Modulation of H(+),K(+)-ATPase activity by the molecular chaperone ERp57 highly expressed in gastric parietal cells.

ERp57 is a ubiquitous ER chaperone that has disulfide isomerase activity. Here, we found that both ERp57 and gastric H(+),K(+)-ATPase are expressed in a sample derived from the apical canalicular membranes of parietal cells. Overexpression of ERp57 in HEK293 cells stably expressing H(+),K(+)-ATPase significantly increased the ATPase activity without changing the expression level of H(+),K(+)-ATPase. Interestingly, overexpression of a catalytically inactive mutant of ERp57 (C57S/C60S/C406S/C409S) in the cells also increased H(+),K(+)-ATPase activity. In contrast, knockdown of endogenous ERp57 in H(+),K(+)-ATPase-expressing cells significantly decreased ATPase activity without changing the expression level of H(+),K(+)-ATPase. Overexpression and knockdown of ERp57 had no significant effect on the expression and function of Na(+),K(+)-ATPase. These results suggest that ERp57 positively regulates H(+),K(+)-ATPase activity apart from its chaperoning function.

Cloning, bacterial expression, and unique structure of adenosylhomocysteine hydrolase-like protein 1, or inositol 1,4,5-triphosphate receptor-binding protein from mouse kidney.

Adenosylhomocysteine hydrolase (SAHase)-like protein 1 (SAH-L), also called inositol 1,4,5-triphosphate receptor-binding protein (IRBIT) is a novel protein involved in fish embryo development and calcium release in mammalian cells through protein-protein interactions. To better understand its reaction mechanism, purified protein is indispensable. Here we describe a simple purification procedure and the unique properties of SAH-L. The cDNA was isolated from mouse kidney by RT-PCR and inserted into various pETtrade mark vectors. Escherichia coli harboring a plasmid coding for SAH-L with a C-terminal His-tag could solely produce a soluble protein. SAH-L purified through a Ni(2+) column gave M(r)s of 59,000 and 190,000 by SDS-PAGE and gel filtration, respectively, which is suggestive of a trimer, but chemical cross-linking experiments demonstrated a dimer. The incompatible M(r) values implicate an irregular structure of SAH-L. In fact, SAH-L was partially purified in a form lacking the 31 N-terminal residues, and was found to be extremely susceptible to proteases in the region around residue 70. The N-terminal polypeptide (residues 1-98) was also expressed as a soluble form and was trypsin-sensitive. Circular dichroism revealed a low alpha-helix content but not a randomly extended structure. Interestingly, SAH-L contained tightly bound NAD(+) despite showing no SAHase activity. The characterized properties of SAH-L and its N-terminal fragment present the notion that the structure of the protease-sensitive N-terminal region is relatively loose and flexible rather than compact, and which protrudes from the major SAHase-like domain. This structure is supposed to be favorable to interact with the IP(3) receptor.

Enzymatic and biochemical properties of a novel human serine dehydratase isoform.

A cDNA clone similar to human serine dehydratase (SDH) is deposited in the GenBank/EMBL databases, but its structural and functional bases remain unknown. Despite the occurrence of mRNA, the expected protein level was found to be low in cultured cells. To learn about physicochemical properties of the protein, we expressed the cDNA in Escherichia coli, and compared the expressed protein with that of a hepatic SDH. The purified protein showed l-serine and l-threonine dehydratase activity, demonstrating to be an isoform of SDH. However, their Km and Vmax constants were different in a range of two-order. Removal of Pro128 from the hepatic SDH consisting of 328 residues, which is missing in the corresponding position of the isoform consisting of 329 residues, significantly changed the Michaelis constants and Kd value for pyridoxal 5'-phosphate, whereas addition of a proline residue to the isoform was without effect. These findings suggest the difference in the structures of the active sites of the two enzymes. Another striking feature was that the expressed level of the isoform in E. coli was 7-fold lower than that of the hepatic SDH. Substitution of Val for Leu287 in the isoform dramatically increased the protein level. The high yield of the mutated isoform was also confirmed by the in vitro transcription and translation experiment. The poor expression of the isoform could be explained by the more stable secondary structure of the mRNA than that of the hepatic SDH mRNA. The present findings may provide a clue as to why the protein level in cultured cells is low.

Catalytic mechanism of S-adenosylhomocysteine hydrolase: roles of His 54, Asp130, Glu155, Lys185, and Aspl89.

