Binding of pyridine nucleotide coenzymes to the beta-subunit of the voltage-sensitive K+ channel

The beta-subunit of the voltage-sensitive K(+) (K(v)) channels belongs to the aldo-keto reductase superfamily, and the crystal structure of K(v)beta2 shows NADP bound in its active site. Here we report that K(v)beta2 displays a high affinity for NADPH (K(d) = 0.1 micrometer) and NADP(+) (K(d) = 0.3 micrometer), as determined by fluorometric titrations of the recombinant protein. The K(v)beta2 also bound NAD(H) but with 10-fold lower affinity. The site-directed mutants R264E and N333W did not bind NADPH, whereas, the K(d)(NADPH) of Q214R was 10-fold greater than the wild-type protein. The K(d)(NADPH) was unaffected by the R189M, W243Y, W243A, or Y255F mutation. The tetrameric structure of the wild-type protein was retained by the R264E mutant, indicating that NADPH binding is not a prerequisite for multimer formation. A C248S mutation caused a 5-fold decrease in K(d)(NADPH), shifted the pK(a) of K(d)(NADPH) from 6.9 to 7.4, and decreased the ionic strength dependence of NADPH binding. These results indicate that Arg-264 and Asn-333 are critical for coenzyme binding, which is regulated in part by Cys-248. The binding of both NADP(H) and NAD(H) to the protein suggests that several types of K(v)beta2-nucleotide complexes may be formed in vivo.     

13C and deuterium isotope effects suggest an aldol cleavage mechanism for L-ribulose-5-phosphate 4-epimerase

On the basis of (13)C and deuterium isotope effects, L-ribulose-5-phosphate 4-epimerase catalyzes the epimerization of L-ribulose 5-phosphate to D-xylulose 5-phosphate by an aldol cleavage to the enediolate of dihydroxyacetone and glycolaldehyde phosphate, followed by rotation of the aldehyde group and condensation to the epimer at C-4. With the wild-type enzyme, (13)C isotope effects were 1.85% at C-3 and 1.5% at C-4 at pH 7, with the values increasing to 2.53 and 2.05% at pH 5.5, respectively. H97N and Y229F mutants at pH 7 gave values of 3.25 and 2.53% at C-3 and 2. 69 and 1.99% at C-4, respectively. Secondary deuterium isotope effects at C-3 were 2.5% at pH 7 and 3.1% at pH 5.5 with the wild-type enzyme, and 4.1% at pH 7 with H97N. At C-4, the corresponding values were 9.6, 14, and 19%. These data suggest that H97N shows no commitments, while the wild-type enzyme has an external commitment of approximately 1.4 at pH 7 and an internal commitment independent of pH of approximately 0.6. The Y229 mutant shows only the internal commitment of 0.6. The sequence of the epimerase is similar to those of L-fuculose-1-phosphate and L-rhamnulose-1-phosphate aldolases for residues in the active site of L-fuculose-1-phosphate aldolase, suggesting that Asp76, His95, His97, and His171 of the epimerase may be metal ion ligands, and Ser44, Gly45, Ser74, and Ser75 may form a phosphate binding pocket. The pH profile of V/K for L-ribulose 5-phosphate is bell-shaped with pK values of 5.94 and 8.24. The CD spectra of L-ribulose 5-phosphate and D-xylulose 5-phosphate differ sufficiently that the epimerization reaction can be followed at 300 nm.     

Mitochondrial nicotinamide nucleotide transhydrogenase: NADPH binding increases and NADP binding decreases the acidity and susceptibility to modification of cysteine-893

