993) Nature 364, 82-84). In this study, we show that interaction of n-alkanols and general anesthetics with PKCalpha results in dramatically different effects on membrane-associated compared with lipid-independent enzyme activity. Furthermore, the effects on membrane-associated PKCalpha differ markedly depending on whether activity is induced by diacylglycerol or phorbol ester and also on n-alkanol chain length. PKCalpha contains two distinct phorbol ester binding regions of low and high affinity for the activator, respectively (Slater, S. J., Ho, C., Kelly, M. B., Larkin, J. D., Taddeo, F. J., Yeager, M. D., and Stubbs, C. D. (1996) J. Biol. Chem. 271, 4627-4631). Short chain n-alkanols competed for low affinity phorbol ester binding to the enzyme, resulting in reduced enzyme activity, whereas high affinity phorbol ester binding was unaffected. Long chain n-alkanols not only competed for low affinity phorbol ester binding but also enhanced high affinity phorbol ester binding. Furthermore, long chain n-alkanols enhanced phorbol ester induced PKCalpha activity. This effect of long chain n-alkanols was similar to that of diacylglycerol, although the n-alkanols alone were weak activators of the enzyme. The cellular effects of n-alkanols and general anesthetics on PKC-mediated processes will therefore depend in a complex manner on the locality of the enzyme (e.g. cytoskeletal or membrane-associated) and activator type, apart from any isoform-specific differences. Furthermore, effects mediated by interaction with the region on the enzyme possessing low affinity for phorbol esters represent a novel mechanism for the regulation of PKC activity.     

Riboflavin 5'-hydroxymethyl oxidation. Molecular cloning, expression, and glycoprotein nature of the 5'-aldehyde-forming enzyme from Schizophyllum commune

Vitamin B2-aldehyde-forming enzyme catalyzes oxidation of the 5'-hydroxymethyl of riboflavin to the formyl group. We have purified the enzyme from the culture media of Schizophyllum commune (ATCC 38719) by modifying the procedure of Tachibana and Oka (Tachibana, S., and Oka, M. (1981) J. Biol. Chem. 256, 6682-6685) for cell-free extract. By SDS-polyacrylamine gel electrophoresis, the enzyme appears to be 78 kDa. The enzyme has a blocked amino terminus, so fragments were obtained by cleaving the purified enzyme with lysyl endopeptidase. Selected peptides were sequenced from their amino termini. We have isolated a full-length cDNA clone using a DNA hybridization probe amplified by polymerase chain reaction with two degenerate oligonucleotide primers, the design of which was based on one of the partial amino acid sequences. From the cDNA clone, it is evident that the enzyme has a Ser/Thr-rich fragment near the COOH-terminal Asp. The enzyme was determined to be a glycoprotein; however, O-deglucosylation only slightly affects activity. Computer searches showed that the B2-aldehyde-forming enzyme has little homology with other proteins, but domain motifs may reflect N-myristoylation of a dehydrogenase with a signature similar to 4Fe-4S ferredoxins. The enzyme cDNA was subcloned into a Pichia expression vector pPIC9K to produce a recombinant protein which exhibited B2-aldehyde-forming enzyme activity.     

Cytochrome c peroxidase from Methylococcus capsulatus Bath

A bacterial cytochrome c peroxidase was purified from the obligate methanotroph Methylococcus capsulatus Bath in either the fully oxidized or the half reduced form depending on the purification procedure. The cytochrome was a homo-dimer with a subunit mol mass of 35.8 kDa and an isoelectric point of 4.5. At physiological temperatures, the enzyme contained one high-spin, low-potential (Em7 = -254 mV) and one low-spin, high-potential (Em7 = +432 mM ) heme. The low-potential heme center exhibited a spin-state transition from the penta-coordinated, high-spin configuration to a low-spin configuration upon cooling the enzyme to cryogenic temperatures. Using M. capsulatus Bath ferrocytochrome c555 as the electron donor, the KM and Vmax for peroxide reduction were 510 +/- 100 nM and 425 +/- 22 mol ferrocytochrome c555 oxidized min-1 (mole cytochrome c peroxidase)-1, respectively.     

