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J D Robertus

Publications and source records attributed to J D Robertus.

At least 37 records · Page 2Linked to original sources
PubMed1996

Molecular cloning and kinetic characterization of a flavin-containing monooxygenase from Saccharomyces cerevisiae.

An open reading frame from yeast coding for a homologue of flavin containing monooxygenase (FMO) has been cloned into several Escherichia coli expression vectors. A His10 peptide attached to the amino terminus produced a high yield of soluble protein when coexpressed with GroEL and GroES. The protein was purified on an affinity column and characterized. The protein binds one mole per mole of flavin but the binding is relatively weak and 50 microM exogenous FAD is used to maintain full occupancy. The yeast enzyme, like mammalian enzymes, exhibits NADPH oxidase activity. The enzyme does not catalyze the oxidation of amines, but thiols, including glutathione, cysteine, and cysteamine, show substrate activity. The Km values for these are 7.0, 9.9, and 1.3 mM, respectively; kcat values are 94, 246, and 94 per min, respectively. The enzyme apparently does not accept xenobiotic compounds but may be involved in maintaining cellular reducing potential, probably through its action on cysteamine. This activity may represent the initial role of the FMO family of enzymes, giving rise to the multigene family of drug metabolizing enzymes seen in modern mammals.

Cloning, Molecular↗
PubMed1996

Structure and activity of an active site substitution of ricin A chain.

The A chain of ricin (RTA) is an N-glycosidase which inactivates ribosomes by removing a single adenine base from a conserved region of rRNA. X-ray structures and site-directed mutagenesis revealed that Arg 180 interacts with the target adenine hydrogen bonding with N3. It may fully or partially protonate that atom as part of the hydrolysis mechanism. Arg 180 was previously converted to His (R180H) and shown to greatly reduce activity. Here R180H is shown to reduce overall activity 500-fold against Artemia salina ribosomes. A 2.2 A crystal structure reveals the mutation causes a rearrangement of the active site cleft, with Tyr 80 moving to block access to the adenine recognition site. His 180 forms a strong aromatic interaction with Trp 211, Tyr 80, and Tyr 123. A complex is formed with 250 mM AMP. The nucleotide binds in the active site region, but in an apparently nonproductive orientation. His 180 cannot bond to N3 and is screened from the substrate analog by the intervening Tyr 80. It may be that natural polynucleotide substrates, using additional interactions, can displace Tyr 80 and effect a productive binding.

Adenosine Monophosphate↗
PubMed1996

Major structural differences between pokeweed antiviral protein and ricin A-chain do not account for their differing ribosome specificity.

Pokeweed antiviral protein (PAP) and the A-chain of ricin (RTA) are two members of a family of ribosome-inactivating proteins (RIPS) that are characterised by their ability to catalytically depurinate eukaryotic ribosomes, a modification that makes the ribosomes incapable of protein synthesis. In contrast to RTA, PAP can also inactivate prokaryotic ribosomes. In order to investigate the reason for this differing ribosome specificity, a series of PAP/RTA hybrid proteins was prepared to test for their ability to depurinate prokaryotic and eukaryotic ribosomes. Information from the X-ray structures of RTA and PAP was used to design gross polypeptide switches and specific peptide insertions. Initial gross polypeptide swaps created hybrids that had altered ribosome inactivation properties. Preliminary results suggest that the carboxy-terminus of the RIPs (PAP 219-262) does not contribute to ribosome recognition, whereas polypeptide swaps in the amino-terminal half of the proteins did affect ribosome inactivation. Structural examination identified three loop regions that were different in both structure and composition within the amino-terminal region. Directed substitution of RTA sequences into PAP at these sites, however, had little effect on the ribosome inactivation characteristics of the mutant PAPs, suggesting that the loops were not crucial for prokaryotic ribosome recognition. On the basis of these results we have identified regions of RIP primary sequence that may be important in ribosome recognition. The implications of this work are discussed.

Antiviral Agents↗
PubMed1996

Crystallization of the NAD-dependent 5,10-methylenetetrahydrofolate dehydrogenase from Saccharomyces cerevisiae.

Saccharomyces cerevisiae possesses three isozymes of 5,10-methylenetetrahydrofolate dehydrogenase (MTD). The NAD-dependent enzyme is the first monofunctional form found in eukaryotes. Here we report its crystallization in a form suitable for high-resolution structure. The space group is P4(2)2(1)2 with cell constants a = b = 75.9, c = 160.0 A, and there is one 36 kDa molecule in the asymmetric unit. Crystals diffract to 2.9 A resolution.

Crystallization↗
PubMed1996

Chitinases, chitosanases, and lysozymes can be divided into procaryotic and eucaryotic families sharing a conserved core.