The mitochondrial nicotinamide nucleotide transhydrogenase is a dimeric enzyme of monomer Mr 110,000. It is located in the inner mitochondrial membrane and catalyzes hydride ion transfer between NAD(H) and NADP(H) in a reaction that is coupled to proton translocation across the inner membrane. The amino acid sequence and the nucleotide binding sites of the enzyme have been determined [Yamaguchi, M., Hatefi, Y., Trach, K., & Hoch, J.A. (1988) J. Biol. Chem. 263, 2761-2767; Wakabayashi, S., & Hatefi, Y. (1987) Biochem. Int. 15, 915-924]. N-Ethylmaleimide, as well as other sulfhydryl group modifiers, inhibits the transhydrogenase. The presence of NADP in the incubation mixture suppressed the inhibition rate by N-ethylmaleimide, and the presence of NADPH greatly increased it. NAD and NADH had little or no effect. The NADPH effect was concentration dependent and saturable, with a half-maximal NADPH concentration effect close to the Km of the enzyme for NADPH. Study of the effect of pH on the N-ethylmaleimide inhibition rate showed that NADPH binding by the enzyme lowers the apparent pKa of the N-ethylmaleimide-sensitive group by 0.4 of a pH unit and NADP binding raises this pKa by 0.4 of a pH unit, thus providing a rationale for the effects of NADP and NADPH on the N-ethylmaleimide inhibition rate. With the use of N-[3H]ethylmaleimide, the modified sulfhydryl group involved in the NADP(H)-modulated inhibition of the transhydrogenase was identified as that belonging to Cys-893, which is located 113 residues upstream of the tyrosyl residue modified by [p-(fluorosulfonyl)benzoyl]-5'-adenosine at the putative NADP(H) binding site of the enzyme (see above references).(ABSTRACT TRUNCATED AT 250 WORDS)     

Introduction of a C-terminal aromatic sequence from snake venom phospholipases A2 into the porcine pancreatic isozyme dramatically changes the interfacial kinetics.

Porcine pancreatic phospholipase A2 (PLA2) was modified by single and multiple site-directed mutations at sites thought to be involved in interfacial binding. Charged and polar residues in the C-terminal region were replaced by aromatic residues on the basis of an analogy with snake venom PLA2s, which display high affinity for a zwitterionic interface. The PLA2 variants constructed were N117W, N117W/D119Y and K116Y/N117W/D119Y. Titration with micelles of a zwitterionic substrate suggests that the variants N117W and K116Y/N117W/D119Y possess improved ability to bind to the micellar substrate interface, relative to the wild-type enzyme. Improved interfacial binding was confirmed by direct binding studies with micelles of a zwitterionic substrate analogue, indicating up to five times higher affinity for both variants. Interfacial binding is not improved for the variant N117W/D119Y. Maximal enzyme velocities (Vapp./max) with the zwitterionic substrate were between 25 and 75% of that of the wild-type enzyme. However, competitive inhibition and direct binding studies with a strong inhibitor revealed that the affinity for substrate present at the interface (Km*) is perturbed by the mutations made. For the variant N117W, the slight decrease observed in Vapp./max is most likely made up of a 24-fold reduction in catalytic turnover (kcat) and 18-fold improved substrate binding (Km*).     

Role of the flexible loop of hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus in enzyme catalysis

The hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase), a type I PRTase, from Tritrichomonas foetus, is a potential target for antitritrichomonal chemotherapy. Structural data on all the type I PRTases reveal a highly flexible, 11-14-amino acid loop, presumably covering the active site. With the exception of a highly conserved Ser-Tyr dipeptide, the other amino acids constituting the loop vary widely among different PRTases. The roles of the conserved Ser73 and Tyr74 residues in the loop and the dynamics of the loop in T. foetus HGXPRTase were investigated using site-directed mutants, stop-flow kinetics, chemical modification, and two-dimensional (1)H-(15)N heteronuclear NMR relaxation experiments. S73A, Y74F, and Y74E mutants of HGXPRTase exhibited a 5-7-fold increase in K(m) for guanine and a 3-5-fold increase in K(m) for PRPP compared to that of the wild type, reflecting the decreased affinity of binding for the two substrates. The k(cat)'s for these mutant-catalyzed reactions, however, do not change appreciably from that of the wild-type enzyme. Stopped-flow fluorescence with a Y74W mutant showed no apparent quenching by adding either PRPP or GMP alone. When both PRPP and guanine were added together, however, the fluorescence was rapidly quenched, followed by a slow recovery as the enzyme-catalyzed reaction progressed, suggesting movement of the loop during catalysis. In the presence of 9-deazaguanine and PRPP, the rapidly quenched fluorescence was not recovered, suggesting a closed loop form. The accessibility of Trp74 in the flexible loop of the mutant enzyme was also analyzed using N-bromosuccinimide (NBS), which reacts specifically with the tryptophan residue. NBS reacted with the only tryptophan in the Y74W mutant enzyme and rendered the enzyme inactive. GMP or PRPP alone failed to protect the enzyme from NBS inactivation. However, the presence of both 9-deazaguanine and PPRP protected the enzyme, allowing it to retain up to 70% of its activity. An S75H mutant, labeled with [(15)N]histidine, was used in the (1)H-(15)N NMR study. Spectra obtained in the presence of enzyme substrates indicated an apparent stabilization of the loop only in the presence of 9-deazaguanine and PRPP. These experimental results thus clearly demonstrated stabilization of the flexible loop upon binding of both PRPP and guanine and suggested its involvement in enzyme catalysis.     