Hepatic Ins(1,3,4,5)P4 3-phosphatase is compartmentalized inside endoplasmic reticulum

In pursuit of the physiological role of inositol 1,3,4,5-tetrakisphosphate 3-phosphatase, which also attacks inositol pentakisphosphate and inositol hexakisphosphate with much higher affinity (Nogimori, K., Hughes, P.J., Glennon, M.C., Hodgson, M.E., Putney, J.W., Jr., and Shears, S.B. (1991) J. Biol. Chem. 266, 16499-16506), we have studied the subcellular distribution of the enzyme in liver. Initially, we had to overcome the problem that potent endogenous inhibitor(s) compromise the detection of this enzyme in vitro (Hodgson, M.E., and Shears, S.B. (1990) Biochem. J. 267, 831-834). We partially purified these inhibitor(s) by anion-exchange chromatography and gel filtration; inhibitory activity co-eluted with standard inositol hexakisphosphate and was depleted by treatment with phytase. Thus, subcellular fractions were pretreated with phytase before assay of 3-phosphatase activity. Our experiments revealed that the hepatic 3-phosphatase was nearly exclusively restricted to the endoplasmic reticulum, and there was little or no activity in either the cytosol, plasma membranes, mitochondria, or nuclei. Detergent treatment of microsomes indicated that there was 93 +/- 2% latency to mannose-6-phosphatase, an intraorganelle enzyme activity (Vanstapel, F., Pua, K., and Blanckaert, N. (1986) Eur. J. Biochem. 156, 73-77). Similar latencies were found for the hydrolysis of inositol 1,3,4,5-tetrakisphosphate (95 +/- 1%), inositol 1,3,4,5,6-pentakisphosphate (94 +/- 1%), and inositol hexakisphosphate (93 +/- 2%). Treatment of microsomes with either sodium carbonate or phosphatidylcholine-specific phospholipase C, to release luminal contents, led to solubilization of approximately 90% of 3-phosphatase activity. Thus, hepatic 3-phosphatase has a highly restricted access to inositol polyphosphates in vivo that needs to be accounted for in the determination of the physiological role of this enzyme.     

Directed mutagenesis studies of the metal binding site at the subunit interface of Escherichia coli inorganic pyrophosphatase

Recent crystallographic studies on Escherichia coli inorganic pyrophosphatase (E-PPase) have identified three Mg2+ ions/enzyme hexamer in water-filled cavities formed by Asn24, Ala25, and Asp26 at the trimer-trimer interface (Kankare, J., Salminen, T., Lahti, R., Cooperman, B., Baykov, A. A., and Goldman, A. (1996) Biochemistry 35, 4670-4677). Here we show that D26S and D26N substitutions decrease the stoichiometry of tight Mg2+ binding to E-PPase by approximately 0.5 mol/mol monomer and increase hexamer stability in acidic medium. Mg2+ markedly decelerates the dissociation of enzyme hexamer into trimers at pH 5.0 and accelerates hexamer formation from trimers at pH 7.2 with wild type E-PPase and the N24D variant, in contrast to the D26S and D26N variants, when little or no effect is seen. The catalytic parameters describing the dependences of enzyme activity on substrate and Mg2+ concentrations are of the same magnitude for wild type E-PPase and the three variants. The affinity of the intertrimer site for Mg2+ at pH 7.2 is intermediate between those of two Mg2+ binding sites found in the E-PPase active site. It is concluded that the metal ion binding site found at the trimer-trimer interface of E-PPase is a high affinity site whose occupancy by Mg2+ greatly stabilizes the enzyme hexamer but has little effect on catalysis.     

Characterization of a new staphylococcal gene, vgaB, encoding a putative ABC transporter conferring resistance to streptogramin A and related compounds

The Staphylococcus aureus plasmid gene, vgaB, conferring resistance to streptogramin (SgA) and related compounds (PIIA, virginiamycin M, mikamycin A, synergistin A, Dalfopristin) was cloned and sequenced. This gene potentially encodes a 552-aa protein, VgaB, of 61,327 Da, which exhibits a significant similarity with the ATP-binding domains of numerous proteins. VgaB has two ATP-binding domains containing each of the A and the B motifs described by Walker et al. [Walker, J.E., Saraste, M., Runswick, M.J., Gay, N.J., 1982. Distantly related sequences in the alpha- and beta-subunits of ATP synthase, myosin, kinases and other ATP-requiring enzymes and a common nucleotide binding fold. EMBO J., 1, 945-951], but does not include TM hydrophobic domains. The 155-amino-acid sequence between the two ATP-binding domains of VgaB is richer in Glu than the rest of the protein. The vgaB gene was found in 21 of the 52 SgA(R) and independent wt staphylococci investigated. In each of the 21 staphylococci, vgaB was carried on a plasmid of 50-90 kb also harboring the vatB gene encoding an acetyltransferase inactivating SgA. In all plasmids, vgaB and vatB have the same relative positions.     

