Identification, Functional Characterization, and Chromosomal Localization of USP15, a Novel Human Ubiquitin-Specific Protease Related to the UNP Oncoprotein, and a Systematic Nomenclature for Human Ubiquitin-Specific Proteases

We have identified a novel gene, USP15, encoding a human ubiquitin-specific protease (USP). The USP15 protein consists of 952 amino acids with a predicted molecular mass of 109.2 kDa and contains the highly conserved Cys and His boxes present in all members of the UBP family of deubiquitinating enzymes. USP15 shares 60.5% sequence identity and 76% sequence similarity with the human homolog (UNP/Unph/USP4) of the mouse Unp proto-oncogene. Recombinant USP15 demonstrated ubiquitin-specific protease activity against engineered linear fusions of ubiquitin to beta-galactosidase and glutathione S-transferase. USP15 can also cleave the ubiquitin-proline bond, a property previously unique to Unp/UNP. Chromosomal mapping by fluorescence in situ hybridization and radiation hybrid analyses localized the USP15 gene to chromosome band 12q14, a different location than that of UNP (3p21.3). Analysis of expressed sequence tag databases reveals evidence of alternate polyadenylation sites in the USP15 gene and also indicates that the gene may possess an exon/intron structure similar to that of the Unp gene, suggesting they have descended from a common ancestor. A systematic nomenclature for the human USPs is proposed.     

In vivo Structure/Function analysis of the Drosophila fat facets deubiquitinating enzyme gene

The Drosophila Fat facets protein is a deubiquitinating enzyme required for patterning the developing compound eye. Ubiquitin, a 76-amino-acid polypeptide, serves as a tag to direct proteins to the proteasome, a protein degradation complex. Deubiquitinating enzymes are a large group of proteins that cleave ubiquitin-protein bonds. Fat facets belongs to a class of deubiquitinating enzymes called Ubps that share a conserved catalytic domain. Fat facets is unique among them in its large size and also because Fat facets is thought to deubiquitinate a specific substrate thereby preventing its proteolysis. Here we asked which portions of the Fat facets protein are essential for its function. P-element constructs that express partial Fat facets proteins were tested for function. In addition, the DNA sequences of 12 mutant fat facets alleles were determined. Finally, regions of amino acid sequence similarity in 18 Drosophila Ubps revealed by the Genome Project were identified. The results indicate functions for specific conserved amino acids in the catalytic region of Fat facets and also indicate that regions of the protein both N- and C-terminal to the catalytic region are required for Fat facets function.     

Allosteric inhibition of PTP1B activity by selective modification of a non-active site cysteine residue

The fluorogenic reagent 4-(aminosulfonyl)-7-fluoro-2,1,3-benzoxadiazole (ABDF) attenuates the functional activity of the protein tyrosine phosphatase PTP1B by reacting selectively with a single cysteine residue, leaving other cysteines in the protein unmodified. This modification reduces Vmax without substantially affecting substrate binding (Km), indicative of an allosteric mode of inhibition. Consistent with this, the cysteine residue modified by ABDF, Cys 121, lies outside the catalytic site but makes interactions with residues that contact His 214, which has been shown to be important for catalysis. Cys 121 is highly conserved among phosphatases, and ABDF also inhibits TC-PTP and LAR. These findings illustrate that targeting cysteine residues outside catalytic sites may be exploited in allosterically regulating enzymes. Moreover, these results suggest a new strategy for inhibiting a promising diabetes target.     

