Secreted expression of pseudozymogen forms of recombinant matriptase in Pichia pastoris

Matriptase is a transmembrane serine protease expressed in vertebrates. This enzyme is synthesized as a zymogen form and is converted to an active form by cleavage at the N-terminus of the serine protease catalytic domain. In a mammalian cell-based expression system, we have produced pseudozymogen forms of recombinant matriptase (r-matriptase) that are activated by cleavage with a recombinant enterokinase (r-EK) in vitro. In the present study, four different pseudozymogen forms of r-matriptase containing a site for activation by r-EK and a hexahistidine tag (His₆-tag) were expressed in and secreted by Pichia pastoris, a methylotrophic yeast. The pseudozymogens with His₆-tag at their C-termini formed multimers linked by intermolecular disulfide bonds. After treatment with r-EK, they exhibited no detectable hydrolytic activity toward a chromogenic substrate. A pseudozymogen form of matriptase catalytic domain with N-terminal His₆-tag (designated His₆t-S-CD) was secreted as a monomer. His₆t-S-CD after r-EK treatment exhibited activity comparable to that of the activated form of an r-matriptase expressed in mammalian cells. His₆t-S-CD could be purified from culture medium in milligram quantities. The expression in the yeast offers an efficient method of producing larger amounts of r-matriptase.     

Expression and characterization of recombinant gamma-tryptase

Tryptases are trypsin-like serine proteases whose expression is restricted to cells of hematopoietic origin, notably mast cells. gamma-Tryptase, a recently described member of the family also known as transmembrane tryptase (TMT), is a membrane-bound serine protease found in the secretory granules or on the surface of degranulated mast cells. The 321 amino acid protein contains an 18 amino acid propeptide linked to the catalytic domain (cd), followed by a single-span transmembrane domain. gamma-Tryptase is distinguished from other human mast cell tryptases by the presence of two unique cysteine residues, Cys(26) and Cys(145), that are predicted to form an intra-molecular disulfide bond linking the propeptide to the catalytic domain to form the mature, membrane-anchored two-chain enzyme. We expressed gamma-tryptase as either a soluble, single-chain enzyme with a C-terminal His tag (cd gamma-tryptase) or as a soluble pseudozymogen activated by enterokinase cleavage to form a two-chain protein with an N-terminal His tag (tc gamma-tryptase). Both recombinant proteins were expressed at high levels in Pichia pastoris and purified by affinity chromatography. The two forms of gamma-tryptase exhibit comparable kinetic parameters, indicating the propeptide does not contribute significantly to the substrate affinity or activity of the protease. Substrate and inhibitor library screening indicate that gamma-tryptase possesses a substrate preference and inhibitor profile distinct from that of beta-tryptase. Although the role of gamma-tryptase in mast cell function is unknown, our results suggest that it is likely to be distinct from that of beta-tryptase.     

Expression, purification and structural characterization of recombinant hepcidin, an antimicrobial peptide identified in Japanese flounder, Paralichthys olivaceus

The cysteine-rich peptide hepcidin is an antimicrobial peptide and iron transport regulator that has been found in vertebrates including birds, fish and mammals. To elucidate the structure and biological function of fish hepcidin, which is difficult to produce synthetically, we have cloned several plasmid constructs encoding hepcidin from Japanese flounder, Paralichthys olivaceus, and tested expression of recombinant peptides, each with an N-terminal hexahistidine (6xHis) tag, in inclusion bodies or the periplasmic space of Escherichia coli. Hepcidin expressed in inclusion bodies was reduced, and subsequently refolded using a dilution technique with a cysteine redox system. The oxidized His-hepcidin monomer was separated from protein multimers and mass spectrometry analysis showed that the peptide was of the predicted size and contained four disulfide bonds. Removal of the 6xHis tag was attempted using enzymatic cleavage by Factor Xa and tobacco etch virus (TEV) protease or chemical cleavage by hydroxylamine. The Factor Xa cleavage was unsuccessful and hydroxylamine cleavage resulted in aggregation of cleaved peptide. TEV protease cleavage was successful but immediately resulted in hexamer formation despite varying reaction conditions (redox, non-redox, pH, temperature, target protein concentration, type of buffer). However, the recombinant His-hepcidin fusion peptide monomer showed considerable antimicrobial activity. NMR-based studies showed that hepcidin contained a rare vicinal disulfide linkage at the top of a loop structure and a short beta-sheet structure encompassing residues 7-13 and 19-25 that is stabilized by three disulfide bonds.     