S-adenosylhomocysteine hydrolase (AdoHcyase) catalyzes the hydrolysis of S-adenosylhomocysteine (AdoHcy) to form adenosine and homocysteine. The crystal structure of the K185N mutated enzyme, which has weak catalytic activity (0.1%), has been determined at 2.8 A resolution and supports the previously predicted mechanism [Takata, Y., Yamada, T., Huang, Y., Komoto, J., Gomi, T., Ogawa, H., Fujioka, M., & Takusagawa, F. (2002). Catalytic mechanism of S-adenosylhomocysteine hydrolase. Site-directed mutagenesis of Asp-130, Lys-185, Asp-189, and Asn-190. J. Biol. Chem. 277, 22670-22676]. The mutated enzyme has an intermediate structure between the open and closed conformation, observed in the substrate-free enzyme and in the inhibitor complexes, respectively. H54, H300, and H352 were mutated to asparagine, respectively, to identify the roles of the histidine residues in catalysis. The kinetic data of H54N, H300N, and H354N mutated enzymes suggest that H54 is the amino acid residue that acts as a general acid/base to cleave the C5'-S(D) bond of AdoHcy. The E155Q mutated enzyme retained a large portion of the catalytic activity (31%), while the E155D mutated enzyme lost most of it (0.3%). The NADH accumulation measurements of the mutated enzymes indicated that the C3'-oxidation and the C4'-proton abstraction are a concerted event and the C5'-S(D) bond cleavage is an independent event. The C4'-proton exchange measurements indicate that the enzyme has an open conformation when AdoHcy is converted to 3'-keto-4', 5'-dehydro-Ado in the active site. With the results of this study and those of the previous studies, a detailed catalytic mechanism of AdoHcyase is described. K185 facilitates the C3'-oxidation, D130 abstracts the C4'-proton, D189, and E155 act as a communicator between the concerted C3'-oxidation and C4'-proton abstraction, and H54 plays as a general acid to cleave the C5'-S(D) bond of AdoHcy.

Some biochemical and histochemical properties of human liver serine dehydratase.

In rat, serine dehydratase (SDH) is abundant in the liver and known to be a gluconeogenic enzyme, while there is little information about the biochemical property of human liver serine dehydratase because of its low content and difficulty in obtaining fresh materials. To circumvent these problems, we purified recombinant enzyme from Escherichia coli, and compared some properties between human and rat liver serine dehydratases. Edman degradation showed that the N-terminal sequence of about 75% of human serine dehydratase starts from MetSTART-Met2-Ser3- and the rest from Ser3-, whereas the N-terminus of rat enzyme begins from the second codon of MetSTART-Ala2-. The heterogeneity of the purified preparation was totally confirmed by mass spectrometry. Accordingly, this observation in part fails to follow the general rule that the first Met is not removed when the side chain of the penultimate amino acid is bulky such as Met, Arg, Lys, etc. There existed the obvious differences in the local structures between the two enzymes as revealed by limited-proteolysis experiments using trypsin and Staphylococcus aureus V8 protease. The most prominent difference was found histochemically: expression of rat liver serine dehydratase is confined to the periportal region in which many enzymes involved in gluconeogenesis and urea cycle are known to coexist, whereas human liver serine dehydratase resides predominantly in the perivenous region. These findings provide an additional support to the previous notion suggested by physiological experiments that contribution of serine dehydratase to gluconeogenesis is negligible or little in human liver.

Deletion and purification studies to elucidate the structure of the Actinobacillus actinomycetemcomitans cytolethal distending toxin.

Cytolethal distending toxin (CDT) is one of the exotoxins produced by Actinobacillus actinomycetemcomitans, an agent of localized aggressive periodontitis. We constructed N-terminal deletion mutants of CdtA using an Escherichia coli expression system and found that ADelta19-47, with a deletion from Asn-19 to Pro-47, showed comparable CDT activity but no apparent heterogeneity of CdtA. The wild-type CDT (wtCDT) and the mutant CDT (ADelta19-47CDT) were purified to homogeneity by introducing a histidine tag into the C-terminal end of CdtB. Both purified wtCDT and purified ADelta19-47CDT showed strong CDT activity and a tripartite structure composed of CdtA (subunit A), 31 kDa CdtB (subunit B), and 18.5 kDa CdtC (subunit C) in nearly a 1:1:1 stoichiometry. Importantly, subunit A was identified as heterogeneous with three CdtA variants in wtCDT, but homogeneous in ADelta19-47CDT. Purified CDTs also showed high stability that was absolutely dependent on the presence of sucrose in the buffer. In conclusion, the region from the Asn-19 to Pro-47 of CdtA contributes to the heterogeneous production of CdtA, but is dispensable for the toxin activity. Furthermore, this study describes an effective protocol for the purification of a native rather than reconstituted CDT, and clarifies the subunit composition of the active CDT holotoxin.