Structural insights into the second step of RNA-dependent cysteine biosynthesis in archaea: crystal structure of Sep-tRNA:Cys-tRNA synthase from Archaeoglobus fulgidus

In the ancient organisms, methanogenic archaea, lacking the canonical cysteinyl-tRNA synthetase, Cys-tRNA(Cys) is produced by an indirect pathway, in which O-phosphoseryl-tRNA synthetase ligates O-phosphoserine (Sep) to tRNA(Cys) and Sep-tRNA:Cys-tRNA synthase (SepCysS) converts Sep-tRNA(Cys) to Cys-tRNA(Cys). In this study, the crystal structure of SepCysS from Archaeoglobus fulgidus has been determined at 2.4 A resolution. SepCysS forms a dimer, composed of monomers bearing large and small domains. The large domain harbors the seven-stranded beta-sheet, which is typical of the pyridoxal 5'-phosphate (PLP)-dependent enzymes. In the active site, which is located near the dimer interface, PLP is covalently bound to the side-chain of the conserved Lys209. In the proximity of PLP, a sulfate ion is bound by the side-chains of the conserved Arg79, His103, and Tyr104 residues. The active site is located deep within the large, basic cleft to accommodate Sep-tRNA(Cys). On the basis of the surface electrostatic potential, the amino acid residue conservation mapping, the position of the bound sulfate ion, and the substrate amino acid binding manner in other PLP-dependent enzymes, a binding model of Sep-tRNA(Cys) to SepCysS was constructed. One of the three strictly conserved Cys residues (Cys39, Cys42, or Cys247), of one subunit may play a crucial role in the catalysis in the active site of the other subunit.     

Essential features of the catalytic core of peptidyl-alpha-hydroxyglycine alpha-amidating lyase

Bioactive peptides frequently terminate with an essential alpha-amide that is generated from a COOH-terminal Gly in a two-step enzymatic process occurring within the lumen of the secretory pathway. The first enzyme, peptidylglycine alpha-hydroxylating monooxygenase, is a member of the copper- and ascorbate-dependent monooxygenase family. The second enzyme, peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL, EC 4.3.2.5), has no known homologues. Examination of the catalytic core of PAL (PALcc) using trypsin, BNPS skatole, and COOH-terminally truncated proteins failed to identify stable subdomains. Treatment of PALcc with divalent metal ion chelators inactivated the enzyme and increased its protease and thermal sensitivity, suggesting a structural role for bound metal. Purified PALcc contained 0.7 +/- 0.4 mol of zinc/mol of enzyme. Since the four Cys residues in PALcc form two disulfide bonds, potential Zn ligands include conserved Asp, Glu, and His residues. The secretion and activity of PALcc bearing mutations in each conserved Asp, Glu, and His residue were evaluated. Mutation of three conserved Asp residues and two conserved His residues yielded a protein that could not be secreted, suggesting that these residues play a structural role. Analysis of mutants that were efficiently secreted identified three His residues along with single Asp residue that may play a role in catalysis. These essential residues occur in a pattern unique to PAL.     

Contribution of active site residues to substrate hydrolysis by USP2: insights into catalysis by ubiquitin specific proteases

The ubiquitin-specific protease (USP) structural class represents the largest and most diverse family of deubiquitinating enzymes (DUBs). Many USPs assume important biological roles and emerge as potential targets for therapeutic intervention. A clear understanding of USP catalytic mechanism requires a functional evaluation of the proposed key active site residues. Crystallographic data of ubiquitin aldehyde adducts of USP catalytic cores provided structural details on the catalytic triad residues, namely the conserved Cys and His, and a variable putative third residue, and inferred indirect structural roles for two other conserved residues (Asn and Asp), in stabilizing via a bridging water molecule the oxyanion of the tetrahedral intermediate (TI). We have expressed the catalytic domain of USP2 and probed by site-directed mutagenesis the role of these active site residues in the hydrolysis of peptide and isopeptide substrates, including a synthetic K48-linked diubiquitin substrate for which a label-free, mass spectrometry based assay has been developed to monitor cleavage. Hydrolysis of ubiquitin-AMC, a model substrate, was not affected by the mutations. Molecular dynamics simulations of USP2, free and complexed with the TI of a bona fide isopeptide substrate, were carried out. We found that Asn271 is structurally poised to directly stabilize the oxyanion developed in the acylation step, while being structurally supported by the adjacent absolutely conserved Asp575. Mutagenesis data functionally confirmed this structural role independent of the nature (isopeptide vs peptide) of the bond being cleaved. We also found that Asn574, structurally located as the third member of the catalytic triad, does not fulfill this role functionally. A dual supporting role is inferred from double-point mutation and structural data for the absolutely conserved residue Asp575, in oxyanion hole formation, and in maintaining the correct alignment and protonation of His557 for catalytic competency.     

