Engineering the disulphide bond patterns of secretory phospholipases A2 into porcine pancreatic isozyme. The effects on folding, stability and enzymatic properties.

Secretory phospholipases A2 (PLA2s) are small homologous proteins rich in disulphide bridges. These PLA2s have been classified into several groups based on the disulphide bond patterns found [Dennis, E. A. (1997) Trends Biochem. Sci. 22, 1-2]. To probe the effect of the various disulphide bond patterns on folding, stability and enzymatic properties, analogues of the secretory PLA2s were produced by protein engineering of porcine pancreatic PLA2. Refolding experiments indicate that small structural variations play an important role in the folding of newly made PLA2 analogues. Introduction of a C-terminal extension together with disulphide bridge 50-131 gives rise to an enzyme that displays full enzymatic activity having increased conformational stability. In contrast, introduction of a small insertion between positions 88 and 89 together with disulphide bridge 86-89 decreases the catalytic activity significantly, but does not change the stability. Both disulphide bridges 11-77 and 61-91 are important for the kinetic properties and stability of the enzyme. Disulphide bridge 11-77, but not 61-91, was found to be essential to resist tryptic breakdown of native porcine pancreatic PLA2.     

The internal Cys-207 of sorghum leaf NADP-malate dehydrogenase can form mixed disulphides with thioredoxin

The role of the internal Cys-207 of sorghum NADP-malate dehydrogenase (NADP-MDH) in the activation of the enzyme has been investigated through the examination of the ability of this residue to form mixed disulphides with thioredoxin mutated at either of its two active-site cysteines. The h-type Chlamydomonas thioredoxin was used, because it has no additional cysteines in the primary sequence besides the active-site cysteines. Both thioredoxin mutants proved equally efficient in forming mixed disulphides with an NADP-MDH devoid of its N-terminal bridge either by truncation, or by mutation of its N-terminal cysteines. They were poorly efficient with the more compact WT oxidised NADP-MDH. Upon mutation of Cys-207, no mixed disulphide could be formed, showing that this cysteine is the only one, among the four internal cysteines, which can form mixed disulphides with thioredoxin. These experiments confirm that the opening of the N-terminal disulphide loosens the interaction between subunits, making Cys-207, located at the dimer contact area, more accessible.     

A de novo designed N-terminal disulphide bridge stabilizes the Trichoderma reesei endo-1,4-beta-xylanase II

We have successfully engineered a disulphide bridge into the N-terminal region of Trichoderma reesei endo-1,4-beta-xylanase II (XYNII) by substituting Thr-2 and Thr-28 with cysteine. The T2C:T28C mutational changes increased the half-life in thermal inactivation of this mesophilic enzyme from approximately 40 s to approximately 20 min at 65 degrees C, and from less than 10 s to approximately 6 min at 70 degrees C. Therefore, the N-terminal disulphide bridge enables the use of XYNII at substantially higher temperatures than permitted by its native mesophilic counterpart. Altogether, thermostability increased by about 15 degrees C. The kinetic properties of the mutant XYNII were maintained at the level of the wild type enzyme. Our findings demonstrated that a properly designed disulphide bridge, here within the N-terminal region of XYNII, can be very effective in resisting thermal inactivation.     

How thioredoxin can reduce a buried disulphide bond

We present a study of the interaction between thioredoxin and the model enzyme pI258 arsenate reductase (ArsC) from Staphylococcus aureus. ArsC catalyses the reduction of arsenate to arsenite. Three redox active cysteine residues (Cys10, Cys82 and Cys89) are involved. After a single catalytic arsenate reduction event, oxidized ArsC exposes a disulphide bridge between Cys82 and Cys89 on a looped-out redox helix. Thioredoxin converts oxidized ArsC back towards its initial reduced state. In the absence of a reducing environment, the active-site P-loop of ArsC is blocked by the formation of a second disulphide bridge (Cys10-Cys15). While fully reduced ArsC can be recovered by exposing this double oxidized ArsC to thioredoxin, the P-loop disulphide bridge is itself inaccessible to thioredoxin. To reduce this buried Cys10-Cys15 disulphide-bridge in double oxidized ArsC, an intra-molecular Cys10-Cys82 disulphide switch connects the thioredoxin mediated inter-protein thiol-disulphide transfer to the buried disulphide. In the initial step of the reduction mechanism, thioredoxin appears to be selective for oxidized ArsC that requires the redox helix to be looped out for its interaction. The formation of a buried disulphide bridge in the active-site might function as protection against irreversible oxidation of the nucleophilic cysteine, a characteristic that has also been observed in the structurally similar low molecular weight tyrosine phosphatase.     