Decolourization and Detoxification of Methyl Red by Aerobic Bacteria from A Wastewater Treatment Plant

Bacterial cultures from a wastewater treatment plant degraded a toxic azo dye (methyl red) by decolourization. Complete decolourization using a mixed-culture was achieved at pH 6, 30 °C within 6 h at 5 mg/l methyl red concentration, and 16 h at 20—30 mg/l. Four bacterial species were isolated that were capable of growth on methyl red as the sole carbon source, and two were identified, namely Vibrio logei and Pseudomonas nitroreducens. The Vibrio species showed the highest methyl red degradation activity at the optimum conditions of pH 6--7, and 30—35 °C. Analysis by NMR showed that previously reported degradation products 2-aminobenzoic acid and N,N-dimethyl-1,4-phenylenediamine were not observed. The decolourized dye was not toxic to a monkey kidney cell line (COS-7) at a concentration of 250 μM. This revised version was published online in July 2006 with corrections to the Cover Date.     

Site-directed mutagenesis studies of rat steroid 5alpha-reductase (isozyme-1): mutation of residues in the cofactor binding and C-terminal regions

We have investigated the roles of highly conserved glycine (G175, G185), negatively charged (E188, D165) and histidine residues (H233, H237) in rat steroid 5alpha-reductase (isozyme-1), on NADPH, testosterone (T) binding and enzyme activity. The mutations G175R and G175S result in a two- to threefold increase in K(m)(NADPH) and an approximately fourfold decrease in the V(max) with no change in K(m)(T). The mutation G185W resulted in a fivefold decrease in K(m)(NADPH) and an eightfold decrease in V(max), with no change in K(m)(T), whereas the mutations E188Q and D165N both resulted in inactive enzyme. Steady-state kinetic measurements showed that the mutation H233R resulted in an approximately 40-fold decrease in V(max), an approximately 20-fold increase in K(m)(T) and no alteration in K(m)(NADPH), whereas the mutation H237R resulted in virtually inactive enzyme. The results suggest that the conserved glycines are not essential for cofactor binding and activity, and that the negatively charged residues may contribute to enzyme stability, whereas the C-terminal histidines appear to be involved in substrate binding and catalytic activity.     

Conversion of the Escherichia coli cytochrome b562 to an archetype cytochrome b: a mutant with bis-histidine ligation of heme iron

A mutant of the Escherichia coli cytochrome b(562) has been created in which the heme-ligating methionine (Met) at position 7 has been replaced with a histidine (His) (M7H). This protein is a double mutant that also has the His 63 to asparagine (H63N) mutation, which removes a solvent-exposed His. While the H63N mutation has no measurable effect on the cytochrome, the M7H mutation converts the atypical His/Met heme ligation in cytochrome b(562) to the classic cytochrome b-type bis-His ligation. This mutation has little effect on the K(d) of heme binding but significantly reduces the chemical and thermal stability of the mutant cytochrome relative to the wild type (wt). Both proteins have similar absorbance (Abs) and electron paramagnetic resonance (EPR) properties characteristic of 6-coordinate low-spin heme. The Abs spectra of the oxidized and reduced bis-His cytochrome are slightly blue-shifted relative to the wt, and the alpha Abs band of ferrous M7H mutant is unusually split. The M7H mutation decreases the midpoint potential of the bound heme by 260 mV at pH 7 and considerably alters the pH dependence of the E(m), which becomes dominated by a single pK(red) = 6.8.     