Barley chitinase, bacterial chitosanase, and lysozymes from goose (GEWL), phage (T4L) and hen (HEWL) all hydrolyse related polysaccharides. The proteins share no significant amino-acid similarities, but have a structurally invariant core consisting of two helices and a three-stranded beta-sheet which form the substrate-binding and catalytic cleft. These enzymes represent a superfamily of hydrolases which are likely to have arisen by divergent evolution. Based on structural criteria, we divide the hydrolase superfamily into a bacterial family (chitosanase and T4L) and a eucaryotic family represented by chitinase and GEWL. Both families contain the core but have differing N- and C-terminal domains. Inclusion of chitinase and chitosanase in the superfamily suggests the archetypal catalytic mechanism of the group is an inverting mechanism. The retaining mechanism of HEWL is unusual.

Amino Acid Sequence↗
PubMed1996

X-ray structure of an anti-fungal chitosanase from streptomyces N174.

We report the 2.4 A X-ray crystal structure of a protein with chitosan endo-hydrolase activity isolated from Streptomyces N174. The structure was solved using phases acquired by SIRAS from a two-site methyl mercury derivative combined with solvent flattening and non-crystallographic two-fold symmetry averaging, and refined to an R-factor of 18.5%. The mostly alpha-helical fold reveals a structural core shared with several classes of lysozyme and barley endochitinase, in spite of a lack of shared sequence. Based on this structural similarity we postulate a putative active site, mechanism of action and mode of substrate recognition. It appears that Glu 22 acts as an acid and Asp 40 serves as a general base to activate a water molecule for an SN2 attack on the glycosidic bond. A series of amino-acid side chains and backbone carbonyl groups may bind the polycationic chitosan substrate in a deep electronegative binding cleft.

Binding Sites↗
PubMed1995

Site-directed mutagenesis of intersubunit boundary residues in histidine decarboxylase, a pH-dependent allosteric enzyme.

Histidine decarboxylase (HDC) from Lactobacillus 30a forms a trimer around a central cavity or well. Three active sites are formed around the well at the interface of each of two adjacent molecules. HDC exhibits cooperative kinetics at pH 7.6 and can be described in terms of a two-state, T and R, model. At pH 4.8, protons stabilize HDC in the R form. Asp 198 and Asp 53, from a neighboring molecule, are the core of the pH-sensitive mechanism controlling the shift in quaternary state. Eight site-directed mutations have been made to analyze the region. Several mutants, including the conversion of Asp 53 to Asn, cause HDC to exhibit sigmoidal kinetics even at pH 4.8. Others lock the enzyme into the T state. Kinetic analysis suggests that kcat values for T and R states are similar. The Km for the T state, near 8 mM, exceeds that for the R state by 40-fold and shows HDC is primarily regulated by altering its affinity for substrate.

Allosteric Site↗
PubMed1995

The refined crystal structure of an endochitinase from Hordeum vulgare L. seeds at 1.8 A resolution.

Class II chitinases (EC 3.2.1.14) are plant defense proteins. They hydrolyze chitin, an insoluble beta-1,4-linked polymer of N-acetylglucosamine (NAG), which is a major cell-wall component of many fungal hyphae. We previously reported the three-dimensional structure of the 26 kDa class II endochitinase from barley seeds at 2.8 A resolution, determined using multiple isomorphous replacement (MIR) methods. Here, we report the crystallographic refinement of this chitinase structure against data to 1.8 A resolution using rounds of hand rebuilding coupled with molecular dynamics (X-PLOR). The final model has an R-value of 18.1% for the 5.0 to 1.8 A data shell and 19.8% for the 10.0 to 1.8 A shell, and root-mean-square deviations from standard bond lengths and angles of 0.017 A and 2.88 degrees, respectively. The 243 residue molecule has one beta-sheet, ten alpha-helices and three disulfide bonds; 129 water molecules are included in the final model. We show structural comparisons confirming that chitinase secondary structure resembles lysozyme at the active site region. Based on substrate binding to lysozyme, we have built a hypothetical model for the binding of a hexasaccharide into the pronounced active site cleft of chitinase. This provides the first view of likely substrate interactions from this family of enzymes; the model is consistent with a lysozyme-like mechanism of action in which Glu67 acts as proton donor and Glu89 is likely to stabilize the transition state oxycarbonium ion. These binding site residues, and many hydrophobic residues are conserved in a range of plant chitinases. This endochitinase structure will serve as a model for other plant chitinases, and that catalytic models based on this structure will be applicable to the entire enzyme family.

Acetylglucosamine↗
PubMed1994

Introduction of a disulfide bond into ricin A chain decreases the cytotoxicity of the ricin holotoxin.

