Interchange of catalytic activity within the 2-enoyl-coenzyme A hydratase/isomerase superfamily based on a common active site template.

The structures and chemical pathways associated with the members of the 2-enoyl-CoA hydratase/isomerase enzyme superfamily are compared to show that a common active site design provides the members of this family with a CoA binding site, an expandable acyl binding pocket, an oxyanion hole for binding/polarizing the thioester C=O, and multiple active site stations for the positioning of acidic and basic amino acid side chains for use in proton shuttling. It is hypothesized that this active site template can be tailored to catalyze a wide range of chemical transformations through strategic positioning of acid/base residues among the active site stations. To test this hypothesis, the active site of one member of the 2-enoyl-CoA hydratase/isomerase family, 4-chlorobenzoyl-CoA dehalogenase, was altered by site-directed mutagenesis to include the two glutamate residues functioning in acid/base catalysis in a second family member, crotonase. Catalysis of the syn hydration of crotonyl-CoA, absent in the wild-type 4-chlorobenzoyl-CoA dehalogenase, was shown to occur with the structurally modified 4-chlorobenzoyl-CoA dehalogenase at kcat = 0.06 s-1 and Km = 50 microM.     

The primary structure of human ribonuclease/angiogenin inhibitor (RAI) discloses a novel highly diversified protein superfamily with a common repetitive module.

Immunological screening of a lambda gt11 library, constructed from HeLa mRNA, yielded several ribonuclease/angiogenin inhibitor (RAI) cDNA clones containing 900-bp inserts. Northern blot analysis revealed that the length of the RAI mRNA is approximately 1.9 kb. Construction and screening of a eukaryotic cDNA expression library (HeLa) containing preferentially complete cDNA inserts led to the isolation of a full length clone. The complete nucleotide sequence was determined. The C-terminal amino acid sequence deduced from the cDNA is identical to the peptide sequence obtained from a CNBr fragment of RAI, confirming the identity of the clone. The deduced primary structure of RAI consists of eight homologous tandem repeats with remarkable periodicity of leucine and cysteine residues. Each repeat is derived from the duplication of a leucine-rich 28-amino-acid module. This prototype module is closely related to a repetitive 24-amino-acid motif of unclear function, previously found in proteins involved in important biological processes such as blood coagulation, embryonic development, cell morphogenesis and signal transduction. Although homologous, the RAI modules show distinct differences in length and amino acid composition to the modules of this group of proteins, demonstrating their high potential of variability, necessary for adaptation to very diverse roles. Based on our results we propose that these repetitive modules are a common structural feature of a novel protein superfamily whose members exert their function by highly specific protein-protein interactions.     

Copper-regulated trafficking of the Menkes disease copper ATPase is associated with formation of a phosphorylated catalytic intermediate

The Menkes protein (MNK; ATP7A) is a copper-transporting P-type ATPase that is defective in the copper deficiency disorder, Menkes disease. MNK is localized in the trans-Golgi network and transports copper to enzymes synthesized within secretory compartments. However, in cells exposed to excessive copper, MNK traffics to the plasma membrane where it functions in copper efflux. A conserved feature of all P-type ATPases is the formation of an acyl-phosphate intermediate, which occurs as part of the catalytic cycle during cation transport. In this study we investigated the effect of mutations within conserved catalytic regions of MNK on intracellular localization and trafficking from the trans-Golgi network (TGN). Our findings suggest that mutations that block formation of the phosphorylated catalytic intermediate also prevent copper-induced relocalization of MNK from the TGN. Furthermore, mutations in the phosphatase domain, which resulted in hyperphosphorylation of MNK, caused constitutive trafficking from the TGN to the plasma membrane. A similar effect on trafficking was observed with a phosphatase mutation in the closely related copper ATPase, ATP7B, affected in Wilson disease. These findings suggest that the copper-induced trafficking of the Menkes and Wilson disease copper ATPases is associated with the phosphorylated intermediate that is formed during the catalysis of these pumps. Our findings describe a novel mechanism for regulating the subcellular location of a transport protein involving the recognition of intermediate conformations during catalysis.     