Purification, reconstitution, and steady-state kinetics of the trans-membrane 17 beta-hydroxysteroid dehydrogenase 2

Human membrane 17 beta-hydroxysteroid dehydrogenase 2 is an enzyme essential in the conversion of the highly active 17beta-hydroxysteroids into their inactive keto forms in a variety of tissues. 17 beta-hydroxysteroid dehydrogenase 2 with 6 consecutive histidines at its N terminus was expressed in Sf9 insect cells. This recombinant protein retained its biological activity and facilitated the enzyme purification and provided the most suitable form in our studies. Dodecyl-beta-D-maltoside was found to be the best detergent for the solubilization, purification, and reconstitution of this enzyme. The overexpressed integral membrane protein was purified with a high catalytic activity and a purity of more than 90% by nickel-chelated chromatography. For reconstitution, the purified protein was incorporated into dodecyl-beta-D-maltoside-destabilized liposomes prepared from l-alpha-phosphatidylcholine. The detergent was removed by adsorption onto polystyrene beads. The reconstituted enzyme had much higher stability and catalytic activity (2.6 micromol/min/mg of enzyme protein with estradiol) than the detergent-solubilized and purified protein (0.9 micromol/min/mg of enzyme protein with estradiol). The purified and reconstituted protein (with a 2-kDa His tag) was proved to be a homodimer, and its functional molecular mass was calculated to be 90.4 +/- 1.2 kDa based on glycerol gradient analytical ultracentrifugation and chemical cross-linking study. The kinetic studies demonstrated that 17 beta-hydroxysteroid dehydrogenase 2 was an NAD-preferring dehydrogenase with the K(m) of NAD being 110 +/- 10 microM and that of NADP 9600 +/- 100 microM using estradiol as substrate. The kinetic constants using estradiol, testosterone, dihydrotestosterone, and 20 alpha-dihydroprogesterone as substrates were also determined.     

Thioredoxin reductase two modes of catalysis have evolved.

Thioredoxin reductase (EC 1.6.4.5) is a widely distributed flavoprotein that catalyzes the NADPH-dependent reduction of thioredoxin. Thioredoxin plays several key roles in maintaining the redox environment of the cell. Like all members of the enzyme family that includes lipoamide dehydrogenase, glutathione reductase and mercuric reductase, thioredoxin reductase contains a redox active disulfide adjacent to the flavin ring. Evolution has produced two forms of thioredoxin reductase, a protein in prokaryotes, archaea and lower eukaryotes having a Mr of 35 000, and a protein in higher eukaryotes having a Mr of 55 000. Reducing equivalents are transferred from the apolar flavin binding site to the protein substrate by distinct mechanisms in the two forms of thioredoxin reductase. In the low Mr enzyme, interconversion between two conformations occurs twice in each catalytic cycle. After reduction of the disulfide by the flavin, the pyridine nucleotide domain must rotate with respect to the flavin domain in order to expose the nascent dithiol for reaction with thioredoxin; this motion repositions the pyridine ring adjacent to the flavin ring. In the high Mr enzyme, a third redox active group shuttles the reducing equivalent from the apolar active site to the protein surface. This group is a second redox active disulfide in thioredoxin reductase from Plasmodium falciparum and a selenenylsulfide in the mammalian enzyme. P. falciparum is the major causative agent of malaria and it is hoped that the chemical difference between the two high Mr forms may be exploited for drug design.     

Biochemical characterization of CA IX, one of the most active carbonic anhydrase isozymes