Wild type ricin A chain (RTA) contains two cysteine residues (Cys171 and Cys259). Cys259 forms the interchain disulfide bond of ricin holotoxin with Cys4 of ricin B chain (RTB). We have used site-directed mutagenesis of RTA cDNA to convert Cys171 to Ser and to introduce a disulfide bond into RTA by converting Ser215 and Met255 to Cys residues. Mutant RTA was expressed in Escherichia coli and directed to the oxidizing environment of the periplasmic space where the Cys215-Cys255 disulfide bond was formed. The disulfide-containing RTA mutant had an in vitro catalytic activity similar to that of an identical form of recombinant RTA that lacked the S215C and M255C mutations. In the presence of glutathione and protein disulfide isomerase, this RTA variant reassociated with RTB to form ricin holotoxin. Incubation of this holotoxin with increasing concentrations of dithiothreitol showed that the interchain disulfide bond joining RTA and RTB was more readily reduced than the intrachain disulfide bond in RTA. Ricin in which the RTA moiety contained the disulfide bond was 15-18-fold less cytotoxic to HeLa or Vero cells than ricin in which the RTA did not contain the stabilizing disulfide cross-link. Since these ricin molecules had identical RTB cell binding and RTA catalytic activities, we suggest that the observed reduction in cytotoxicity caused by the introduced disulfide bond resulted from a constraint on the unfolding of RTA, indicating that such unfolding is necessary for the membrane translocation of RTA during its entry into the cytosol.

Animals↗
PubMed1994

Ricin: structure, mode of action, and some current applications.

Ricin is an abundant protein component of Ricinus communis seeds (castor beans) that is exquisitely toxic to mammalian cells. It consists of an enzymic polypeptide that catalyzes the N-glycosidic cleavage of a specific adenine residue from 28S ribosomal RNA, joined by a single disulfide bond to a galactose (cell)-binding lectin. The enzymatic activity renders ribosomes containing depurinated 28S RNA incapable of protein synthesis. The bipartite molecular structure of ricin allows it to bind to the mammalian cell surface, enter via endocytic uptake, and deliver the catalytically active polypeptide into the cell cytosol where it irreversibly inhibits protein synthesis causing cell death. Because of its cytotoxic potency, modified ricin is being used for the selective killing of unwanted cells and for the toxigenic ablation of cell lineages in transgenic organisms.

Animals↗
PubMed1993

The 2.5 A structure of pokeweed antiviral protein.

The pokeweed antiviral protein (PAP), isolated from the leaves of Phytolacca americana, is one of a family of plant and bacterial ribosome-inhibiting proteins (RIPs) which act as specific N-glycosidases on rRNA. Here we report the three-dimensional structure of PAP determined to 2.5 A resolution by X-ray crystallography. After 14 rounds of refinement, the R factor is 0.17 for 5.0 to 2.5 A data. The protein is homologous with the A chain of ricin and exhibits a very similar folding pattern. The positions of key active site residues are also similar. We also report the 2.8 A structure of PAP complexed with a substrate analog, formycin 5'-monophosphate. As seen previously in ricin, the formycin ring is stacked between invariant tyrosines 72 and 123. Arg179 bonds to N-3 which is thought to be important in catalysis.

Amino Acid Sequence↗
PubMed1993

Crystallization of a chitosanase from Streptomyces N174.

Chitosanases are produced by many soil fungi and bacteria to degrade chitosan present in fungal cell walls. Here, we report the crystallization of a 29,500 dalton protein with chitosan endo-hydrolase activity isolated from Streptomyces N174. The crystals were grown by vapor diffusion. They are mechanically strong and diffract to at least 1.9 A resolution. The crystals belong to the monoclinic space group P2(1) with unit cell parameters a = 56.4 A, b = 59.6 A, c = 86.1 A and beta = 96.6 degrees. Cell parameters and crystal density are consistent with two chitosanase molecules per asymmetric unit.

Crystallization↗
PubMed1993

Site-directed alteration of three active-site residues of a pyruvoyl-dependent histidine decarboxylase.

The active site of histidine decarboxylase (HDC) from Lactobacillus 30a contains a pyruvoyl cofactor sitting at the interface of two molecules in a trimer. Although exhibiting hyperbolic kinetics at pH 4.8, near its optimum, HDC is cooperative at pH 7.6, indicating that the units of the trimer communicate. A Hill plot analysis shows that HDC, at pH 7.6, can be described by a two-state model. The tense (T) state has an apparent Km for histidine of 50 mM, while the relaxed (R) state has a Km of 5 mM. To explore the catalytic mechanism, three of the cross-boundary active-site residues were altered by site-directed mutagenesis and their effects observed. Ile-59 is known to act as lid on the substrate binding pocket; it was converted to Ala (I59A) and to Val (I59V). The former was inactive, attesting to the importance of this residue in the mechanism. The I59V mutant showed a decrease in Km and in kcat at pHs 4.8 and 7.6. Ile-59 appears to help orient substrate properly for catalysis; decreasing its size expands the binding site. This may allow the substrate to bind more readily, but in a number of conformations which are not optimal for catalysis. Conversion of Tyr-62 to Phe (Y62F) had no effect on catalysis but raised the Km 7-fold at pH 4.8. Asp-63 appears to form an ion pair to the substrate imidazolium. Conversion to the neutral amide (D63N) had no effect on the kcat, but raised the Km 240-fold at pH 4.8. This is consistent with the notion that the ion pair is up to 3 kcal/mol stronger than a simple hydrogen bond with the substrate. The mutant had no detectable activity at pH 7.6.