Catalytic mechanism of guanidinoacetate methyltransferase: crystal structures of guanidinoacetate methyltransferase ternary complexes.

Guanidinoacetate methyltransferase (GAMT) is the enzyme that catalyzes the last step of creatine biosynthesis. The enzyme is found in abundance in the livers of all vertebrates. The intact GAMT from recombinant rat liver has been crystallized with an inhibitor S-adenosylhomocysteine (SAH) and a substrate guanidinoacetate (GAA), and with SAH and an inhibitor guanidine (GUN). These ternary complex structures have been determined at 2.0 A resolution. GAMT has an alpha/beta open-sandwich structure, and the N-terminal section (residues 1-42) covers the active site entrance so that the active site is not visible. SAH has extensive interactions with GAMT through H-bonds and hydrophobic interactions. The guanidino groups of GAA and GUN form two pairs of H-bonds with E45 and D134, respectively. The carboxylate group of GAA interacts with the backbone amide groups of L170 and T171. A model structure of GAMT containing the two substrates (SAM and GAA) was built by attaching a methyl group (C(E)) on S(D) of the bound SAH. On the basis of this model structure, a catalytic mechanism of GAMT is proposed. The active site entrance is opened when the N-terminal section is moved out. GAA and SAM enter the active site and interact with the amino acid residues on the surface of the active site by polar and nonpolar interactions. O(D1) of D134 and C(E) of SAM approach N(E) of GAA from the tetrahedral directions. The O(D1)...N(E) and C(E)...N(E) distances are 2.9 and 2.2 A, respectively. It is proposed that three slightly negatively charged carbonyl oxygen atoms (O of T135, O of C168, and O(B) of GAA) around O(D1) of D134 increase the pK(a) of O(D1) so that O(D1) abstracts the proton on N(E). A strong nucleophile is generated on the deprotonated N(E) of GAA, which abstracts the methyl group (C(E)) from the positively charged S(D) of SAM, and creatine (methyl-GAA) and SAH (demethyl-SAM) are produced. E45, D134, and Y221 mutagenesis studies support the proposed mechanism. A mutagenesis study and the inhibitory mechanism of guanidine analogues support the proposed mechanism.

Solution structure of epiregulin and the effect of its C-terminal domain for receptor binding affinity.

Epiregulin (EPR), a novel member of epidermal growth factor (EGF) family, is a ligand for ErbB-1 and ErbB-4 receptors. The binding affinity of EPR for the receptors is lower than those of other EGF-family ligands. The solution structure of EPR was determined using two-dimensional nuclear magnetic resonance spectroscopy. The secondary structure in the C-terminal domain of EPR is different from other EGF-family ligands because of the lack of hydrogen bonds. The structural difference in the C-terminal domain may provide an explanation for the reduced binding affinity of EPR to the ErbB receptors.

Monoclinic guanidinoacetate methyltransferase and gadolinium ion-binding characteristics.