The impact of commercially available purinergic ligands on purinergic signalling research

Pannexin1 is a prime candidate to represent an ATP release channel. The pannexin1 channel can be activated by extracellular ATP through purinergic receptors P2X7 or P2Y. Recent studies have shown that the Pannexin1 channel is inhibited by its own permeant ion, ATP, and also by P2X7 receptor agonists and antagonists. However, the dose dependence of this inhibition indicated that significant inhibition was prominent at ATP concentrations higher than required for activation of purinergic receptors, including P2X7 and P2Y2. The inhibitory effect of ATP is largely decreased when R75 in the first extracellular loop of Pannexin1 is mutated to alanine, indicating that ATP regulates this channel presumably through binding. To further investigate the structural property of the putative ATP binding site, we performed alanine-scanning mutagenesis of the extracellular loops of pannexin1. Mutations on W74, S237, S240, I247 and L266 in the extracellular loops 1 and 2 severely impaired the inhibitory effect of BzATP, indicating that they might be the essential amino acids in the putative binding site. Mutations on R75, S82, S93, L94, D241, S249, P259 and I267 moderately (≥50%) decreased BzATP sensitivity, suggesting their supporting roles in the binding. Mutations of other residues did not change the BzATP potency compared to wild-type except for some nonfunctional mutants. These data demonstrate that several amino acid residues on the extracellular loops of Pannexin1 mediate ATP sensitivity. Cells expressing mutant Panx1W74A exhibited an enhanced release of ATP, consistent with the removal of a negative feedback loop controlling ATP release.     

丹霞柿，广东柿属(柿科)一新组合及其一新异名

通过查阅模式标本后发现丹霞南烛(Lyonia danxiaensis R. H. MiaoW. Q. Liu)具有聚伞花序,花部为4数,雄蕊16枚且成对着生,花丝非膝曲状等特征与珍珠花属(Lyonia)不符,而与柿科(Ebenaceae)柿属(Diospyros)一致,因此提出一新组合:丹霞柿[Diospyros danxiaensis (R. H. MiaoW. Q. Liu) Y. H. TongN. H. Xia]。且经标本比对后发现,新近发表的彭华柿(Diospyros penghuae W. B. Liao, Q. FanW. Y. Zhao)与丹霞柿实为同种,在此也予以合并。     

Engineering the properties of a cold active enzyme through rational redesign of the active site.

In an effort to explore the effects of local flexibility on the cold adaptation of enzymes, we designed point mutations aiming to modify side-chain flexibility at the active site of the psychrophilic alkaline phosphatase from the Antarctic strain TAB5. The mutagenesis targets were residues Trp260 and Ala219 of the catalytic site and His135 of the Mg2+ binding site. The replacement of Trp260 by Lys in mutant W260K, resulted in an enzyme less active than the wild-type in the temperature range 5-25 degrees C. The additional replacement of Ala219 by Asn in the double mutant W260K/A219N, resulted in a drastic increase in the energy of activation, which was reflected in a considerably decreased activity at temperatures of 5-15 degrees C and a significantly increased activity at 20-25 degrees C. Further substitution of His135 by Asp in the triple mutant W260K/A219N/H135D restored a low energy of activation. In addition, the His135-->Asp replacement in mutants H135D and W260K/A219N/H135D resulted in considerable stabilization. These results suggest that the psychrophilic character of mutants can be established or masked by very slight variations of the wild-type sequence, which may affect active site flexibility through changes in various conformational constraints.     