Characterization of the activity and folding of the glutathione transferase from Escherichia coli and the roles of residues Cys(10) and His(106)

GSTs (glutathione transferases) are an important class of enzymes involved in cellular detoxification. GSTs are found in all classes of organisms and are implicated in resistance towards drugs, pesticides, herbicides and antibiotics. The activity, structure and folding, particularly of eukaryotic GSTs, have therefore been widely studied. The crystal structure of EGST (GST from Escherichia coli) was reported around 10 years ago and it suggested Cys(10) and His(106) as potential catalytic residues. However, the role of these residues in catalysis has not been further investigated, nor have the folding properties of the protein been described. In the present study we investigated the contributions of residues Cys(10) and His(106) to the activity and stability of EGST. We found that EGST shows a complex equilibrium unfolding profile, involving a population of at least two partially folded intermediates, one of which is dimeric. Mutation of residues Cys(10) and His(106) leads to stabilization of the protein and affects the apparent steady-state kinetic parameters for enzyme catalysis. The results suggest that the imidazole ring of His(106) plays an important role in the catalytic mechanism of the enzyme, whereas Cys(10) is involved in binding of the substrate, glutathione. Engineering of the Cys(10) site can be used to increase both the stability and GST activity of EGST. However, in addition to GST activity, we discovered that EGST also possesses thiol:disulfide oxidoreductase activity, for which the residue Cys(10) plays an essential role. Further, tryptophan quenching experiments indicate that a mixed disulfide is formed between the free thiol group of Cys(10) and the substrate, glutathione.     

Solution structure of a CCHHC domain of neural zinc finger factor-1 and its implications for DNA binding

The structure of a CCHHC zinc-binding domain from neural zinc finger factor-1 (NZF-1) has been determined in solution though the use of NMR methods. This domain is a member of a family of domains that have the Cys-X(4)-Cys-X(4)-His-X(7)-His-X(5)-Cys consensus sequence. The structure determination reveals a novel fold based around a zinc(II) ion coordinated to three Cys residues and the second of the two conserved His residues. The other His residue is stacked between the metal-coordinated His residue and a relatively conserved aromatic residue. Analysis of His to Gln sequence variants reveals that both His residues are required for the formation of a well-defined structure, but neither is required for high-affinity metal binding at a tetrahedral site. The structure suggests that a two-domain protein fragment and a double-stranded DNA binding site may interact with a common two-fold axis relating the two domains and the two half-sites of the DNA-inverted repeat.     

Structural elements responsible for transglutaminase activity of protein disulphide isomerases and thioredoxins

According to recent results both protein disulphide isomerase (PDI) and thioredoxin (Trx) enzymes have transglutaminase activity which can be linked to the thioredoxin box found in these proteins. Analysis of known protein disulphide isomerase and thioredoxin sequences has revealed the presence of conserved Cys, His and Asp residues required for transglutaminases to catalyze the incorporation of primary amines into protein-bound glutamine residues. The available 3D structures of PDIs and Trxs show that these residues are in close proximity to achieve transglutamylation of substrate proteins. The shared activities of the members of the large protein disulphide isomerase, thioredoxin and transglutaminase enzyme families reviewed here may have general biological significance in the regulation of cellular and tissue processes.     