Carbonic anhydrase IX (CA IX) is an exceptional member of the CA protein family; in addition to its classical role in pH regulation, it has also been proposed to participate in cell proliferation, cell adhesion, and tumorigenic processes. To characterize the biochemical properties of this membrane protein, two soluble recombinant forms were produced using the baculovirus-insect cell expression system. The recombinant proteins consisted of either the CA IX catalytic domain only (CA form) or the extracellular domain, which included both the proteoglycan and catalytic domains (PG + CA form). The produced proteins lacked the small transmembrane and intracytoplasmic regions of CA IX. Stopped-flow spectrophotometry experiments on both proteins demonstrated that in the excess of certain metal ions the PG + CA form exhibited the highest catalytic activity ever measured for any CA isozyme. Investigations on the oligomerization and stability of the enzymes revealed that both recombinant proteins form dimers that are stabilized by intermolecular disulfide bond(s). Mass spectrometry experiments showed that CA IX contains an intramolecular disulfide bridge (Cys(119)-Cys(299)) and a unique N-linked glycosylation site (Asn(309)) that bears high mannose-type glycan structures. Parallel experiments on a recombinant protein obtained by a mammalian cell expression system demonstrated the occurrence of an additional O-linked glycosylation site (Thr(78)) and characterized the nature of the oligosaccharide structures. This study provides novel information on the biochemical properties of CA IX and may help characterize the various cellular and pathophysiological processes in which this unique enzyme is involved.     

High-level production and optimization of monodispersity of 11beta-hydroxysteroid dehydrogenase type 1

11beta-Hydroxysteroid dehydrogenase type 1 (11beta-HSD1) is an intraluminally oriented, endoplasmic reticulum (ER)-bound enzyme catalyzing the interconversion between inactive cortisone and hormonally active cortisol. Heterologous production of 11beta-HSD1, devoid of its N-terminal transmembrane segment, is possible but yields only small amounts of soluble protein. Here we show that the soluble portion of recombinant 11beta-HSD1 produced in E. coli is found mainly as multimeric aggregates in the absence of detergent, and to a large extent associated with the endogenous chaperonin GroEL and other E. coli proteins. By co-overexpressing GroEL/ES and adding an 11beta-HSD1 inhibitor during protein synthesis, we have increased the accumulation of soluble 11beta-HSD1 by more than one order of magnitude. Using monodispersity as a screening criterion, we have also optimized the purification process by evaluating various solubilizing systems for the chromatographic steps, finally obtaining stable monodisperse preparations of both human and guinea pig 11beta-HSD1. By analytical ultracentrifugation, we could demonstrate that 11beta-HSD1 mainly exists as a dimer in the solubilized state. Moreover, active site titration of human 11beta-HSD1 revealed that at least 75% of the protein in a typical preparation represents active enzyme. Equilibrium unfolding experiments indicate that addition of inhibitor and the cofactor NADP(H) can stabilize the conformational stability of this enzyme in an additive manner. The outlined procedure may provide a general method for preparing similar proteins to oligomeric homogeneity and with retained biological activity.     

H2O2-induced intermolecular disulfide bond formation between receptor protein-tyrosine phosphatases

Receptor protein-tyrosine phosphatase alpha (RPTPalpha) belongs to the subfamily of receptor-like protein-tyrosine phosphatases that are characterized by two catalytic domains of which only the membrane-proximal one (D1) exhibits appreciable catalytic activity. The C-terminal catalytic domain (D2) regulates RPTPalpha catalytic activity by controlling rotational coupling within RPTPalpha dimers. RPTPalpha-D2 changes conformation and thereby rotational coupling within RPTPalpha dimers in response to changes in the cellular redox state. Here we report a decrease in motility of RPTPalpha from cells treated with H2O2 on non-reducing SDS-polyacrylamide gels to a position that corresponds to RPTPalpha dimers, indicating intermolecular disulfide bond formation. Using mutants of all individual cysteines in RPTPalpha and constructs encoding the individual protein-tyrosine phosphatase domains, we located the intermolecular disulfide bond to the catalytic Cys-723 in D2. Disulfide bond formation and dimer stabilization showed similar levels of concentration and time dependence. However, treatment of lysates with dithiothreitol abolished intermolecular disulfide bonds but not stable dimer formation. Intermolecular disulfide bond formation and rotational coupling were also found using a chimera of the extracellular domain of RPTPalpha fused to the transmembrane and intracellular domain of the leukocyte common antigen-related protein (LAR). These results suggest that H2O2 treatment leads to oxidation of the catalytic Cys in D2, which then rapidly forms a disulfide bond with the D2 catalytic Cys of the dyad-related monomer, rendering an inactive RPTP dimer. Recovery from oxidative stress first leads to the reduction of the disulfide bond followed by a slower refolding of the protein to the active conformation.     