Constitutive activation and uncoupling of the atrial natriuretic peptide receptor by mutations at the dimer interface. Role of the dimer structure in signalling

The crystal packing of the extracellular hormone binding domain of the atrial natriuretic peptide (ANP) receptor contains two possible dimer pairs, the head-to-head (hh) and tail-to-tail (tt) dimer pairs associated through the membrane-distal and membrane-proximal subdomains, respectively. The tt-dimer structure has been proposed previously (van den Akker, F., Zhang, X., Miyagi, M., Huo, X., Misono, K. S., and Yee, V. C. (2000) Nature 406, 101-104). However, no direct evidence is available to identify the physiological dimer form. Here we report site-directed mutagenesis studies of residues at the two alternative dimer interfaces in the full-length receptor expressed on COS cells. The Trp74 to Arg mutation (W74R) or D71R at the hh-dimer interface caused partial constitutive guanylate cyclase activation, whereas mutation F96D or H99D caused receptor uncoupling. In contrast, mutation Y196D or L225D at the tt-interface had no such effect. His99 modification at the hh-dimer interface by ethoxyformic anhydride abolished ANP binding. These results suggest that the hh-dimer represents the physiological structure. Recently, we determined the crystal structure of ANPR complexed with ANP and proposed a hormone-induced rotation mechanism mediating transmembrane signaling (H. Ogawa, Y. Qiu, C. M. Ogata, and K. S. Misono, submitted for publication). The observed effects of mutations are consistent with the ANP-induced structural change identified from the crystal structures with and without ANP and support the proposed rotation mechanism for ANP receptor signaling.     

Implications of the reactive thiol and the proximal non-proline cis-peptide bond in the Structure and function of Vibrio harveyi luciferase

The role of a highly reactive cysteine residue, Cys106, in Vibrio harveyi luciferase in modulating the substrate-enzyme interactions and in turn affecting the enzyme activity has been extensively investigated over the past three decades. Replacing Cys106 with valine dramatically hinders the ability of luciferase to stabilize the C4a-hydroperoxyflavin intermediate [Abu-Soud, H. M., Clark, A. C., Francisco, W. A., Baldwin, T. O., and Raushel, F. M. (1993) J. Biol. Chem. 268, 7699-7706] and consume aldehyde substrate [Xi, L., Cho, K.-W., Herndon, M. E., and Tu, S.-C. (1990) J. Biol. Chem. 265, 4200-4203], therefore markedly decreasing enzyme activity. On the basis of the structure-activity relationship of flavin analogues and the location of the phosphate binding site of flavin mononucleotide (FMN) coupled with molecular modeling, the functional part of the isoalloxazine ring of FMN, the thiol side chain of Cys106, the methyl group of Ala75, and the unique non-prolyl cis-peptide bond between Ala74 and Ala75 were found to be closely packed [Lin, L. Y., Sulea, T., Szittner, R., Vassilyev, V., Purisima, E. O., and Meighen, E. A. (2001) Protein Sci. 10, 1563-1571]. Here, by mutating Ala75 to Gly, we restored key wild-type properties to the C106V mutant, in particular, high enzyme activity and a stable C4a-hydroperoxyflavin intermediate, demonstrating that the primary reason for the dark phenotype of the C106V mutant was the unfavorable steric interaction between Val106 and Ala75 side chains, which could in turn disturb the cis-oriented amide linkage of Ala74 and Ala75. Moreover, significant red shifts in light emission of 3-10 nm were measured for luciferases carrying Val106 with the spectrum of the double mutant C106V/A75G now red shifted to that of Photobacterium phosphoreum luciferase, which also has Val and Gly at positions 106 and 75, respectively. These results strengthen the validity of the binding geometry of the modeled flavin with the re-face of the pyrimidine end of the isoalloxazine ring next to Cys106 and implicate the Ala74-Ala75 cis-peptide as a key component in the bioluminescence reaction.     

Substrate inhibition of acetylcholinesterase: residues affecting signal transduction from the surface to the catalytic center.