Vibrio spp. secrete proaerolysin as a folded dimer without the need for disulphide bond formation

Proaerolysin is an extracellular dimeric protein that is secreted across the inner and outer membranes of Aeromonas spp. in separate steps. To investigate the role of protein folding in the second step, one or more cysteine residues were introduced and the mutant proaerolysins were expressed in Aeromonas hydrophila and Aeromonas salmonicida , as well as Vibrio cholerae . Replacing Met-41 with Cys resulted in expression of a protein that could form a dimer in which the monomers were linked together by a disulphide bridge. A double mutant was also made, in which Gly-202 and Ile-445 were replaced with cysteine in order to allow the formation of an intrachain disulphide bridge when the molecule was correctly folded. The M41C covalent dimer and G202C/I445C proaerolysin with the new intrachain bridge were both easily detected inside the bacteria, and they later appeared in the culture supernatants. Small amounts of incorrectly folded proaerolysin were also observed in the cells, but they were not secreted. We conclude that proaerolysin folds and dimerizes before being released from the cell, and that correct folding is a requirement for secretion to occur. The proton ionophore CCCP reduced release of the folded proteins. Unoxidized protein was secreted by cells grown in β-mercaptoethanol and by a dsbA mutant of V. cholerae , indicating that disulphide bond formation may not be essential for release.     

Inactivation of horse liver mitochondrial aldehyde dehydrogenase by disulfiram. Evidence that disulfiram is not an active-site-directed reagent.

The inhibition of mitochondrial (pI 5) horse liver aldehyde dehydrogenase by disulfiram (tetraethylthiuram disulphide) was investigated to determine if the drug was an active-site-directed inhibitor. Stoichiometry of inhibition was determined by using an analogue, [35S]tetramethylthiuram disulphide. A 50% loss of the dehydrogenase activity was observed when only one site per tetrameric enzyme was modified, and complete inactivation was not obtained even after seven sites per tetramer were modified. Modification of only two sites accounted for a loss of 75% of the initial catalytic activity. The number of functioning active sites per tetrameric enzyme, as determined by the magnitude of the pre-steady-state burst of NADH formation, did not decrease until approx. 75% of the catalytic activity was lost. These data indicate that disulfiram does not modify the essential nucleophilic amino acid at the active site of the enzyme. The data support an inactivation mechanism involving the formation of a mixed disulphide with a non-essential cysteine residue, resulting in a lowered specific activity of the enzyme.     

An amino acid sequence in the active site of lipoamide dehydrogenase from pig heart

1. The two cysteine residues forming the disulphide bridge that comprises part of the active site of lipoamide dehydrogenase from pig heart were specifically labelled with iodo[2-(14)C]acetic acid. 2. A tryptic peptide containing these carboxymethylcysteine residues was isolated from digests of reduced and S-carboxymethylated lipoamide dehydrogenase and its amino acid sequence of 23 residues was determined. 3. The sequence is highly homologous with a similar sequence containing the active-site disulphide bridge of lipoamide dehydrogenase derived from the 2-oxoglutarate dehydrogenase complex of Escherichia coli (Crookes strain) and it is probable that, as in the bacterial enzyme, the disulphide bridge forms an intrachain loop containing six residues. The results indicate that the bacterial and mammalian proteins have a common genetic origin. 4. Amino acid sequences containing six other unique carboxymethylcysteine residues were also partly determined. 5. The analysis of the primary structure thus far is consistent with the view that the enzyme (mol.wt. approx. 110000) is composed of two identical polypeptide chains.     