Molecular characterization of the human Calpha-formylglycine-generating enzyme

Calpha-formylglycine (FGly) is the catalytic residue in the active site of sulfatases. In eukaryotes, it is generated in the endoplasmic reticulum by post-translational modification of a conserved cysteine residue. The FGly-generating enzyme (FGE), performing this modification, is an endoplasmic reticulum-resident enzyme that upon overexpression is secreted. Recombinant FGE was purified from cells and secretions to homogeneity. Intracellular FGE contains a high mannose type N-glycan, which is processed to the complex type in secreted FGE. Secreted FGE shows partial N-terminal trimming up to residue 73 without loosing catalytic activity. FGE is a calcium-binding protein containing an N-terminal (residues 86-168) and a C-terminal (residues 178-374) protease-resistant domain. The latter is stabilized by three disulfide bridges arranged in a clamp-like manner, which links the third to the eighth, the fourth to the seventh, and the fifth to the sixth cysteine residue. The innermost cysteine pair is partially reduced. The first two cysteine residues are located in the sequence preceding the N-terminal protease-resistant domain. They can form intramolecular or intermolecular disulfide bonds, the latter stabilizing homodimers. The C-terminal domain comprises the substrate binding site, as evidenced by yeast two-hybrid interaction assays and photocross-linking of a substrate peptide to proline 182. Peptides derived from all known human sulfatases served as substrates for purified FGE indicating that FGE is sufficient to modify all sulfatases of the same species.     

Sequence of xynC and properties of XynC, a major component of the Clostridium thermocellum cellulosome.

The nucleotide sequence of the Clostridium thermocellum F1 xynC gene, which encodes the xylanase XynC, consists of 1,857 bp and encodes a protein of 619 amino acids with a molecular weight of 69,517. XynC contains a typical N-terminal signal peptide of 32 amino acid residues, followed by a 165-amino-acid sequence which is homologous to the thermostabilizing domain. Downstream of this domain was a family 10 catalytic domain of glycosyl hydrolase. The C terminus separated from the catalytic domain by a short linker sequence contains a dockerin domain responsible for cellulosome assembly. The N-terminal amino acid sequence of XynC-II, the enzyme purified from a recombinant Escherichia coli strain, was in agreement with that deduced from the nucleotide sequence although XynC-II suffered from proteolytic truncation by a host protease(s) at the C-terminal region. Immunological and N-terminal amino acid sequence analyses disclosed that the full-length XynC is one of the major components of the C. thermocellum cellulosome. XynC-II was highly active toward xylan and slightly active toward p-nitrophenyl-beta-D-xylopyranoside, p-nitrophenyl-beta-D-cellobioside, p-nitrophenyl-beta-D-glucopyranoside, and carboxymethyl cellulose. The Km and Vmax values for xylan were 3.9 mg/ml and 611 micromol/min/mg of protein, respectively. This enzyme was optimally active at 80 degrees C and was stable up to 70 degrees C at neutral pHs and over the pH range of 4 to 11 at 25 degrees C.     

The tetratricopeptide repeat domain and a C-terminal region control the activity of Ser/Thr protein phosphatase 5.

Protein Ser/Thr phosphatase 5 is a 58-kDa protein containing a catalytic domain structurally related to the catalytic subunits of protein phosphatases 1, 2A, and 2B and an extended N-terminal domain with three tetratricopeptide repeats. The activity of this enzyme is stimulated 4-14-fold in vitro by polyunsaturated fatty acids and anionic phospholipids. The structural basis for lipid activation of protein phosphatase 5 was examined by limited proteolysis and site-directed mutagenesis. Trypsinolysis removed the tetratricopeptide repeat domain and increased activity to approximately half that of lipid-stimulated, full-length enzyme. Subtilisin removed the tetratricopeptide repeat domain and 10 residues from the C terminus, creating a catalytic fragment with activity that was equal to or greater than that of lipid-stimulated, full-length enzyme. Catalytic fragments generated by proteolysis were no longer stimulated by lipid, and degradation of the tetratricopeptide repeat domain was decreased by association with lipid. A truncated mutant missing 13 C-terminal residues was also insensitive to lipid and was as active as full-length, lipid-stimulated enzyme. These results suggest that the C-terminal and N-terminal domain act in a coordinated manner to suppress the activity of protein phosphatase 5 and mediate its activation by lipid. These regions may be targets for the regulation of protein phosphatase 5 activity in vivo.     