Amino acids located within and around the 'active site gorge' of human acetylcholinesterase (AChE) were substituted. Replacement of W86 yielded inactive enzyme molecules, consistent with its proposed involvement in binding of the choline moiety in the active center. A decrease in affinity to propidium and a concomitant loss of substrate inhibition was observed in D74G, D74N, D74K and W286A mutants, supporting the idea that the site for substrate inhibition and the peripheral anionic site overlap. Mutations of amino acids neighboring the active center (E202, Y337 and F338) resulted in a decrease in the catalytic and the apparent bimolecular rate constants. A decrease in affinity to edrophonium was observed in D74, E202, Y337 and to a lesser extent in F338 and Y341 mutants. E202, Y337 and Y341 mutants were not inhibited efficiently by high substrate concentrations. We propose that binding of acetylcholine, on the surface of AChE, may trigger sequence of conformational changes extending from the peripheral anionic site through W286 to D74, at the entrance of the 'gorge', and down to the catalytic center (through Y341 to F338 and Y337). These changes, especially in Y337, could block the entrance/exit of the catalytic center and reduce the catalytic efficiency of AChE.     

BTI1, an azoreductase with pH-dependent substrate specificity

The group II azoreductase BTI1 utilizes NADPH to directly cleave azo bonds in water-soluble azo dyes, including quenchers of fluorescence. Unexpectedly, optimal reduction was dye specific, ranging from a pH of <5.5 for Janus green B, to pH 6.0 for methyl red, methyl orange, and BHQ-10, to pH >8.3 for flame orange.     

cDNA cloning of an alginate lyase from a marine gastropod Aplysia kurodai and assessment of catalytically important residues of this enzyme

Herbivorous marine gastropods such as abalone and sea hare ingest brown algae as a major diet and degrade the dietary alginate with alginate lyase (EC 4.2.2.3) in their digestive fluid. To date alginate lyases from Haliotidae species such as abalone have been well characterized and the primary structure analyses have classified abalone enzymes into polysaccharide-lyase-family 14 (PL-14). However, other gastropod enzymes have not been so well investigated and only partial amino-acid sequences are currently available. To improve the knowledge for primary structure and catalytic residues of gastropod alginate lyases, we cloned the cDNA encoding an alginate lyase, AkAly30, from an Aplysiidae species Aplysia kurodai and assessed its catalytically important residues by site-directed mutagenesis. Alginate lyase cDNA fragments were amplified by PCR followed by 5′- and 3′-RACE from A. kurodai hepatopancreas cDNA. The finally cloned cDNA comprised 1313 bp which encoded an amino-acid sequence of 295 residues of AkAly30. The deduced sequence comprised an initiation methionine, a putative signal peptide for secretion (18 residues), a propeptide-like region (9 residues), and a mature AkAly30 domain (267 residues) which showed ∼40% amino-acid identity with abalone alginate lyases. An Escherichia coli BL21(DE3)-pCold I expression system for recombinant AkAly30 (recAkAly30) was constructed and site-directed mutagenesis was performed to assess catalytically important amino-acid residues which had been suggested in abalone and Chlorella virus PL-14 enzymes. Replacements of K99, S126, R128, Y140 and Y142 of recAkAly30 by Ala and/or Phe greatly decreased its activity as in the case of abalone and/or Chlorella virus enzymes. Whereas, H213 that was essential for Chlorella virus enzyme to exhibit the activity at pH 10.0 was originally replaced by N120 in AkAly30. The reverse replacement of N120 by His in recAkAly30 increased the activity at pH 10.0 from 8 U/mg to 93 U/mg; however, the activity level at pH 7.0, i.e., 774.8 U/mg, was still much higher than that at pH 10.0. This indicates that N120 is not directly related to the pH dependence of AkAly30 unlike H213 of vAL-1.     