The CR3 motif of Rrp44p is important for interaction with the core exosome and exosome function

The 10-subunit RNA exosome is involved in a large number of diverse RNA processing and degradation events in eukaryotes. These reactions are carried out by the single catalytic subunit, Rrp44p/Dis3p, which is composed of three parts that are conserved throughout eukaryotes. The exosome is named for the 3′ to 5′ exoribonuclease activity provided by a large C-terminal region of the Rrp44p subunit that resembles other exoribonucleases. Rrp44p also contains an endoribonuclease domain. Finally, the very N-terminus of Rrp44p contains three Cys residues (CR3 motif) that are conserved in many eukaryotes but have no known function. These three conserved Cys residues cluster with a previously unrecognized conserved His residue in what resembles a metal-ion-binding site. Genetic and biochemical data show that this CR3 motif affects both endo- and exonuclease activity in vivo and both the nuclear and cytoplasmic exosome, as well as the ability of Rrp44p to associate with the other exosome subunits. These data provide the first direct evidence that the exosome-Rrp44p interaction is functionally important and also provides a molecular explanation for the functional defects when the conserved Cys residues are mutated.     

Ubiquitin-specific proteases from Arabidopsis thaliana: cloning of AtUBP5 and analysis of substrate specificity of AtUBP3, AtUBP4, and AtUBP5 using Escherichia coli in vivo and in vitro assays

A cDNA for a new ubiquitin-specific protease (UBP), AtUBP5, was identified from Arabidopsis thaliana flower mRNA using an oligonucleotide made against the conserved UBP cysteine (Cys) box. The 924-amino-acid AtUBP5 contains the regions characteristic of all UBPs and has 35% identity and 53% similarity overall to a mammalian UBP (Unp), resulting from additional significant similarity outside these regions. AtUBP5 has 48% identity and 58% similarity overall to two uncharacterized Arabidopsis genomic sequences but is distinct outside the UBP conserved regions from two other previously published Arabidopsis UBPs, AtUBP3 and -4. Using in vivo Escherichia coli assays, which allow co-expression of GSTAtUBPs and substrates, we show that all three UBPs were active. AtUBP5 was active without 311 amino acids N-terminal to the active site cysteine, or without 233 nonconserved amino acids between the Cys and His boxes, or without both, indicating the core region was sufficient. In in vivo and in vitro assays, GSTAtUBP3, -4, and -5 exhibited preference for specific Ub-Ub linkages, suggesting accessibility and/or conformation is important and demonstrating that these enzymes cleave post-translationally. A chimeric UBP consisting of the AtUBP5 Cys box with AtUBP3 amino acids was active and exhibited AtUBP3 specificity, indicating that the modular nature of UBPs and specificity for cleavage sites is not determined by the Cys box.     

Analysis of essential histidine residues of maize branching enzymes by chemical modification and site-directed mutagenesis

Incubation of maize branching enzyme, mBEI and mBEII, with 100 μM diethylpyrocarbonate (DEPC) rapidly inactivated the enzymes. Treatment of the DEPC-inactivated enzymes with 100–500 mM hydroxylamine restored the enzyme activities. Spectroscopic data indicated that the inactivation of BE with DEPC was the result of histidine modification. The addition of the substrate amylose or amylopectin retarded the enzyme inactivation by DEPC, suggesting that the histidine residues are important for substrate binding. In maize BEII, conserved histidine residues are in catalytic regions 1 (His320) and 4 (His508). His320 and His508 were individually replaced by Ala via site-directed mutagenesis to probe their role in catalysis. Expression of these mutants inE. coli showed a significant decrease of the activity and the mutant enzymes hadK _m values 10 times higher than the wild type. Therefore, residues His320 and His508 do play an important role in substrate binding.     