DNA-binding activity of the ERp57 C-terminal domain is related to a redox-dependent conformational change

ERp57, a member of the protein-disulfide isomerase family, although mainly localized in the endoplasmic reticulum is here shown to have a nuclear distribution. We previously showed the DNA-binding properties of ERp57, its association with the internal nuclear matrix, and identified the C-terminal region, containing the a' domain, as being directly involved in the DNA-binding activity. In this work, we demonstrate that its DNA-binding properties are strongly dependent on the redox state of the a' domain active site. Site-directed mutagenesis experiments on the first cysteine residue of the -CGHC-thioredoxin-like active site lead to a mutant domain (C406S) lacking DNA-binding activity. Biochemical studies on the recombinant domain revealed a conformational change associated with the redox-dependent formation of a homodimer, having two disulfide bridges between the cysteine residues of two a' domain active sites. The formation of intermolecular disulfide bridges rather than intramolecular oxidation of active site cysteines is important to generate species with DNA-binding properties. Thus, in the absence of any dedicated motif within the protein sequence, this structural rearrangement might be responsible for the DNA-binding properties of the C-terminal domain. Moreover, NADH-dependent thioredoxin reductase is active on intermolecular disulfides of the a' domain, allowing the control of dimeric protein content as well as its DNA-binding activity. A similar behavior was also observed for whole ERp57.     

Fusion of a cleavable signal peptide to the ectodomain of neutral endopeptidase (EC 3.4.24.11) results in the secretion of an active enzyme in COS-1 cells

Neutral endopeptidase (EC 3.4.24.11) is an integral membrane protein found at the plasma membrane of many cell types and is especially abundant at the apical "brush border" membrane of the kidney proximal tubules. The enzyme consists of a short amino-terminal cytosolic domain of 27 amino acids, a single hydrophobic sequence which is believed to be responsible for anchoring the enzyme in the plasma membrane, and a large extracellular domain containing the active site. This model is consistent with the proposed function of neutral endopeptidase, which is believed to play an important role in the inactivation of small regulatory peptides at the cell surface. Site-directed mutagenesis has allowed the identification of 1 glutamic acid and 2 histidine residues essential for catalysis. All are located near the COOH terminus of the protein. We do not know, however, whether other segments of the protein are involved in the structure of the active site. The exact role of the cytosolic and transmembrane domains is also unknown. In this report, we have induced the secretion of a soluble form of recombinant neutral endopeptidase in COS-1 cells by fusing in-frame, the cDNA encoding the signal peptide of a secreted protein (pro-opiomelanocortin) to the cDNA sequences of the complete ectodomain of neutral endopeptidase. Characterization of the secreted recombinant protein indicated that it has the same catalytic properties as the membrane-bound recombinant enzyme or as the enzyme extracted from kidney brush border membranes. Thus the extracellular domain alone is sufficient for conferring full catalytic activity to neutral endopeptidase.     

Comparative study of His- and Non-His-tagged CLIC proteins,reveals changes in their enzymatic activity

The chloride intracellular ion channel protein (CLIC) family are a unique set of ion channels that can exist as soluble and integral membrane proteins. New evidence has emerged that demonstrates CLICs' possess oxidoreductase enzymatic activity and may function as either membrane-spanning ion channels or as globular enzymes. To further characterize the enzymatic profile of members of the CLIC family and to expand our understanding of their functions, we expressed and purified recombinant CLIC1, CLIC3, and a non-functional CLIC1-Cys24A mutant using a Histidine tag, bacterial protein expression system. We demonstrate that the presence of the six-polyhistidine tag at the amino terminus of the proteins led to a decrease in their oxidoreductase enzymatic activity compared to their non-His-tagged counterparts, when assessed using 2-hydroxyethyl disulfide as a substrate. These results strongly suggest the six-polyhistidine tag alters CLIC's structure at the N-terminus, which also contains the enzyme active site. It also raises the need for caution in use of His-tagged proteins when assessing oxidoreductase protein enzymatic function.     

