Evolution of an enzyme active site: The structure of a new crystal form of muconate lactonizing enzyme compared with mandelate racemase and enolase

Muconate lactonizing enzyme (MLE), a component of the ��-ketoadipate pathway of Pseudomonas putida, is a member of a family of related enzymes (the ���enolase superfamily���) that catalyze the abstraction of the ��-proton of a carboxylic acid in the context of different overall reactions. New untwinned crystal forms of MLE were obtained, one of which diffracts to better than 2.0-��� resolution. The packing of the octameric enzyme in this crystal form is unusual, because the asymmetric unit contains three subunits. The structure of MLE presented here contains no bound metal ion, but is very similar to a recently determined Mn2+-bound structure. Thus, absence of the metal ion does not perturb the structure of the active site. The structures of enolase, mandelate racemase, and MLE were superimposed. A comparison of metal ligands suggests that enolase may retain some characteristics of the ancestor of this enzyme family. Comparison of other residues involved in catalysis indicates two unusual patterns of conservation: (i) that the position of catalytic atoms remains constant, although the residues that contain them are located at different points in the protein fold; and (ii) that the positions of catalytic residues in the protein scaffold are conserved, whereas their identities and roles in catalysis vary.

Locking in alternate conformations of the integrin ��L��2 I domain with disulfide bonds reveals functional relationships among integrin domains

We used integrin ��L��2 heterodimers containing I domains locked open (active) or closed (inactive) with disulfide bonds to investigate regulatory interactions among domains in integrins. mAbs to the ��L I domain and ��2 I-like domain inhibit adhesion of wild-type ��L��2 to intercellular adhesion molecule-1. However, with ��L��2 containing a locked open I domain, mAbs to the I domain were subdivided into subsets (i) that did not inhibit, and thus appear to inhibit by favoring the closed conformation, and (ii) that did inhibit, and thus appear to bind to the ligand binding site. Furthermore, ��L��2 containing a locked open I domain was completely resistant to inhibition by mAbs to the ��2 I-like domain, but became fully susceptible to inhibition after disulfide reduction with DTT. This finding suggests that the I-like domain indirectly contributes to ligand binding by regulating opening of the I domain in wild-type ��L��2. Conversely, locking the I domain closed partially restrained conformational change of the I-like domain by Mn2+, as measured with mAb m24, which we map here to the ��2 I-like domain. By contrast, locking the I domain closed or open did not affect constitutive or Mn2+-induced exposure of the KIM127 epitope in the ��2 stalk region. Furthermore, locked open I domains, in ��L��2 complexes or expressed in isolation on the cell surface, bound to intercellular adhesion molecule-1 equivalently in Mg2+ and Mn2+. These results suggest that Mn2+ activates ��L��2 by binding to a site other than the I domain, most likely the I-like domain of ��2.

Monitoring the structure of Escherichia coli RNase P RNA in the presence of various divalent metal ions

Lead(II)-induced cleavage can be used as a tool to probe conformational changes in RNA. In this report, we have investigated the conformation of M1 RNA, the catalytic subunit of Escherichia coli RNase P, by studying the lead(II)-induced cleavage pattern in the presence of various divalent metal ions. Our data suggest that the overall conformation of M1 RNA is very similar in the presence of Mg2+, Mn2+, Ca2+, Sr2+ and Ba2+, while it is changed compared to the Mg2+-induced conformation in the presence of other divalent metal ions, Cd2+ for example. We also observed that correct folding of some M1 RNA domains is promoted by Pb2+, while folding of other domain(s) requires the additional presence of other divalent metal ions, cobalt(III) hexamine or spermidine. Based on the suppression of Pb2+ cleavage at increasing concentrations of various divalent metal ions, our findings suggest that different divalent metal ions bind with different affinities to M1 RNA as well as to an RNase P hairpin���loop substrate and yeast tRNAPhe. We suggest that this approach can be used to obtain information about the relative binding strength for different divalent metal ions to RNA in general, as well as to specific RNA divalent metal ion binding sites. Of those studied in this report, Mn2+ is generally among the strongest RNA binders.