Cysteine proteases of positive strand RNA viruses and chymotrypsin-like serine proteases. A distinct protein superfamily with a common structural fold

Evidence is presented, based on sequence comparison and secondary structure prediction, of structural and evolutionary relationship between chymotrypsin-like serine proteases, cysteine proteases of positive strand RNA viruses (3C proteases of picornaviruses and related enzymes of como-, nepo- and potyviruses) and putative serine protease of a sobemovirus. These observations lead to re-identification of principal catalytic residues of viral proteases. Instead of the pair of Cys and His, both located in the C-terminal part of 3C proteases, a triad of conserved His, Asp(Glu) and Cys(Ser) has been identified, the first two residues resident in the N-terminal, and Cys in the C-terminal beta-barrel domain. These residues are suggested to form a charge-transfer system similar to that formed by the catalytic triad of chymotrypsin-like proteases. Based on the structural analogy with chymotrypsin-like proteases, the His residue previously implicated in catalysis, together with two partially conserved Gly residues, is predicted to constitute part of the substrate-binding pocket of 3C proteases. A partially conserved ThrLys/Arg dipeptide located in the loop preceding the catalytic Cys is suggested to confer the primary cleavage specificity of 3C toward Glx/Gly(Ser) sites. These observations provide the first example of relatedness between proteases belonging, by definition, to different classes.     

Mechanistic similarity and diversity among the guanidine-modifying members of the pentein superfamily

The pentein superfamily is a mechanistically diverse superfamily encompassing both noncatalytic proteins and enzymes that catalyze hydrolase, dihydrolase and amidinotransfer reactions on guanidine substrates. Despite generally low sequence identity, they possess a conserved structural fold and display common mechanistic themes in catalysis. The structurally characterized catalytic penteins possess a conserved core of residues that include a Cys, His and two polar, guanidine-binding residues. All known catalytic penteins use the core Cys to attack the substrate's guanidine moiety to form a covalent thiouronium adduct and all cleave one or more of the guanidine C―N bonds. The mechanistic information compiled to date supports the hypothesis that this superfamily may have evolved divergently from a catalytically promiscuous ancestor.     

Heterogeneity of the covalent structure of the blue copper protein umecyanin from horseradish roots

The covalent structure of umecyanin has been determined by a combination of classical Edman degradation sequence analysis and plasma desorption, laser desorption, and electrospray ionization mass spectrometry. The preparation appeared to contain two isoforms having either a valine (75%) or an isoleucine (25%) residue at position 48. The polypeptide chain of 115 amino acids is strongly heterogeneous at its C-terminal end as a result of proteolytic cleavages at several places within the last 10 residues. The major fraction of the umecyanin preparation is only 106 residues long. The C-terminal tail 107–115 contains mainly alanine and glycine residues and a single hydroxyproline residue. In the native protein there is a disulfide bridge between Cys 91 and Cys 57, but in the apoprotein there is a disulfide shift that involves Cys 91 and one of the four copper binding residues (Cys 85). The three other ligand binding residues are His 44, His 90, and Gin 95. This tetrad of amino acids is the same as occurs in other type 1 copper proteins from plants such as cucumber peeling cupredoxin and lacquer tree stellacyanin. The umecyanin isoforms are glycoproteins with a glycan core having the same carbohydrate composition as that of horseradish peroxidase, a fact that is convincingly supported thanks to the high accuracy of the electrospray mass spectrometric technique. We suggest that the glycan may play a role in the association of the protein to the cellular membrane, but the precise functional role of umecyanin remains to be determined. We also discuss the evolutionary position of umecyanin in relation to the type 1 copper proteins in general.     

Potential ligands to the [2Fe-2S] Rieske cluster of the cytochrome bc1 complex of Rhodobacter capsulatus probed by site-directed mutagenesis.