The thiol groups of mouse immunoglobulin A. Incomplete formation of the C alpha 1-domain disulphide bridge.

The BALB/c IgA (immunoglobulin A) myeloma protein M167 contained on average 5.7 free SH groups per IgA dimer. These groups were preponderantly on the heavy chains and comprised two distinct populations: 3.3 exposed SH groups per dimer in the Fc region, and 2.4 buried SH groups per dimer in the Fd region, detectable o only after denaturation. To locate the cysteine residues involved, labelled peptides were purified from thermolysin digests of radioalkylated IgA by high-performance liquid chromatography. From the amino acid compositions of the peptides, the exposed thiol groups were assigned to Cys-307 in the C alpha 2 domain, which thus existed in the reduced form to an extent exceeding 80%. This residue may allow attachment of secretory component to dimer IgA in the mouse to proceed via thiol-disulphide exchange. The buried thiol groups were assigned to Cys-150 and Cys-208, in the C alpha 1 domain, each being in the reduced form to the extent of approx. 30%. This pair of residues would normally give rise to the characteristic intradomain disulphide bridge. It appears that disulphide formation is not a crucial event during folding of the C alpha 1 domain in IgA biosynthesis. The sequence in the region 140-151 was re-investigated, and residue 142 was shown to be serine, not cysteine, helping explain the lack of heavy-chain-light chain bonding in BALB/c mouse IgA. A disulphide-bond model for mouse IgA is proposed on the basis of these assignments and other features of the mouse alpha-chain sequence.     

Improving the thermostability and activity of Melanocarpus albomyces cellobiohydrolase Cel7B

Two different types of approach were taken to improve the hydrolytic activity towards crystalline cellulose at elevated temperatures of Melanocarpus albomyces Cel7B (Ma Cel7B), a single-module GH-7 family cellobiohydrolase. Structure-guided protein engineering was used to introduce an additional tenth disulphide bridge to the Ma Cel7B catalytic module. In addition, a fusion protein was constructed by linking a cellulose-binding module (CBM) and a linker from the Trichoderma reesei Cel7A to the C terminus of Ma Cel7B. Both approaches proved successful. The disulphide bridge mutation G4C/M70C located near the N terminus, close to the entrance of the active site tunnel of Ma Cel7B, led to improved thermostability (ΔT _m = 2.5°C). By adding the earlier found thermostability-increasing mutation S290T (ΔT _m = 1.5°C) together with the disulphide bridge mutation, the unfolding temperature was increased by 4°C (mutant G4C/M70C/S290T) compared to that of the wild-type enzyme, thus showing an additive effect on thermostability. Both disulphide mutants had increased activity towards microcrystalline cellulose (Avicel) at 75°C, apparently solely because of their improved thermostability. The addition of a CBM also improved the thermostability (ΔT _m = 2.5°C) and caused a clear (sevenfold) increase in the hydrolysis activity of Ma Cel7B towards Avicel at 70°C.     

Primary structure of the active site region of fungal cutinase,an enzyme involved in phytopathogenesis

Cutinase, a fungal extracellular enzyme involved in phytopathogenesis, was labeled by treatment with [³H]diisopropylfluorophosphate and by reduction of the only disulphide with dithioerythritol followed by treatment with iodo[1-¹⁴C]acetamide. A tryptic peptide containing both the active serine and one of the cys involved in the disulphide bridge was isolated and the primary structure was determined to be: Glu-Met-Leu-Gly-Leu-Phe-Gln-Gln-Ala-Asn-Thr-Lys-Cys-Pro-Asp-Ala-Thr-Leu-Ile-Ala-Gly-Gly-Tyr-Ser-Gln-Gly-(Ala)-Ala-Leu-Ala. This active site has very little homology with the active serine containing regions of other enzymes.     