Re-design of Rhodobacter sphaeroides dimethyl sulfoxide reductase. Enhancement of adenosine N1-oxide reductase activity

The periplasmic DMSO reductase from Rhodobacter sphaeroides f. sp. denitrificans has been expressed in Escherichia coli BL21(DE3) cells in its mature form and with the R. sphaeroides or E. coli N-terminal signal sequence. Whereas the R. sphaeroides signal sequence prevents formation of active enzyme, addition of a 6x His-tag at the N terminus of the mature peptide maximizes production of active enzyme and allows for affinity purification. The recombinant protein contains 1.7-1.9 guanines and greater than 0.7 molybdenum atoms per molecule and has a DMSO reductase activity of 3.4-3.7 units/nmol molybdenum, compared with 3.7 units/nmol molybdenum for enzyme purified from R. sphaeroides. The recombinant enzyme differs from the native enzyme in its color and spectrum but is indistinguishable from the native protein after redox cycling with reduced methyl viologen and Me2SO. Substitution of Cys for the molybdenum-ligating Ser-147 produced a protein with DMSO reductase activity of 1.4-1.5 units/nmol molybdenum. The mutant protein differs from wild type in its color and absorption spectrum in both the oxidized and reduced states. This substitution leads to losses of 61-99% of activity toward five substrates, but the adenosine N1-oxide reductase activity increases by over 400%.     

Inhibition of human NTPDase 2 by modification of an intramembrane cysteine by p-chloromercuriphenylsulfonate and oxidative cross-linking of the transmembrane domains

Human NTPDase 2 is a cell surface integral membrane glycoprotein that is anchored to the membranes by two transmembrane domains while the bulk of the protein containing the active site faces the extracellular milieu. It contains 10 conserved cysteine residues in the extracellular domain that are involved in disulfide bond formation and one free cysteine residue, C26, which is located in the N-terminal transmembrane domain. The human NTPDase 2 activity is inactivated by membrane perturbation that disrupts interaction of the transmembrane domains and is inhibited by p-chloromercuriphenylsulfonate (pCMPS), a sulfhydryl reagent. In this report, we show that C26 is the target of pCMPS modification, since a mutant in which C26 was replaced with a serine was no longer inhibited by pCMPS. Mutants in which cysteine residues are placed in the C-terminal transmembrane domain near the extracellular surface were still modified by pCMPS, but the degree of inhibition of their ATPase activity was lower than that of the wild-type enzyme. Thus, loss of the ATPase activity of human NTPDase 2 in the presence of pCMPS probably results from the disturbance of both transmembrane domain interaction and its active site. Inhibition of human NTPDase 2 activity by pCMPS and membrane perturbation is attenuated when the enzyme is cross-linked by glutaraldehyde. On the other hand, NTPDase 2 dimers formed from oxidative cross-linking of the wild-type enzyme and mutants containing a single cysteine residue in the C-terminal transmembrane domain displayed reduced ATPase activity. A similar reduction in activity was also obtained upon intramolecular disulfide formation in mutants that contain a cysteine residue in each of the two transmembrane domains. These results indicate that the mobility of the transmembrane helices is necessary for maximal catalysis.     

Attachment of the N-terminal domain of Salmonella typhimurium AhpF to Escherichia coli thioredoxin reductase confers AhpC reductase activity but does not affect thioredoxin reductase activity

AhpF of Salmonella typhimurium, the flavoprotein reductase required for catalytic turnover of AhpC with hydroperoxide substrates in the alkyl hydroperoxide reductase system, is a 57 kDa protein with homology to thioredoxin reductase (TrR) from Escherichia coli. Like TrR, AhpF employs tightly bound FAD and redox-active disulfide center(s) in catalyzing electron transfer from reduced pyridine nucleotides to the disulfide bond of its protein substrate. Homology of AhpF to the smaller (35 kDa) TrR protein occurs in the C-terminal part of AhpF; a stretch of about 200 amino acids at the N-terminus of AhpF contains an additional redox-active disulfide center and is required for catalysis of AhpC reduction. We have demonstrated that fusion of the N-terminal 207 amino acids of AhpF to full-length TrR results in a chimeric protein (Nt-TrR) with essentially the same catalytic efficiency (k(cat)/K(m)) as AhpF in AhpC reductase assays; both k(cat) and the K(m) for AhpC are decreased about 3-4-fold for Nt-TrR compared with AhpF. In addition, Nt-TrR retains essentially full TrR activity. Based on results from two mutants of Nt-TrR (C129, 132S and C342,345S), AhpC reductase activity requires both centers while TrR activity requires only the C-terminal-most disulfide center in Nt-TrR. The high catalytic efficiency with which Nt-TrR can reduce thioredoxin implies that the attached N-terminal domain does not block access of thioredoxin to the TrR-derived Cys342-Cys345 center of Nt-TrR nor does it impede the putative conformational changes that this part of Nt-TrR is proposed to undergo during catalysis. These studies indicate that the C-terminal part of AhpF and bacterial TrR have very similar mechanistic properties. These findings also confirm that the N-terminal domain of AhpF plays a direct role in AhpC reduction.     