A Steep Dependence of Inward-Rectifying Potassium Channels on Cytosolic Free Calcium Concentration Increase Evoked by Hyperpolarization in Guard Cells1

Inactivation of inward-rectifying K+ channels (IK,in) by a rise in cytosolic free [Ca2+] ([Ca2+]i) is a key event leading to solute loss from guard cells and stomatal closure. However, [Ca2+]i action on IK,in has never been quantified, nor are its origins well understood. We used membrane voltage to manipulate [Ca2+]i (A. Grabov and M.R. Blatt [1998] Proc Natl Acad Sci USA 95: 4778���4783) while recording IK,in under a voltage clamp and [Ca2+]i by Fura-2 fluorescence ratiophotometry. IK,in inactivation correlated positively with [Ca2+]i and indicated a Ki of 329 �� 31 nm with cooperative binding of four Ca2+ ions per channel. IK,in was promoted by the Ca2+ channel antagonists Gd3+ and calcicludine, both of which suppressed the [Ca2+]i rise, but the [Ca2+]i rise was unaffected by the K+ channel blocker Cs+. We also found that ryanodine, an antagonist of intracellular Ca2+ channels that mediate Ca2+-induced Ca2+ release, blocked the [Ca2+]i rise, and Mn2+ quenching of Fura-2 fluorescence showed that membrane hyperpolarization triggered divalent release from intracellular stores. These and additional results point to a high signal gain in [Ca2+]i control of IK,in and to roles for discrete Ca2+ flux pathways in feedback control of the K+ channels by membrane voltage.

A Chloroplast DNA Helicase II from Pea That Prefers Fork-Like Replication Structures

A DNA helicase, called chloroplast DNA (ctDNA) helicase II, was purified to apparent homogeneity from pea (Pisum sativum). The enzyme contained intrinsic, single-stranded, DNA-dependent ATPase activity and an apparent molecular mass of 78 kD on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The DNA helicase was markedly stimulated by DNA substrates with fork-like replication structures. A 5���-tailed fork was more active than the 3���-tailed fork, which itself was more active than substrates without a fork. The direction of unwinding was 3��� to 5��� along the bound strand, and it failed to unwind blunt-ended duplex DNA. DNA helicase activity required only ATP or dATP hydrolysis. The enzyme also required a divalent cation (Mg2+>Mn2+>Ca2+) for its unwinding activity and was inhibited at 200 mm KCl or NaCl. This enzyme could be involved in the replication of ctDNA. The DNA major groove-intercalating ligands nogalamycin and daunorubicin were inhibitory to unwinding (Ki approximately 0.85 ��m and 2.2 ��m, respectively) and ATPase (Ki approximately 1.3 ��m and 3.0 ��m, respectively) activities of pea ctDNA helicase II, whereas ellipticine, etoposide (VP-16), and camptothecin had no effect on the enzyme activity. These ligands may be useful in further studies of the mechanisms of chloroplast helicase activities.

Localized Changes in Peroxidase Activity Accompany Hydrogen Peroxide Generation during the Development of a Nonhost Hypersensitive Reaction in Lettuce1