Preparation of fluorescence quenched libraries containing interchain disulphide bonds for studies of protein disulphide isomerases

Protein disulphide isomerase is an enzyme that catalyses disulphide redox reactions in proteins. In this paper, fluorogenic and interchain disulphide bond containing peptide libraries and suitable substrates, useful in the study of protein disulphide isomerase, are described. In order to establish the chemistry required for the generation of a split-synthesis library, two substrates containing an interchain disulphide bond, a fluoroescent probe and a quencher were synthesized. The library consists of a Cys residue flanked by randomized amino acid residues at both sides and the fluoroescent Abz group at the amino terminal. All the 20 natural amino acids except Cys were employed. The library was linked to PEGA‒beads via methionine so that the peptides could be selectively removed from the resin by cleavage with CNBr. A disulphide bridge was formed between the bead‒linked library and a peptide containing the quenching chromophore (Tyr(NO₂)) and Cys(pNpys) activated for reaction with a second thiol. The formation and cleavage of the interchain disulphide bonds in the library were monitored under a fluoroescence microscope. Substrates to investigate the properties of protein disulphide isomerase in solution were also synthesized. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Streptomyces griseus trypsin is stabilized against autolysis by the cooperation of a salt bridge and cation-pi interaction

Streptomyces griseus trypsin (SGT) is a bacterial trypsin that lacks the conserved disulphide bond surrounding the autolysis loop. We investigated the molecular mechanism by which SGT is stabilized against autolysis. The autolysis loop connects to another surface loop via a salt bridge (Glu146-Arg222), and the Arg222 residue also forms a cation-pi interaction with Tyr217. Elimination of these bonds by site-directed mutagenesis showed that the surface salt bridge at Glu146-Arg222 is the main force stabilizing the enzyme against autolysis. The effect of the cation-pi interaction at Tyr217-Arg222 is small, however, its presence increases the half-life by about five hours and enhances the protein stability more than three-fold considering the catalytic activity in the presence of the salt bridge. The melting temperature also showed cooperation between the salt bridge and cation-pi interaction. These findings show that S. griseus trypsin is stabilized against autolysis through a cooperative network of a salt bridge and cation-pi interaction, which compensate for the absence of the conserved C136-C201 disulphide bond.     

Purification and physical-chemical properties of acetyl-CoA:arylamine N-acetyltransferase from pigeon liver

Acetyl-CoA:arylamine N-acetyltransferase (EC 2.3.1.5) from pigeon liver was purified by protamine sulfate precipitation, ion exchange chromatography on DEAE-A-25 Sephadex, gel filtration on Sephadex G-75, amethopterin-AH-Sepharose 4B affinity chromatography, and finally, gel filtration on Sephadex G-100. The enzyme preparation was homogeneous as judged by ultracentrifugation studies, SDS-polyacrylamide gel electrophoresis and gel filtration. The N-terminal amino acid was detected to be histidine and the complete amino acid composition is reported. The enzyme contains one disulfide bridge and two cysteine residues/mol monomer. The isoelectric point was estimated to be 4.8. The molecular weight was determined to be 32900 by high-speed sedimentation equilibrium analysis, 33000 by Sephadex G-100 gel filtration and 31600 by SDS-disc gel electrophoresis. The sedimentation coefficient from conventional sedimentation velocity runs was 3.1 S observed by ultraviolet optics. 'Active enzyme centrifugation' showed a sedimentation constant of 5.0 and 4.8 S for the purified enzyme and crude extract from pigeon liver, respectively, indicating that the enzyme forms a dimer under conditions of catalysis. It could be demonstrated that the inhibitor amethopterin was noncompetitive with respect to the acetyl donor and the acetyl acceptor. Acetyl-CoA:arylamine N-acetyltransferase was examined in different organs of pigeon. The enzyme was not inducible by 1,3-phenylenediamine and hexobarbital in vivo.     

Structure of glutathione reductase from Escherichia coli at 1.86 A resolution: comparison with the enzyme from human erythrocytes.