Substrate recognition by "password" in p-hydroxybenzoate hydroxylase

The flavin of p-hydroxybenzoate hydroxylase (PHBH) adopts two conformations [Gatti, D. L., Palfey, B. A., Lah, M.-S., Entsch, B., Massey, V., Ballou, D. P., and Ludwig, M. L. (1994) Science 266, 110-114; Schreuder, H. A., Mattevi, A., Obmolova, G., Kalk, K. H., Hol, W. G. J., van der Bolt, F. J. T., and van Berkel, W. J. H. (1994) Biochemistry 33, 10161-10170]. Kinetic studies detected the movement of the flavin from the buried conformation to the exposed conformation caused by the binding of NADPH prior to its reaction with the flavin. The pH dependence of the rate constant for flavin reduction in wild-type PHBH and the His72Asn mutant indicates that the deprotonation of bound p-hydroxybenzoate is also required for flavin movement, and is accomplished by the same internal proton transport network previously found to be involved in substrate oxidation. The linkage of substrate deprotonation to flavin movement constitutes a novel mode of molecular recognition in which the enzyme tests the suitability of aromatic substrates before committing to the catalytic cycle.     

Relationship between left ventricular dysfunction studied by multigated radionuclide angiography and carotid stenosis examined by Doppler

For investigating neuronal information processing at the cellular level, a technique which visualizes the voltage distribution within single neurons in situ would be extremely useful. Voltage-sensitive dyes are, in principle, capable of reporting membrane potential [Cohen, L.B. and Salzberg, B.M., Rev. Physiol. Biochem. Pharmacol., 83 (1978) 35-88; Grinvald, A., Lieke, E.E., Frostig, R.D. and Hildesheim, R., J. Neurosci., 14 (1994) 2545-2568; Kleinfeld, D., Delaney, K.R., Fee, M.S., Flores, J.A., Tank, D.W. and Gelperin, A., J. Neurophysiol., 72 (1994) 1402-1419]. However, their application to single cells internally is technically difficult [Antic, S. and Zecevic, D., J. Neurosci., 15 (1995) 1392-1405; Grinvald, A., Salzberg, B.M., Lev-Ram, V. and Hildesheim, R., Biophys. J., 51 (1987) 643-651; Kogan, A., Ross, W.N., Zecevic, D. and Lasser-Ross, N., Brain Res., 700 (1995) 235-239; Zecevic, D., Nature, 381 (1996) 322-325]. An alternative strategy consists in applying the dye from the outside to all cells in the tissue, while manipulating a single cell by current injection [Krauthamer, V. and Ross, W.N., J. Neurosci., 4 (1984) 673-682; Ross, W.N. and Krauthamer, V., J. Neurosci., 4 (1984) 659-672]. Here, we modify this technique to further enhance spatial at the cost of temporal resolution [Borst, A., Z. Naturforsch., 50 (1995) 435-438]. Applied to rat cerebellar slices we demonstrate that the potential spread in individual Purkinje cells can be imaged up to even fine dendritic branches. The acquired optical signals suggest that steadily hyperpolarized Purkinje cells are electrically compact. When permanently depolarized, the somatic input resistance is significantly diminished, yet the spatial voltage drop along the dendrites remains unchanged. As demonstrated by compartmental modeling, this hints to a concentration of outward rectifying currents at the soma of the cells.     

Cloning, sequencing, and overexpression of the genes encoding coenzyme B12-dependent glycerol dehydratase of Citrobacter freundii

The genes encoding coenzyme B12-dependent glycerol dehydratase of Citrobacter freundii were cloned and overexpressed in Escherichia coli. The B12-free enzyme was purified to homogeneity. It consists of three types of subunits whose N-terminal sequences are in accordance with those deduced from the open reading frames dhaB, dhaC, and dhaE, coding for subunits of 60,433 (alpha), 21,487 (beta), and 16,121 (gamma) Da, respectively. The enzyme complex has the composition alpha2beta2gamma2. Amino acid alignments with the subunits of the recently sequenced diol dehydratase of Klebsiella oxytoca (T. Tobimatsu, T. Hara, M. Sakaguchi, Y. Kishimoto, Y. Wada, M. Isoda, T. Sakai, and T. Toraya, J. Biol. Chem. 270:7142-7148, 1995) revealed identities between 51.8 and 70.9%.     

Sulfhydryl reagents and cAMP-dependent kinase increase the sensitivity of the inositol 1,4,5-trisphosphate receptor in hepatocytes