Peroxidase activity was characterized in lettuce (Lactuca sativa L.) leaf tissue. Changes in the activity and distribution of the enzyme were examined during the development of a nonhost hypersensitive reaction (HR) induced by Pseudomonas syringae (P. s.) pv phaseolicola and in response to an hrp mutant of the bacterium. Assays of activity in tissue extracts revealed pH optima of 4.5, 6.0, 5.5 to 6.0, and 6.0 to 6.5 for the substrates tetramethylbenzidine, guaiacol, caffeic acid, and chlorogenic acid, respectively. Inoculation with water or with wild-type or hrp mutant strains of P. s. pv phaseolicola caused an initial decline in total peroxidase activity; subsequent increases depended on the hydrogen donor used in the assay. Guaiacol peroxidase recovered more rapidly in tissues undergoing the HR, whereas changes in tetramethylbenzidine peroxidase were generally similar in the two interactions. In contrast, increases in chlorogenic acid peroxidase were significantly higher in tissues inoculated with the hrp mutant. During the HR, increased levels of Mn2+/2,4-dichlorophenol-stimulated NADH and NADPH oxidase activities, characteristic of certain peroxidases, were found in intercellular fluids and closely matched the accumulation of H2O2 in the apoplast. Histochemical analysis of peroxidase distribution by electron microscopy revealed a striking, highly localized increase in activity within the endomembrane system and cell wall at the sites of bacterial attachment. However, no clear differences in peroxidase location were observed in tissue challenged by the wild-type strain or the hrp mutant. Our results highlight the significance of the subcellular control of oxidative reactions leading to the generation of reactive oxygen species, cell wall alterations, and the HR.

Purification and Characterization of NAD-Isocitrate Dehydrogenase from Chlamydomonas reinhardtii1

NAD-isocitrate dehydrogenase (NAD-IDH) from the eukaryotic microalga Chlamydomonas reinhardtii was purified to electrophoretic homogeneity by successive chromatography steps on Phenyl-Sepharose, Blue-Sepharose, diethylaminoethyl-Sephacel, and Sephacryl S-300 (all Pharmacia Biotech). The 320-kD enzyme was found to be an octamer composed of 45-kD subunits. The presence of isocitrate plus Mn2+ protected the enzyme against thermal inactivation or inhibition by specific reagents for arginine or lysine. NADH was a competitive inhibitor (Ki, 0.14 mm) and NADPH was a noncompetitive inhibitor (Ki, 0.42 mm) with respect to NAD+. Citrate and adenine nucleotides at concentrations less than 1 mm had no effect on the activity, but 10 mm citrate, ATP, or ADP had an inhibitory effect. In addition, NAD-IDH was inhibited by inorganic monovalent anions, but l-amino acids and intermediates of glycolysis and the tricarboxylic acid cycle had no significant effect. These data support the idea that NAD-IDH from photosynthetic organisms may be a key regulatory enzyme within the tricarboxylic acid cycle.

Aluminum Induces a Decrease in Cytosolic Calcium Concentration in BY-2 Tobacco Cell Cultures1

Al toxicity is a major problem that limits crop productivity on acid soils. It has been suggested that Al toxicity is linked to changes in cellular Ca homeostasis and the blockage of plasma membrane Ca2+-permeable channels. BY-2 suspension-cultured cells of tobacco (Nicotiana tabacum L.) exhibit rapid cell expansion that is sensitive to Al. Therefore, the effect of Al on changes in cytoplasmic free Ca concentration ([Ca2+]cyt) was followed in BY-2 cells to assess whether Al perturbed cellular Ca homeostasis. Al exposure resulted in a prolonged reduction in [Ca2+]cyt and inhibition of growth that was similar to the effect of the Ca2+ channel blocker La3+ and the Ca2+ chelator ethyleneglycol-bis(��-aminoethyl ether)-N,N���-tetraacetic acid. The Ca2+ channel blockers verapamil and nifedipine did not induce a decrease in [Ca2+]cyt in these cells and also failed to inhibit growth. Al and La3+, but not verapamil or nifedipine, reduced the rate of Mn2+ quenching of Indo-1 fluorescence, which is consistent with the blockage of Ca2+- and Mn2+-permeable channels. These results suggest that Al may act to block Ca2+ channels at the plasma membrane of plant cells and this action may play a crucial role in the phytotoxic activity of the Al ion.