Evidence for the formation of a covalent thiosulfinate intermediate with peroxiredoxin in the catalytic mechanism of sulfiredoxin

The typical 2-Cys peroxiredoxins are thiol-peroxidases involved in the physiology of hydrogen peroxide not only as a toxic but also as a signaling molecule. Coordination of these functions depends on the sulfinylation of the catalytic Cys, a modification reversed by ATP-dependent sulfiredoxin, which specifically reduces the sulfinic acid group of overoxidized 2-Cys peroxiredoxins into a sulfenic acid. Sulfiredoxin was originally proposed to operate by covalent catalysis, with formation of a peroxiredoxin-sulfiredoxin intermediate linked by a thiosulfinate bond between the catalytic Cys of both partners, a hypothesis rejected by a study of the human enzyme. To settle the argument, we investigated the catalytic mechanism of Saccharomyces cerevisiae sulfiredoxin, by the characterization of the nature and kinetics of formation of the protein species formed between sulfiredoxin and its substrate in the presence of ATP, using mutants of the non-essential Cys residues of both proteins. We observed the formation of a dithiothreitol-reducible peroxiredoxin-sulfiredoxin species using SDS-PAGE and Western blot analysis, and its mass was shown to correspond to a thiosulfinate complex by high resolution mass spectrometry coupled to liquid chromatography. We next measured indirectly and directly a rate constant of formation of the thiosulfinate species of approximately 2 min(-1), for both wild-type and mutant sulfiredoxins, at least equal to the steady-state rate constant of the reaction, with a stoichiometry of 1:1 relative to peroxiredoxin. Taken altogether, our results strongly argue in favor of the formation of a covalent thiosulfinate peroxiredoxin-sulfiredoxin species as an intermediate on the catalytic pathway.     

Evolutionary genomics of the HAD superfamily: understanding the structural adaptations and catalytic diversity in a superfamily of phosphoesterases and allied enzymes

The HAD (haloacid dehalogenase) superfamily includes phosphoesterases, ATPases, phosphonatases, dehalogenases, and sugar phosphomutases acting on a remarkably diverse set of substrates. The availability of numerous crystal structures of representatives belonging to diverse branches of the HAD superfamily provides us with a unique opportunity to reconstruct their evolutionary history and uncover the principal determinants that led to their diversification of structure and function. To this end we present a comprehensive analysis of the HAD superfamily that identifies their unique structural features and provides a detailed classification of the entire superfamily. We show that at the highest level the HAD superfamily is unified with several other superfamilies, namely the DHH, receiver (CheY-like), von Willebrand A, TOPRIM, classical histone deacetylases and PIN/FLAP nuclease domains, all of which contain a specific form of the Rossmannoid fold. These Rossmannoid folds are distinguished from others by the presence of equivalently placed acidic catalytic residues, including one at the end of the first core beta-strand of the central sheet. The HAD domain is distinguished from these related Rossmannoid folds by two key structural signatures, a "squiggle" (a single helical turn) and a "flap" (a beta hairpin motif) located immediately downstream of the first beta-strand of their core Rossmanoid fold. The squiggle and the flap motifs are predicted to provide the necessary mobility to these enzymes for them to alternate between the "open" and "closed" conformations. In addition, most members of the HAD superfamily contains inserts, termed caps, occurring at either of two positions in the core Rossmannoid fold. We show that the cap modules have been independently inserted into these two stereotypic positions on multiple occasions in evolution and display extensive evolutionary diversification independent of the core catalytic domain. The first group of caps, the C1 caps, is directly inserted into the flap motif and regulates access of reactants to the active site. The second group, the C2 caps, forms a roof over the active site, and access to their internal cavities might be in part regulated by the movement of the flap. The diversification of the cap module was a major factor in the exploration of a vast substrate space in the course of the evolution of this superfamily. We show that the HAD superfamily contains 33 major families distributed across the three superkingdoms of life. Analysis of the phyletic patterns suggests that at least five distinct HAD proteins are traceable to the last universal common ancestor (LUCA) of all extant organisms. While these prototypes diverged prior to the emergence of the LUCA, the major diversification in terms of both substrate specificity and reaction types occurred after the radiation of the three superkingdoms of life, primarily in bacteria. Most major diversification events appear to correlate with the acquisition of new metabolic capabilities, especially related to the elaboration of carbohydrate metabolism in the bacteria. The newly identified relationships and functional predictions provided here are likely to aid the future exploration of the numerous poorly understood members of this large superfamily of enzymes.     