Alanine↗
PubMed1993

Crystal structure of an endochitinase from Hordeum vulgare L. seeds.

Higher plants contain multiple constitutively expressed proteins for defense against infection by viruses, bacteria, and fungi. One such class of proteins, the chitinases, are effective antifungal agents because they hydrolyze the insoluble beta-1,4-linked polymer of N-acetylglucosamine (chitin), which is the major component of the mycelial cell wall of many fungi. We report here the three-dimensional, 2.8 A, crystal structure of a 26 kDa endochitinase from barley (Hordeum vulgare L.) seeds. The 243 amino acid residue molecule is rich in alpha-helices and has three disulfide bonds. A prominent elongated cleft runs the length of the molecule, and is presumably the region responsible for substrate binding and catalysis. Endochitinases from various species of plants show a high degree of similarity in their amino acid sequences. It is therefore likely that the barley endochitinase structure can serve as a model for other plant endochitinases and that catalytic models based on that structure will be applicable to the entire enzyme family.

Chitinases↗
PubMed1993

Structure of recombinant ricin A chain at 2.3 A.

The plant cytotoxin ricin is a heterodimer with a cell surface binding (B) chain and an enzymatically active A chain (RTA) known to act as a specific N-glycosidase. RTA must be separated from B chain to attack rRNA. The X-ray structure of ricin has been solved recently; here we report the structure of the isolated A chain expressed from a clone in Escherichia coli. This structure of wild-type rRTA has and will continue to serve as the parent compound for difference Fouriers used to assess the structure of site-directed mutants designed to analyze the mechanism of this medically and commercially important toxin. The structure of the recombinant protein, rRTA, is virtually identical to that seen previously for A chain in the heterodimeric toxin. Some minor conformational changes due to interactions with B chain and to crystal packing differences are described. Perhaps the most significant difference is the presence in rRTA of an additional active site water. This molecule is positioned to act as the ultimate nucleophile in the depurination reaction mechanism proposed by Monzingo and Robertus (1992, J. Mol. Biol. 227, 1136-1145).

Cloning, Molecular↗
PubMed1992

X-ray analysis of substrate analogs in the ricin A-chain active site.

Ricin A-chain is an N-glycosidase that hydrolyzes the adenine ring from a specific adenosine of rRNA. Formycin monophosphate (FMP) and adenyl(3'-->5')guanosine (ApG) were bound to ricin A-chain and their structures elucidated by X-ray crystallography. The formycin ring stacks between tyrosines 80 and 123 and at least four hydrogen bonds are made to the adenine moiety. A residue invariant in this enzyme class, Arg180, appears to hydrogen bond to N-3 of the susceptible adenine. Three hypothetical models for binding a true hexanucleotide substrate, CGAGAG, are proposed. They incorporate adenine binding, shown by crystallography, but also include geometry likely to favor catalysis. For example, efforts have been made to orient the ribose ring in a way that allows solvent attack and oxycarbonium stabilization by the enzyme. The favored model is a simple perturbation of the tetraloop structure determined by nuclear magnetic resonance for similar polynucleotides. The model is attractive in that specific roles are defined for conserved protein residues. A mechanism of action is proposed. It invokes oxycarbonium ion stabilization on ribose by Glu177 in the transition state. Arg180 stabilizes anion development on the leaving adenine by protonation at N-3 and may activate a trapped water molecule that is the ultimate nucleophile in the depurination.

Adenosine↗
PubMed1992

Crystallization of an endochitinase from Hordeum vulgar L. seeds.

Higher plants contain several constitutively expressed proteins for protection against infections by viruses, bacteria and fungi. Here we report the crystallization of a polypeptide with antifungal activity, a 26,000 dalton endochitinase from barley (Hordeum vulgare L.) seeds, in a form suitable for high-resolution X-ray analysis. Crystals were grown by vapor diffusion under several different conditions. The best crystals, obtained with ammonium sulfate as the precipitant, belong to the tetragonal space group P4(1)2(1)2 (P4(3)2(1)2), with cell dimensions a = b = 62.9 A and c = 96.0 A. The cell dimensions are consistent with one endochitinase molecule per asymmetric unit, and the crystals diffract to at least 2.0 A resolution.

Ammonium Sulfate↗