Sulfhydryl reagents such as tert-butyl hydroperoxide (TBHP) have been shown to increase cytosolic Ca2+ concentration ([Ca2+]i) in rat hepatocytes in a way that resembles responses to Ca(2+)-mobilizing hormones (Saikada, I., Thomas, A. P., and Farber, J. L. (1991) J. Biol. Chem. 266, 717-722; Rooney, T. A., Renard, D. C., Sass, E. J., and Thomas, A. P. (1991) J. Biol. Chem. 266, 12272-12282) and to increase the amount of Ca2+ released by inositol 1,4,5-trisphosphate ((1,4,5)IP3) from permeable rat liver cells (Rooney et al., 1991, op. cit.; Missiaen, L., Taylor, C. W., and Berridge, M. J. (1991) Nature 352, 241-244; Renard, D. C., Seitz, M. B., and Thomas, A. P. (1992) Biochem. J. 284, 507-512). The effects of sulfhydryl reagents were studied in fura-2-injected rat and guinea pig hepatocytes and compared with the actions of cAMP (Burgess, G. M., Bird, G. St. J., Obie, J. F., and Putney, J. W., Jr. (1991) J. Biol. Chem. 261, 4772-4781). In rat liver cells, the increases in [Ca2+]i induced by TBHP and thimerosal were prevented by microinjection of the cells with the (1,4,5)IP3 receptor antagonist heparin. In guinea pig hepatocytes, TBHP was not able to increase [Ca2+]i unless the cells were pretreated with angiotensin II to raise endogenous levels of (1,4,5)IP3 or were first injected with a sub-threshold concentration of inositol 2,4,5-trisphosphate ((2,4,5)IP3). The responses to TBHP in (2,4,5)IP3-injected guinea pig cells were also blocked by heparin. In many respects, the actions of TBHP appeared to be similar to those of cAMP, which has previously been shown to increase sensitivity to (1,4,5)IP3 in intact guinea pig hepatocytes (Burgess et al., 1991, op. cit.). TBHP also mimicked the effect of cAMP-dependent kinase (PKA) in permeabilized guinea pig hepatocytes by increasing the amount of Ca2+ released by (1,4,5)IP3. The responses to TBHP and cAMP in (2,4,5)IP3-injected guinea pig hepatocytes differed, however, in that the increase in [Ca2+]i evoked by elevating intracellular cAMP was greatly reduced by Wiptide, an inhibitor of PKA, while Wiptide had no effect on the Ca2+ transients induced by TBHP. This provides evidence that the sensitizing effect of TBHP is not mediated by PKA and is more likely to be a direct effect on the inositol trisphosphate receptor. It is possible, however, that the sulfhydryl reagents and PKA act on a common regulatory site on the receptor protein.     

Dual inhibition of human type 4 phosphodiesterase isostates by (R, R)-(+/-)-methyl 3-acetyl-4-[3-(cyclopentyloxy)-4-methoxyphenyl]-3- methyl-1-pyrrolidinecarboxylate

Purified recombinant human type 4 phosphodiesterase B2B (HSPDE4B2B) exists in both a low- and a high-affinity state that bind (R)-rolipram with Kd's of ca. 500 and 1 nM, respectively [Rocque, W. J., Tian, G., Wiseman, J. S., Holmes, W. D., Thompson, I. Z., Willard, D. H., Patel, I. R., Wisely, G. B., Clay, W. C., Kadwell, S. H., Hoffman, C. R., and Luther, M. A. (1997) Biochemistry 36, 14250-14261]. Since the tissue distribution of the two isostates may be significantly different, development of inhibitors that effectively inhibit both forms may be advantageous pharmacologically. In this study, enzyme inhibition and binding of HSPDE4B2B by (R, R)-(+/-)-methyl 3-acetyl-4-[3-(cyclopentyloxy)-4-methoxyphenyl]-3-methyl-1-pyrrolidin ecarboxylate (1), a novel inhibitor of phosphodiesterase 4 (PDE 4), were investigated. Binding experiments demonstrated high-affinity binding of 1 to HSPDE4B2B with a stoichiometry of 1:1. Inhibition of PDE activity showed only a single transition with an observed Ki similar to the apparent Kd determined by the binding experiments. Deletional mutants of HSPDE4B2B, which have been shown to bind (R)-rolipram with low affinity, were shown to interact with 1 with high affinity, indistinguishable from the results obtained with the full-length enzyme. Bound 1 was completely displaced by (R)-rolipram, and the displacement showed a biphasic transition that resembles the biphasic inhibition of HSPDE4B2B by (R)-rolipram. Theoretical analysis of the two transitions exemplified in the interaction of (R)-rolipram with HSPDE4B2B indicated that the two isostates were nonexchangeable. Phosphorylation at serines 487 and 489 on HSPDE4B2B had no effect on the stoichiometry of binding, the affinity for binding, or the inhibition of the enzyme by 1. These data further illustrate the presence of two isostates in PDE 4 as shown previously for (R)-rolipram binding and inhibition. In contrast to (R)-rolipram, where only one of the two isostates of PDE 4 binds with high affinity, 1 is a potent, dual inhibitor of both of the isostates of PDE 4. Kinetic and thermodynamic models describing the interactions between the nonexchangeable isostates of PDE 4 and its ligands are discussed.