Metabolism of N-methyl-amide by cytochrome P450s: formation and characterization of highly stable carbinol-amide intermediate

We report unambiguous proof of the stability of a carbinol intermediate in the case of P450 metabolism of an N-methylated natural cyclo-peptide, namely tentoxin. Under mild acidic or neutral conditions, the lifetime of carbinol-amide is long enough to be fully characterized. This metabolite has been characterized using specifically labeled (14) C-methyl tentoxin isotopomers, HPLC, HPLC-MS, MS-MS and NMR. Under stronger acidic conditions, the stability of this metabolite vanishes through deformylation. A theoretical mechanistic investigation reveals that the stability is governed by the accessibility of the nitrogen lone pair and its protonation state. For carbinol-amines, even in neutral conditions, the energy barrier for deformylation is low enough to allow rapid deformylation. Carbinol-amide behaves differently. Under neutral conditions, delocalization of the nitrogen lone pair increases the energy barrier of deformylation that is a slow process under such conditions. After protonation, we were able to optimize a deformylation transition that is lower in energy and thus accounts for the lower stability of carbinol-amides observed experimentally in acidic conditions. Finally, by considering the protocol usually used for extraction and analysis of this type of metabolite, carbinol-amide may thus be frequently ignored in drug metabolism pathways.     

X-ray crystal structure of rabbit N-acetylglucosaminyltransferase I: catalytic mechanism and a new protein superfamily

N:-acetylglucosaminyltransferase I (GnT I) serves as the gateway from oligomannose to hybrid and complex N:-glycans and plays a critical role in mammalian development and possibly all metazoans. We have determined the X-ray crystal structure of the catalytic fragment of GnT I in the absence and presence of bound UDP-GlcNAc/Mn(2+) at 1.5 and 1.8 A resolution, respectively. The structures identify residues critical for substrate binding and catalysis and provide evidence for similarity, at the mechanistic level, to the deglycosylation step of retaining beta-glycosidases. The structuring of a 13 residue loop, resulting from UDP-GlcNAc/Mn(2+) binding, provides an explanation for the ordered sequential 'Bi Bi' kinetics shown by GnT I. Analysis reveals a domain shared with Bacillus subtilis glycosyltransferase SpsA, bovine beta-1,4-galactosyl transferase 1 and Escherichia coli N:-acetylglucosamine-1-phosphate uridyltransferase. The low sequence identity, conserved fold and related functional features shown by this domain define a superfamily whose members probably share a common ancestor. Sequence analysis and protein threading show that the domain is represented in proteins from several glycosyltransferase families.     

Light chain of botulinum neurotoxin serotype A: structural resolution of a catalytic intermediate

Botulinum neurotoxin serotype A (BoNT/A, 1296 residues) is a zinc metalloprotease that cleaves SNAP25 to inhibit the fusion of neurotransmitter-carrying vesicles to the plasma membrane of peripheral neurons. BoNT/A is a disulfide-linked di-chain protein composed of an N-terminal, thermolysin-like metalloprotease light chain domain (LC/A, 448 residues) and a C-terminal heavy chain domain (848 residues) that can be divided into two subdomains, a translocation subdomain and a receptor binding subdomain. LC/A cleaves SNAP25 between residues Gln197-Arg198 and, unlike thermolysin, recognizes an extended region of SNAP25 for cleavage. The structure of a recombinant LC/A (1-425) treated with EDTA (No-Zn LC/A) was determined. The overall structure of No-Zn LC/A is similar to that reported for the holotoxin, except that it lacks the Zn ion, indicating that the role of Zn is catalytic not structural. In addition, structures of a noncatalytic mutant LC/A (Arg362Ala/Tyr365Phe) complexed with and without an inhibitor, ArgHX, were determined. The overall structure and the active site conformation for the mutant are the same as wild type. When the inhibitor binds to the active site, the carbonyl and N-hydroxyl groups form a bidentate ligand to the Zn ion and the arginine moiety binds to Asp369, suggesting that the inhibitor-bound structure mimics a catalytic intermediate with the Arg moiety binding at the P1' site. Consistent with this model, mutation of Asp369 to Ala decreases the catalytic activity of LC/A by approximately 600-fold, and the residual activity is not inhibited by ArgHX. These results provide new information on the reaction mechanism and insight into the development of strategies for small molecule inhibitors of BoNTs.     