Location and properties of metal-binding sites on the human prion protein

Although a functional role in copper binding has been suggested for the prion protein, evidence for binding at affinities characteristic of authentic metal-binding proteins has been lacking. By presentation of copper(II) ions in the presence of the weak chelator glycine, we have now characterized two high-affinity binding sites for divalent transition metals within the human prion protein. One is in the N-terminal octapeptide-repeat segment and has a Kd for copper(II) of 10���14 M, with other metals (Ni2+, Zn2+, and Mn2+) binding three or more orders of magnitude more weakly. However, NMR and fluorescence data reveal a previously unreported second site around histidines 96 and 111, a region of the molecule known to be crucial for prion propagation. The Kd for copper(II) at this site is 4 �� 10���14 M, whereas nickel(II), zinc(II), and manganese(II) bind 6, 7, and 10 orders of magnitude more weakly, respectively, regardless of whether the protein is in its oxidized ��-helical (��-PrP) or reduced ��-sheet (��-PrP) conformation. A role for prion protein (PrP) in copper metabolism or transport seems likely and disturbance of this function may be involved in prion-related neurotoxicity.

Differential regulation of beta 1 and beta 2 integrin avidity by chemoattractants in eosinophils.

The CC chemokines regulated on activation normal T expressed and secreted (RANTES) and monocyte chemotactic protein 3 (MCP-3), and the anaphylatoxin C5a, induce activation, degranulation, chemotaxis, and transendothelial migration of eosinophils. Adhesion assays on purified ligands showed differential regulation of beta 1 and beta 2 integrin avidity in eosinophils. Adhesiveness of VLA-4 (alpha 4 beta 1, CD29/CD49d) for vascular cell adhesion molecule 1 or fibronectin was rapidly increased but subsequently reduced by RANTES, MCP-3, or C5a. The deactivation of VLA-4 lead to cell detachment, whereas phorbol 12-myristate 13-acetate induced sustained activation of VLA-4. In contrast, chemoattractants stimulated a prolonged increase in the adhesiveness of Mac-1 (alpha M beta 2, CD11b/CD18) for intercellular adhesion molecule 1. Inhibition by pertussis toxin confirmed signaling via G protein-coupled receptors. Chemoattractants induced transient, while phorbol 12-myristate 13-acetate induced sustained actin polymerization. Disruption of actin filaments by cytochalasins inhibited increases in avidity of VLA-4 but not of Mac-1. Chemoattractants did not upregulate a Mn2+-inducible beta 1 neoepitope defined by the mAb 9EG7, but induced prolonged expression of a Mac-1 activation epitope recognized by the mAb CBRM1/5. This mAb inhibited chemoattractant-stimulated adhesion of eosinophils to intercellular adhesion molecule 1. Thus, regulation of VLA-4 was dependent on the actin cytoskeleton, whereas conformational changes appeared to be crucial for activation of Mac-1. To our knowledge, this is the first demonstration that physiological agonists, such as chemoattractants, can differentially regulate the avidity of a beta 1 and a beta 2 integrin expressed on the same leukocyte.

Ribonuclease P (RNase P) RNA is converted to a Cd(2+)-ribozyme by a single Rp-phosphorothioate modification in the precursor tRNA at the RNase P cleavage site.

To study the cleavage mechanism of bacterial Nase P RNA, we have synthesized precursor tRNA substrates carrying a single Rp- or Sp-phosphorothioate modification at the RNase P cleavage site. Both the Sp- and the Rp-diastereomer reduced the rate of processing by Escherichia coli RNase P RNA at least 1000-fold under conditions where the chemical step is rate-limiting. The Rp-modification had no effect and the Sp-modification had a moderate effect on precursor tRNA ground state binding to RNase P RNA. Processing of the Rp-diastereomeric substrate was largely restored in the presence of the "thiophilic" Cd2+ as the only divalent metal ion, demonstrating direct metal ion coordination to the (pro)-Rp substituent at the cleavage site and arguing against a specific role for Mg(2+)-ions at the pro-Sp oxygen. For the Rp-diastereomeric substrate, Hill plot analysis revealed a cooperative dependence upon [Cd2+] of nH = 1.8, consistent with a two-metal ion mechanism. In the presence of the Sp-modification, neither Mn2+ nor Cd2+ was able to restore detectable cleavage at the canonical site. Instead, the ribozyme promotes cleavage at the neighboring unmodified phosphodiester with low efficiency. Dramatic inhibition of the chemical step by both the Rp- and Sp-phosphorothioate modification is unprecedented among known ribozymes and points to unique features of transition state geometry in the RNase P RNA-catalyzed reaction.