The crystal structure of the dimeric flavoenzyme glutathione reductase from Escherichia coli was determined and refined to an R-factor of 16.8% at 1.86 A resolution. The molecular 2-fold axis of the dimer is local but very close to a possible crystallographic 2-fold axis; the slight asymmetry could be rationalized from the packing contacts. The 2 crystallographically independent subunits of the dimer are virtually identical, yielding no structural clue on possible cooperativity. The structure was compared with the well-known structure of the homologous enzyme from human erythrocytes with 52% sequence identity. Significant differences were found at the dimer interface, where the human enzyme has a disulfide bridge, whereas the E. coli enzyme has an antiparallel beta-sheet connecting the subunits. The differences at the glutathione binding site and in particular a deformation caused by a Leu-Ile exchange indicate why the E. coli enzyme accepts trypanothione much better than the human enzyme. The reported structure provides a frame for explaining numerous published engineering results in detail and for guiding further ones.     

Interchain disulphide bridges of mouse immunoglobulin M.

Mouse IgM (immunoglobulin M) was selectively and partially reduced and treated with iodo[2-14C]acetate to label the interchain disulphide bridges. The carboxymethylation was studied in some detail. The labelled peptides were purified, sequenced and positioned by homology with human IgM. Only peptides originating from three interchain disulphide bridges were labelled, in contrast with the four labelled bridges obtained in human IgM under the same conditions. These peptides are homologous to human bridge peptides forming the heavy-light bridge and two inter-heavy bridges, one present in the CMU2 region and the other in the C-terminal region. The inter-heavy bridge in the Cmu2 region was alone cleaved and radioactively labelled in selectively reduced IgM held together as a pentamer by non-covalen interactions. The same bridge was the only one to be totally cleaved in subunits released after more extensive, though still selective, reduction. In the light of these results a possible arrangement of the disulphide bridges of the mouse IgM.     

High probability of disrupting a disulphide bridge mediated by an endogenous excited tryptophan residue

It is well known that ultraviolet (UV) radiation may reduce or even abolish the biological activity of proteins and enzymes. UV light, as a component of sunlight, is illuminating all light-exposed parts of living organisms, partly composed of proteins and enzymes. Although a considerable amount of empirical evidence for UV damage has been compiled, no deeper understanding of this important phenomenon has yet emerged. The present paper presents a detailed analysis of a classical example of UV-induced changes in three-dimensional structure and activity of a model enzyme, cutinase from Fusarium solani pisi. The effect of illumination duration and power has been investigated. A photon-induced mechanism responsible for structural and functional changes is proposed. Tryptophan excitation energy disrupts a neighboring disulphide bridge, which in turn leads to altered biological activity and stability. The loss of the disulphide bridge has a pronounced effect on the fluorescence quantum yield, which has been monitored as a function of illumination power. A general theoretical model for slow two-state chemical exchange is formulated, which allows for calculation of both the mean number of photons involved in the process and the ratio between the quantum yields of the two states. It is clear from the present data that the likelihood for UV damage of proteins is directly proportional to the intensity of the UV radiation. Consistent with the loss of the disulphide bridge, a complex pH-dependent change in the fluorescence lifetimes is observed. Earlier studies in this laboratory indicate that proteins are prone to such UV-induced radiation damage because tryptophan residues typically are located as next spatial neighbors to disulphide bridges. We believe that these observations may have far-reaching implications for protein stability and for assessing the true risks involved in increasing UV radiation loads on living organisms.     

Elimination of the disulphide bridge in fragment B of diphtheria toxin: effect on membrane insertion, channel formation, and ATP binding

Active diphtheria toxin consists of two disulphide-linked fragments, termed A and B. Fragment B, which contains an internal disulphide bridge, facilitates translocation of the enzymatically active fragment A to the cytosol of eukaryotic cells. In this process cation-selective channels are formed. An in vitro translated full-length mutant lacking the internal disulphide bridge (A-58**) was functionally indistinguishable from its disulphide-containing counterpart (A-58) with respect to trypsin sensitivity, receptor binding, A-fragment translocation, and channel formation. In contrast, the B fragment of A-58** (B-36**) was slightly less trypsin resistant than the S-S-containing B fragment, B-36, and was approximately 300-fold less efficient than B-36 in permeabilizing cells. When first dialysed and then reconstituted with A fragment, B fragment without disulphide bridge yielded a less-active toxin than did wild-type B fragment. We conclude that the disulphide bridge in fragment B is not necessary for toxicity, as earlier believed, and that channel formation may play a role in membrane translocation.     