The Rieske protein of the ubiquinol-cytochrome c oxidoreductase (bc1 complex or b6f complex) contains a [2Fe-2S] cluster which is thought to be bound to the protein via two nitrogen and two sulfur ligands [Britt, R. D., Sauer, K., Klein, M. P., Knaff, D. B., Kriauciunas, A., Yu, C.-A., Yu, L., & Malkin, R. (1991) Biochemistry 30, 1892-1901; Gurbiel, R. J., Ohnishi, T., Robertson, D. E., Daldal, F., & Hoffman, B. M. (1991) Biochemistry 30, 11579-11584]. All available Rieske amino acid sequences have carboxyl termini featuring two conserved regions containing four cysteine (Cys) and two or three histidine (His) residues. Site-directed mutagenesis was applied to the Rieske protein of the photosynthetic bacterium Rhodobacter capsulatus, and the mutants obtained were studied biochemically in order to identify which of these conserved residues are the ligands of the [2Fe-2S] cluster. It was found that His159 (in the R. capsulatus numbering) is not a ligand and that the presence of the Rieske protein in the intracytoplasmic membrane is greatly decreased by alteration of any of the remaining six His or Cys residues. Among these mutations, only the substitution Cys155 to Ser resulted in the synthesis of Rieske protein (in a small amount) which contained a [2Fe-2S] cluster with altered biophysical properties. This finding suggested that Cys155 is not a ligand to the cluster. A comparison of the conserved regions of the Rieske proteins with bacterial aromatic dioxygenases (which contain a spectrally and electrochemically similar [2Fe-2S] cluster) indicated that Cys133, His135, Cys153, and His156 are conserved in both groups of enzymes, possibly as ligands to their [2Fe-2S] clusters. These findings led to the proposal that Cys138 and Cys155, which are not conserved in bacterial dioxygenases, may form an internal disulfide bond which is important for the structure of the Rieske protein and the conformation of the quinol oxidation (Qo) site of the bc1 complex.     

Expression and biochemical characterization of the human enzyme N-terminal asparagine amidohydrolase

The enzymatic deamidation of N-terminal L-Asn by N-terminal asparagine amidohydrolase (NTAN1) is a feature of the ubiquitin-dependent N-end rule pathway of protein degradation, which relates the in vivo half-life of a protein to the identity of its N-terminal residue. Herein, we report the bacterial expression, purification, and biochemical characterization of human NTAN1 (hNTAN1). We show here that hNTAN1 is highly selective for the hydrolysis of N-terminal peptidyl L-Asn but fails to deamidate free L-Asn or L-Gln, N-terminal peptidyl L-Gln, or acetylated N-terminal peptidyl L-Asn. Similar to other N-terminal deamidases, hNTAN1 is shown to possess a critical Cys residue that is absolutely required for catalysis, corroborated in part by abolishment of activity through the Cys75Ala point mutation. We also present evidence that the exposure of a conserved L-Pro at the N-terminus of hNTAN1 following removal of the initiating L-Met is important for the function of the enzyme. The results presented here should assist in the elucidation of molecular mechanisms underlying the neurological defects of NTAN1-deficient mice observed in other studies, and in the discovery of potential physiological substrates targeted by the enzyme in the modulation of protein turnover via the N-end rule pathway.     

Crystal structures of Staphylococcus aureus sortase A and its substrate complex

The cell wall envelope of staphylococci and other Gram-positive pathogens is coated with surface proteins that interact with human host tissues. Surface proteins of Staphylococcus aureus are covalently linked to the cell wall envelope by a mechanism requiring C-terminal sorting signals with an LPXTG motif. Sortase (SrtA) cleaves surface proteins between the threonine (T) and the glycine (G) of the LPXTG motif and catalyzes the formation of an amide bond between threonine at the C-terminal end of polypeptides and cell wall cross-bridges. The active site architecture and catalytic mechanism of sortase A has hitherto not been revealed. Here we present the crystal structures of native SrtA, of an active site mutant of SrtA, and of the mutant SrtA complexed with its substrate LPETG peptide and describe the substrate binding pocket of the enzyme. Highly conserved proline (P) and threonine (T) residues of the LPXTG motif are held in position by hydrophobic contacts, whereas the glutamic acid residue (E) at the X position points out into the solvent. The scissile T-G peptide bond is positioned between the active site Cys(184) and Arg(197) residues and at a greater distance from the imidazolium side chain of His(120). All three residues, His(120), Cys(184), and Arg(197), are conserved in sortase enzymes from Gram-positive bacteria. Comparison of the active sites of S. aureus sortase A and sortase B provides insight into substrate specificity and suggests a universal sortase-catalyzed mechanism of bacterial surface protein anchoring in Gram-positive bacteria.     