A yeast manganese transporter related to the macrophage protein involved in conferring resistance to mycobacteria.

A novel Saccharomyces cerevisiae mutant, unable to grow in the presence of 12.5 mM EGTA, was isolated by replica plating. The phenotype of the mutant is caused by a single amino acid change (Gly149 to Arg) in the essential yeast gene CDC1. The mutant could be suppressed by overexpression of the SMF1 gene, which was isolated as an extragenic high-copy suppressor. The SMF1 gene codes for a highly hydrophobic protein and its deletion renders the yeast cells sensitive to low manganese concentration. In accordance with this observation, the smf1 null mutant exhibits reduced Mn2+ uptake at micromolar concentrations. Using a specific antibody, we demonstrated that Smf1p is located in the yeast plasma membrane. These results suggest that Smf1p is involved in high-affinity Mn2+ uptake. This assumption was also tested by overexpressing the SMF1 gene in the temperature-sensitive mutant of the mitochondrial processing peptidase (MAS1). SMF1 overexpression as well as addition of 1 mM Mn2+ to the growth medium complemented this mutation. This also suggests that in vivo Mas1p is a manganese-dependent peptidase. The yeast Smf1p resembles a protein from Drosophila and mammalian macrophages. The latter was implicated in conferring resistance to mycobacteria. A connection between Mn2+ transport and resistance or sensitivity to mycobacteria is discussed.

Heterometallic hybrids of homometallic human hemoglobins.

Hybridization experiments between normal Hb tetramers (Fe2+ Hb) and those with four metal-substituted hemes (i.e., replacement of Fe2+ by Co2+, Mg2+, Mn2+, Mn3+, Ni2+, or Zn2+) have revealed unexpected behavior. These homometallic Hbs have previously served as models that mimic the deoxy or oxy properties of normal Fe2+ Hb. In this study, hybrids were composed of one alpha 1 beta 1 dimer that is metal-substituted at both hemes, in association with a second dimer alpha 2 beta 2 that has normal Fe2+ hemes. Both metal-substituted subunits are unligated, whereas the two Fe2+ subunits either are both unligated or both ligated with O2, CO, or CN. It was found that four of the metal-substituted Hbs (Mg2+ Hb, Mn2+ Hb, Ni2+ Hb, and Zn2+ Hb) did not form detectable amounts of heterometallic hybrids with normal Fe2+ Hb even though (i) their homometallic parents formed tight tetrameric complexes with stabilities similar to that of Fe2+ Hb and (ii) hybrids with metal substitution at both alpha sites or both beta sites are known to form readily. This striking positional effect was independent of whether the normal Fe2+ hemes were ligated and of which ligand was used. These findings indicate that surprisingly large changes in tetramer behavior can arise from small and subtle perturbations at the heme sites. Possible origins of these effects are considered.

The two single-strand cleavages at each end of Tn10 occur in a specific order during transposition.

During Tn10 transposition, the element is excised from the donor site by double-strand cleavages at the two transposon ends. Double-strand cleavage is a central step in the nonreplicative transposition reaction of many transposons in both prokaryotes and eukaryotes. Evidence is presented to show that the Tn10 double-strand cut is made by an ordered, sequential cleavage of the two strands. The transferred strand is cut first, and then the nontransferred strand is cleaved. The single-strand nicked intermediate is seen to accumulate when Mn2+ is substituted for Mg2+ in the reaction or when certain mutant transposases are used. The fact that the transferred strand is cleaved before the non-transferred strand implies that the order of strand cleavages is not the determining factor that precludes a replicative mechanism of transposition.