Folding and activity of mutant cystathionine β-synthase depends on the position and nature of the purification tag: characterization of the R266K CBS mutant

Cystathionine β-synthase (CBS), a heme-containing pyridoxal-5-phosphate (PLP)-dependent enzyme, catalyzes the condensation of serine and homocysteine to yield cystathionine. Missense mutations in CBS, the most common cause of homocystinuria, often result in misfolded proteins. Arginine 266, where the pathogenic missense mutation R266K was identified, appears to be involved in the communication between heme and the PLP-containing catalytic center. Here, we assessed the effect of a short affinity tag (6xHis) compared to a bulky fusion partner (glutathione S-transferase - GST) on CBS wild type (WT) and R266K mutant enzyme properties. While WT CBS was successfully expressed either in conjunction with a GST or with a 6xHis tag, the mutant R266K CBS had no activity, did not form native tetramers and did not respond to chemical chaperone treatment when expressed with a GST fusion partner. Interestingly, expression of R266K CBS constructs with a 6xHis tag at either end yielded active enzymes. The purified, predominantly tetrameric, R266K CBS with a C-terminal 6xHis tag had ～82% of the activity of a corresponding WT CBS construct. Results from thermal pre-treatment of the enzyme and the denaturation profile of R266K suggests a lower thermal stability of the mutant enzyme compared to WT, presumably due to a disturbed heme environment.     

Sequence and expression of 11β-hydroxysteroid dehydrogenase type 1 cDNA cloned from pig testis

Pig 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1 cDNA was cloned from neonatal pig testis, and 15 nucleotides were found to differ from the sequence in GenBank (Accession No. NM_214248). It was an exclusive clone obtained as pig 11β-HSD type 1, and the sequence of 11β-HSD type 1 cDNA cloned from pig liver was identical to that from testis. Amino acid sequence, deduced from cloned cDNA, also had a conserved triad of catalytically important Ser, Tyr and Lys residues for the short-chain dehydrogenase/reductase family, a membrane-spanning domain consisting of hydrophobic amino acid and a glycine motif in the cofactor binding region. The protein translated from this clone on expression in mammalian HEK293 cells exhibited oxo-reduction activity of cortisone and oxidation activity of cortisol. Furthermore, this oxo-reduction activity of cortisone was stimulated by co-expression of human H6PDH, while oxidation activity of cortisol was suppressed by H6PDH co-expression in HEK293 cells. Based on these results, the sequence of newly cloned cDNA is considered to correspond to an active enzyme form of pig 11β-HSD type 1.     

Reaction mechanism and regulation of mammalian thioredoxin/glutathione reductase

Thioredoxin/glutathione reductase (TGR) is a recently discovered member of the selenoprotein thioredoxin reductase family in mammals. In contrast to two other mammalian thioredoxin reductases, it contains an N-terminal glutaredoxin domain and exhibits a wide spectrum of enzyme activities. To elucidate the reaction mechanism and regulation of TGR, we prepared a recombinant mouse TGR in the selenoprotein form as well as various mutants and individual domains of this enzyme. Using these proteins, we showed that the glutaredoxin and thioredoxin reductase domains of TGR could independently catalyze reactions normally associated with each domain. The glutaredoxin domain is a monothiol glutaredoxin containing a CxxS motif at the active site, which could receive electrons from either the thioredoxin reductase domain of TGR or thioredoxin reductase 1. We also found that the C-terminal penultimate selenocysteine was required for transfer of reducing equivalents from the thiol/disulfide active site of TGR to the glutaredoxin domain. Thus, the physiologically relevant NADPH-dependent activities of TGR were dependent on this residue. In addition, we examined the effects of selenium levels in the diet and perturbations in selenocysteine tRNA function on TGR biosynthesis and found that expression of this protein was regulated by both selenium and tRNA status in liver, but was more resistant to this regulation in testes.