Kinetic study of a substrate cycle involving a chemical step: highly amplified determination of phenolic compounds

A kinetic analysis of a substrate cycle in which one of the two steps was substituted by a chemical reaction has been made. The model is illustrated by the amplified determination, in a continuous assay, of phenolic compounds at low concentrations using the enzyme tyrosinase and β-NADH to reduce the o-quinone product of catalytic activity. Progress curves corresponding to β-NADH disappearance were not linear and followed first-order kinetics. Knowledge of the kinetics of the reaction has allowed us to achieve detection limits as low as 50 nM in a simple 10-min assay. There is no analytical solution to the non-linear differential equation system that describes the kinetics of the reaction, therefore, computer simulations of its dynamic behaviour are also presented, good agreement with the experimental results being obtained. The method is applicable to the measurement of any other metabolite, and its amplification capacity as well as the simplicity of determining kinetic parameters enable it to be implemented in a bioreactor for automation purposes.     

Disulfide structures of highly bridged peptides: a new strategy for analysis.

A new approach is described for analyzing disulfide linkage patterns in peptides containing tightly clustered cystines. Such peptides are very difficult to analyze with traditional strategies, which require that the peptide chain be split between close or adjacent Cys residues. The water-soluble tris-(2-carboxyethyl)-phosphine (TCEP) reduced disulfides at pH 3, and partially reduced peptides were purified by high performance liquid chromatography with minimal thiol-disulfide exchange. Alkylation of free thiols, followed by sequencer analysis, provided explicit assignment of disulfides that had been reduced. Thiol-disulfide exchange occurred during alkylation of some peptides, but correct deductions were still possible. Alkylation competed best with exchange when peptide solution was added with rapid mixing to 2.2 M iodoacetamide. Variants were developed in which up to three alkylating agents were used to label different pairs of thiols, allowing a full assignment in one sequencer analysis. Model peptides used included insulin (three bridges, intra- and interchain disulfides; -Cys.Cys- pair), endothelin and apamin (two disulfides; -Cys.x.Cys- pair), conotoxin GI and isomers (two disulfides; -Cys.Cys- pair), and bacterial enterotoxin (three bridges within 13 residues; two -Cys.Cys- pairs). With insulin, all intermediates in the reduction pathway were identified; with conotoxin GI, analysis was carried out successfully for all three disulfide isomers. In addition to these known structures, the method has been applied successfully to the analysis of several previously unsolved structures of similar complexity. Rates of reduction of disulfide bonds varied widely, but most peptides did not show a strongly preferred route for reduction.     

Kinetics of the formation of thrombin-thrombospondin complexes: involvement of a 77-kDa intermediate