Engineering of an intersubunit disulfide bridge in the iron-superoxide dismutase of Mycobacterium tuberculosis.

With the aim of enhancing interactions involved in dimer formation, an intersubunit disulfide bridge was engineered in the superoxide dismutase enzyme of Mycobacterium tuberculosis. Ser-123 was chosen for mutation to cysteine since it resides at the dimer interface where the serine side chain interacts with the same residue in the opposite subunit. Gel electrophoresis and X-ray crystallographic studies of the expressed mutant confirmed formation of the disulfide bond under nonreducing conditions. However, the mutant protein was found to be less stable than the wild type as judged by susceptibility to denaturation in the presence of guanidine hydrochloride. Decreased stability probably results from formation of a disulfide bridge with a suboptimal torsion angle and exclusion of solvent molecules from the dimer interface.     

Structure of the dimeric N-glycosylated form of fungal β-N-acetylhexosaminidase revealed by computer modeling, vibrational spectroscopy, and biochemical studies

Background

Fungal β-N-acetylhexosaminidases catalyze the hydrolysis of chitobiose into its constituent monosaccharides. These enzymes are physiologically important during the life cycle of the fungus for the formation of septa, germ tubes and fruit-bodies. Crystal structures are known for two monomeric bacterial enzymes and the dimeric human lysosomal β-N-acetylhexosaminidase. The fungal β-N-acetylhexosaminidases are robust enzymes commonly used in chemoenzymatic syntheses of oligosaccharides. The enzyme from Aspergillus oryzae was purified and its sequence was determined.

Results

The complete primary structure of the fungal β-N-acetylhexosaminidase from Aspergillus oryzae CCF1066 was used to construct molecular models of the catalytic subunit of the enzyme, the enzyme dimer, and the N-glycosylated dimer. Experimental data were obtained from infrared and Raman spectroscopy, and biochemical studies of the native and deglycosylated enzyme, and are in good agreement with the models. Enzyme deglycosylated under native conditions displays identical kinetic parameters but is significantly less stable in acidic conditions, consistent with model predictions. The molecular model of the deglycosylated enzyme was solvated and a molecular dynamics simulation was run over 20 ns. The molecular model is able to bind the natural substrate – chitobiose with a stable value of binding energy during the molecular dynamics simulation.

Conclusion

Whereas the intracellular bacterial β-N-acetylhexosaminidases are monomeric, the extracellular secreted enzymes of fungi and humans occur as dimers. Dimerization of the fungal β-N-acetylhexosaminidase appears to be a reversible process that is strictly pH dependent. Oligosaccharide moieties may also participate in the dimerization process that might represent a unique feature of the exclusively extracellular enzymes. Deglycosylation had only limited effect on enzyme activity, but it significantly affected enzyme stability in acidic conditions. Dimerization and N-glycosylation are the enzyme's strategy for catalytic subunit stabilization. The disulfide bridge that connects Cys⁴⁴⁸ with Cys⁴⁸³ stabilizes a hinge region in a flexible loop close to the active site, which is an exclusive feature of the fungal enzymes, neither present in bacterial nor mammalian structures. This loop may play the role of a substrate binding site lid, anchored by a disulphide bridge that prevents the substrate binding site from being influenced by the flexible motion of the loop.     

Photoaffinity labelling of the TSH receptor on FRTL5 cells

An investigation of the properties of TSH receptors on FRTL5 cells using affinity labelling with a 125I-labelled photoactive derivative of TSH is described. Our studies suggest that FRTL5 cells contain 2 principal types of cell surface TSH receptors. One form, probably a precursor, consists of a single polypeptide chain (Mr 120,000) with an intrachain loop of amino acids formed by a disulphide bridge. The other type of receptor consists of a water-soluble A chain (Mr 55,000) linked to an amphiphilic B chain (Mr 35,000) by a disulphide bridge. The 2 chain structure is probably derived from the single chain 120,000 protein by enzymatic cleavage of peptide sequences within the loop of amino acids formed by the intrachain disulphide bridge.