Biosynthesis of pteridines. Reaction mechanism of GTP cyclohydrolase I

GTP cyclohydrolase I catalyses the hydrolytic release of formate from GTP followed by cyclization to dihydroneopterin triphosphate. The enzymes from bacteria and animals are homodecamers containing one zinc ion per subunit. Replacement of Cys110, Cys181, His112 or His113 of the enzyme from Escherichia coli by serine affords catalytically inactive mutant proteins with reduced capacity to bind zinc. These mutant proteins are unable to convert GTP or the committed reaction intermediate, 2-amino-5-formylamino-6-(beta-ribosylamino)-4(3H)-pyrimidinone 5'-triphosphate, to dihydroneopterin triphosphate. The crystal structures of GTP complexes of the His113Ser, His112Ser and Cys181Ser mutant proteins determined at resolutions of 2.5A, 2.8A and 3.2A, respectively, revealed the conformation of substrate GTP in the active site cavity. The carboxylic group of the highly conserved residue Glu152 anchors the substrate GTP, by hydrogen bonding to N-3 and to the position 2 amino group. Several basic amino acid residues interact with the triphosphate moiety of the substrate. The structure of the His112Ser mutant in complex with an undefined mixture of nucleotides determined at a resolution of 2.1A afforded additional details of the peptide folding. Comparison between the wild-type and mutant enzyme structures indicates that the catalytically active zinc ion is directly coordinated to Cys110, Cys181 and His113. Moreover, the zinc ion is complexed to a water molecule, which is in close hydrogen bond contact to His112. In close analogy to zinc proteases, the zinc-coordinated water molecule is suggested to attack C-8 of the substrate affording a zinc-bound 8R hydrate of GTP. Opening of the hydrated imidazole ring affords a formamide derivative, which remains coordinated to zinc. The subsequent hydrolysis of the formamide motif has an absolute requirement for zinc ion catalysis. The hydrolysis of the formamide bond shows close mechanistic similarity with peptide hydrolysis by zinc proteases.     

Influence of key residues on the heterologous extracellular production of fungal ribonuclease U2 in the yeast Pichia pastoris

Ribonuclease U2, secreted by the smut fungus Ustilago sphaerogena, is a cyclizing ribonuclease that displays a rather unusual specificity within the group of microbial extracellular RNases, best represented by RNase T1. Superposition of the three-dimensional structures of RNases T1 and U2 suggests that the RNase U2 His 101 would be the residue equivalent to the RNase T1 catalytically essential His 92. RNase U2 contains three disulfide bridges but only two of them are conserved among the family of fungal extracellular RNases. The non-conserved disulfide bond is established between Cys residues 1 and 54. Mispairing of the disulfide network due to the presence of two consecutive Cys residues (54 and 55) has been invoked to explain the presence of wrongly folded RNase U2 species when produced in Pichia pastoris. In order to study both hypotheses, the RNase U2 H101Q and C1/54S variants have been produced, purified, and characterized. The results obtained support the major conclusion that His 101 is required for proper protein folding when secreted by the yeast P. pastoris. On the other hand, substitution of the first Cys residue for Ser results in a mutant version which is more efficiently processed in terms of a more complete removal of the yeast α-factor signal peptide. In addition, it has been shown that elimination of the Cys 1–Cys 54 disulfide bridge does not interfere with RNase U2 proper folding, generating a natively folded but much less stable protein.