Thrombin forms sodium dodecyl sulfate stable complexes of 77 and greater than 450 kDa with proteins secreted by activated platelets. The kinetics of formation of these complexes were investigated by addition of 125I-thrombin to the supernatant solution of A23187-activated platelets. Complexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis either with or without reduction of disulfide bonds. When analyzed on nonreduced gels, the 77-kDa complex reached a maximum at about 3 min and then declined as the greater than 450-kDa complex increased. On reduced gels (on which there was no greater than 450-kDa complex) the 77-kDa complex approached the level of the greater than 450-kDa complex on nonreduced gels. The half-time of formation was less than 1 min for the 77-kDa complex and about 15 min for the greater than 450-kDa complex. These time courses suggested that the 77-kDa complex was incorporated into the greater than 450-kDa complex as an essential precursor. Formation of complexes was inhibited by a competitive inhibitor or a noncompetitive inhibitor of thrombin, and the pH dependence of formation of both complexes was similar to the pH dependence for catalytic activity of thrombin. Ca2+ inhibited formation of the greater than 450-kDa complex but not of the 77-kDa complex. A model is presented in which thrombin and a secreted protein form a 77-kDa complex by a process that involves the active site of thrombin. The 77-kDa complex is then incorporated into a greater than 450-kDa complex by thiol-disulfide exchange with thrombospondin, a process that is inhibited by Ca2+. Thrombin in the greater than 450-kDa complex had no catalytic activity.     

Double Holliday structure: a possible in vivo intermediate form of general recombination in Escherichia coli

From Escherichia coli cells we purified '8'-shaped dimeric molecules in which two circular DNA molecules of bacteriophage lambda were joined at a homologous site. Some of them had a complex junction which we interpreted as being two closely spaced Holliday structures because of (i) superhelicity of the molecule, (ii) the sedimentation rate of the molecule in sucrose gradients, and (iii) the appearance in the electron microscope. Other 'figure-eights's' had two separate homologous junctions, presumably two Holliday bridges. A possible role for these 'double Holliday structures' in UV-stimulated recA-dependent recombination in vivo is discussed.     

Restin: a novel intermediate filament-associated protein highly expressed in the Reed-Sternberg cells of Hodgkin''s disease.

We have identified a cDNA coding for a protein of 160 kDa which is expressed in in vitro cultured human peripheral blood monocytes. The predicted amino acid sequence contains an alpha-helical rod domain possessing features characteristic of intermediate filament proteins. However, the immunocytochemical staining pattern, abundance and solubility in Triton X-100/high salt buffers suggest that this protein is probably only associated with the intermediate filament network and represents a new type of intermediate filament associated protein. In a survey of normal, inflammatory and human tumour tissue samples, this protein, which we have named restin, was found to be highly expressed in Reed-Sternberg cells, the tumoral cells diagnostic for Hodgkin's disease. We suggest that restin overexpression may be a contributing factor in the progression of Hodgkin's disease.     

Heterocycle formation in vibriobactin biosynthesis: alternative substrate utilization and identification of a condensed intermediate

The iron-chelating peptide vibriobactin of the pathogenic Vibrio cholerae is assembled by a four-subunit nonribosomal peptide synthetase complex, VibE, VibB, VibH, and VibF, using 2,3-dihydroxybenzoate and L-threonine as precursors to two 2,3-dihydroxyphenyl- (DHP-) methyloxazolinyl groups in amide linkage on a norspermidine scaffold. We have tested the ability of the six-domain VibF subunit (Cy-Cy-A-C-PCP-C) to utilize various L-threonine analogues and found the beta-functionalized amino acids serine and cysteine can function as alternate substrates in aminoacyl-AMP formation (adenylation or A domain), aminoacyl-S-enzyme formation (A domain), acylation by 2,3-dihydrobenzoyl- (DHB-) S-VibB (heterocyclization or Cy domain), heterocyclization to DHP-oxazolinyl- and DHP-thiazolinyl-S-enzyme forms of VibF (Cy domain) as well as transfer to DHB-norspermidine at both N(5) and N(9) positions (condensation or C domain) to make the bis(oxazolinyl) and bis(thiazolinyl) analogues of vibriobactin. When L-threonyl-S-pantetheine or L-threonyl-S-(N-acetyl)cysteamine was used as a small-molecule thioester analogue of the threonyl-S-VibF acyl enzyme intermediate, the Cy domain(s) of a CyCyA fragment of VibF generated DHB-threonyl-thioester products of the condensation step but not the methyloxazolinyl thioesters of the heterocyclization step. This clean separation of condensation from cyclization validates a two-stage mechanism for threonyl, seryl, and cysteinyl heterocyclization domains in siderophore and antibiotic synthetases. Full heterocyclization activity could be restored by providing CyCyA with the substrate L-threonyl-S-peptidyl carrier protein (PCP)-C2, suggesting an important role for the protein scaffold component of the heterocyclization acceptor substrate. We also examined heterocyclization donor substrate specificity at the level of acyl group and protein scaffold and observed intolerance for substitution at either position.     