Evaluation by mutagenesis of the roles of His309, His315, and His319 in the coenzyme site of pig heart NADP-dependent isocitrate dehydrogenase

Sequence alignment predicts that His(309) of pig heart NADP-dependent isocitrate dehydrogenase is equivalent to His(339) of the Escherichia coli enzyme, which interacts with the coenzyme in the crystal structure [Hurley et al. (1991) Biochemistry 30, 8671-8688], and porcine His(315) and His(319) are close to that site. The mutant porcine enzymes H309Q, H309F, H315Q, and H319Q were prepared by site-directed mutagenesis, expressed in E. coli, and purified. The H319Q mutant has K(m) values for NADP, isocitrate, and Mn(2+) similar to those of wild-type enzyme, and V(max) = 20.1, as compared to 37.8 micromol of NADPH min(-1) (mg of protein)(-1) for wild type. Thus, His(319) is not involved in coenzyme binding and has a minimal effect on catalysis. In contrast, H315Q exhibits a K(m) for NADP 40 times that of wild type and V(max) = 16.2 units/mg of protein, with K(m) values for isocitrate and Mn(2+) similar to those of wild type. These results implicate His(315) in the region of the NADP site. Replacement of His(309) by Q or F yields enzyme with no detectable activity. The His(309) mutants bind NADPH poorly, under conditions in which wild type and H319Q bind 1.0 mol of NADPH/mol of subunit, indicating that His(309) is important for the binding of coenzyme. The His(309) mutants bind isocitrate stoichiometrically, as do wild-type and the other mutant enzymes. However, as distinguished from the wild-type enzyme, the His(309) mutants are not oxidatively cleaved by metal isocitrate, implying that the metal ion is not bound normally. Since circular dichroism spectra are similar for wild type, H315Q, and H319Q, these amino acid substitutions do not cause major conformational changes. In contrast, replacement of His(309) results in detectable change in the enzyme's CD spectrum and therefore in its secondary structure. We propose that His(309) plays a significant role in the binding of coenzyme, contributes to the proper coordination of divalent metal ion in the presence of isocitrate, and maintains the normal conformation of the enzyme.     

中国橐吾属(菊科-千里光族)的分类学研究(五):君范橐吾、长毛槖吾和南川橐吾原白中一些排印错误的改正

对我国菊科橐吾属(Ligularia Cass.) 3种植物原白中模式标本引证的排印错误进行了改正。君范橐吾(L. lingiana S.W. Liu)原白中错误地将主模式标本引证为赵清盛82946,实际应为赵清盛、牟克平和杨亚斌8294。长毛槖吾(L. changiana S.W. Liu ex Y. L. Chen & Z. Yu Li)(=L. heterophylla C. C. Chang,为L. heterophylla Rupr.的晚出同名)主模式为蔡希陶59771,但L. heterophylla C. C. Chang 的原白中错误地将主模式标本引证为蔡希陶59711;该号标本属于唇形科的灯笼草[Clinopodium polycephalum (Vaniot) C. Y. Wu & Hsuan]。南川橐吾(L. nanchuanica S. W. Liu)原白中引证的副模式标本熊济华和周子林93871实际应为李国凤63871,前者属于桤叶树科的城口桤叶树(Clethra fargesii Franch.)。     

Structure of the Rtt109-AcCoA/Vps75 complex and implications for chaperone-mediated histone acetylation

Yeast Rtt109 promotes nucleosome assembly and genome stability by acetylating K9, K27, and K56 of histone H3 through interaction with either of two distinct histone chaperones, Vps75 or Asf1. We report the crystal structure of an Rtt109-AcCoA/Vps75 complex revealing an elongated Vps75 homodimer bound to two globular Rtt109 molecules to form a symmetrical holoenzyme with a ～12?? diameter central hole. Vps75 and Rtt109 residues that mediate complex formation in the crystals are also important for Rtt109-Vps75 interaction and H3K9/K27 acetylation both in?vitro and in yeast cells. The same Rtt109 residues do not participate in Asf1-mediated Rtt109 acetylation in?vitro or H3K56 acetylation in yeast cells, demonstrating that Asf1 and Vps75 dictate Rtt109 substrate specificity through distinct mechanisms. These studies also suggest that Vps75 binding stimulates Rtt109 catalytic activity by appropriately presenting the H3-H4 substrate within the central cavity of the holoenzyme to promote H3K9/K27 acetylation of new histones before deposition.     

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.