Replacement of two invariant serine residues in chorismate synthase provides evidence that a proton relay system is essential for intermediate formation and catalytic activity

Chorismate synthase is the last enzyme of the common shikimate pathway, which catalyzes the anti-1,4-elimination of the 3-phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate (EPSP) to generate chorismate, a precursor for the biosynthesis of aromatic compounds. Enzyme activity relies on reduced FMN, which is thought to donate an electron transiently to the substrate, facilitating C(3)-O bond breakage. The crystal structure of the enzyme with bound EPSP and the flavin cofactor highlighted two invariant serine residues interacting with a bound water molecule that is close to the C(3)-O of EPSP. In this article we present the results of a mutagenesis study where we replaced the two invariant serine residues at positions 16 and 127 of the Neurospora crassa chorismate synthase with alanine, producing two single-mutant proteins (Ser16Ala and Ser127Ala) and a double-mutant protein (Ser16AlaSer127Ala). The residual activity of the Ser127Ala and Ser16Ala single-mutant proteins was found to be six-fold and 70-fold lower, respectively, than that of the wild-type protein. No residual activity was detected for the Ser16AlaSer127Ala double-mutant protein, and formation of the typical transient intermediate, characteristic for the chorismate synthase-catalysed reaction, was not observed, in contrast to the single-mutant proteins. On the basis of the structure of the enzyme, we propose that Ser16 and Ser127 form part of a proton relay system among the isoalloxazine ring of FMN, histidine 106 and the phosphate group of EPSP that is essential for the formation of the transient intermediate and for substrate turnover.     

Partial complementation of embryo defective mutations: a general strategy to elucidate gene function

The EMB 506 gene has been characterised as essential for embryo development. To provide insights into the role of EMB 506, which is hidden by the embryo defective phenotype, the ABI3 promoter was fused to the EMB 506 cDNA. The expression of such a transgene should provide sufficient protein during embryogenesis to ensure normal embryo development in homozygous emb 506 seeds. We show that homozygous emb 506 seedlings, partially complemented with the ABI3::EMB 506 transgene, can be obtained. Most of the rescued emb 506 plants are able to flower and to set normal seeds, but show mild to severe depigmentation of rosette leaves and/or inflorescences. This effect on chloroplast development indicated a putative chloroplast localisation of the EMB 506 protein, which was demonstrated by GFP-protein fusion. However, EMB 506 cannot be considered as a chloroplast housekeeping protein only, since EMB 506 is not present in all photosynthetic tissues. This study demonstrates the power of this simple strategy, which could be widely applied to other emb mutants and which may reveal similar or additional roles for EMB genes at vegetative stages of the life cycle.     

Methylation of DNA by three N-nitroso compounds: evidence for sequence specific methylation by a common intermediate

Methylation in vitro of calf thymus DNA, a supercoiled plasmid, poly(dG).poly(dC), and poly(dGdC).poly(dGdC) by N-nitroso(acetoxy-methyl)methylamine and N-nitroso(acetoxybenzyl)methylamine in the presence of esterase, and by N-nitrosomethylurea was investigated. Although there were differences in the amounts of 7-methylguanine and O6-methylguanine formed in the various DNA substrates, the methylation pattern was the same for each of these methylating agents. The three compounds reacted identically when methylation of a portion of a 345 bp restriction fragment of the plasmid pBR322 was examined at nucleotide resolution by a sequencing assay. They also showed a tendency to react preferentially with particular guanines. These data suggest that the three N-nitroso compounds methylate DNA via a common intermediate such as the methyl diazonium ion, which exhibits some sequence specificity.