Guanidinoacetate methyltransferase (GAMT) is the enzyme that catalyzes the last step of creatine biosynthesis. The enzyme is found in abundance in the livers of all vertebrates. Recombinant rat liver GAMT truncated at amino acid 37 from the N-terminus has been crystallized with S-adenosylhomocysteine (SAH) in a monoclinic modification and the crystal structure has been determined at 2.8 A resolution. There are two dimers in the crystallographic asymmetric unit. Each dimer has non-crystallographic twofold symmetry and is related to the other dimer by pseudo-4(3) symmetry along the crystallographic b axis. The overall structure of GAMT crystallized in the monoclinic modification is quite similar to the structure observed in the tetragonal modification [Komoto et al. (2002), J. Mol. Biol. 320, 223-235], with the exception of the loop containing Tyr136. In the monoclinic modification, the loops in three of the four subunits have a catalytically unfavorable conformation and the loop of the fourth subunit has a catalytically favorable conformation as observed in the crystals of the tetragonal modification. From the structures in the monoclinic and tetragonal modifications, we can explain why the Y136F mutant enzyme retains considerable catalytic activity while the Y136V mutant enzyme loses the catalytic activity. The crystal structure of a Gd derivative of the tetragonal modification has also been determined. By comparing the Gd-derivative structure with the native structures in the tetragonal and the monoclinic modifications, useful characteristic features of Gd-ion binding for application in protein crystallography have been observed. Gd ions can bind to proteins without changing the native protein structures and Gd atoms produce strong anomalous dispersion signals from Cu Kalpha radiation; however, Gd-ion binding to protein requires a relatively specific geometry.

Catalytic mechanism of glycine N-methyltransferase.

Methyltransfer reactions are some of the most important reactions in biological systems. Glycine N-methyltransferase (GNMT) catalyzes the S-adenosyl-l-methionine- (SAM-) dependent methylation of glycine to form sarcosine. Unlike most SAM-dependent methyltransferases, GNMT has a relatively high value and is weakly inhibited by the product S-adenosyl-l-homocysteine (SAH). The major role of GNMT is believed to be the regulation of the cellular SAM/SAH ratio, which is thought to play a key role in SAM-dependent methyltransfer reactions. Crystal structures of GNMT complexed with SAM and acetate (a potent competitive inhibitor of Gly) and the R175K mutated enzyme complexed with SAM were determined at 2.8 and 3.0 A resolutions, respectively. With these crystal structures and the previously determined structures of substrate-free enzyme, a catalytic mechanism has been proposed. Structural changes occur in the transitions from the substrate-free to the binary complex and from the binary to the ternary complex. In the ternary complex stage, an alpha-helix in the N-terminus undergoes a major conformational change. As a result, the bound SAM is firmly connected to protein and a "Gly pocket" is created near the bound SAM. The second substrate Gly binds to Arg175 and is brought into the Gly pocket. Five hydrogen bonds connect the Gly in the proximity of the bound SAM and orient the lone pair orbital on the amino nitrogen (N) of Gly toward the donor methyl group (C(E)) of SAM. Thermal motion of the enzyme leads to a collision of the N and C(E) so that a S(N)2 methyltransfer reaction occurs. The proposed mechanism is supported by mutagenesis studies.

Crystal structure of guanidinoacetate methyltransferase from rat liver: a model structure of protein arginine methyltransferase.

Guanidinoacetate methyltransferase (GAMT) is the enzyme that catalyzes the last step of creatine biosynthesis. The enzyme is found in abundance in the livers of all vertebrates. Recombinant rat liver GAMT has been crystallized with S-adenosylhomocysteine (SAH), and the crystal structure has been determined at 2.5 A resolution. The 36 amino acid residues at the N terminus were cleaved during the purification and the truncated enzyme was crystallized. The truncated enzyme forms a dimer, and each subunit contains one SAH molecule in the active site. Arg220 of the partner subunit forms a pair of hydrogen bonds with Asp134 at the guanidinoacetate-binding site. On the basis of the crystal structure, site-directed mutagenesis on Asp134, and chemical modification and limited proteolysis studies, we propose a catalytic mechanism of this enzyme. The truncated GAMT dimer structure can be seen as a ternary complex of protein arginine methyltransferase (one subunit) complexed with a protein substrate (the partner subunit) and the product SAH. Therefore, this structure provides insight into the structure and catalysis of protein arginine methyltransferases.

Catalytic mechanism of S-adenosylhomocysteine hydrolase. Site-directed mutagenesis of Asp-130, Lys-185, Asp-189, and Asn-190.

S-Adenosylhomocysteine hydrolase (AdoHcyase) catalyzes the hydrolysis of S-adenosylhomocysteine to form adenosine and homocysteine. On the bases of crystal structures of the wild type enzyme and the D244E mutated enzyme complexed with 3'-keto-adenosine (D244E.Ado*), we have identified the important amino acid residues, Asp-130, Lys-185, Asp-189, and Asn-190, for the catalytic reaction and have proposed a catalytic mechanism (Komoto, J., Huang, Y., Gomi, T., Ogawa, H., Takata, Y., Fujioka, M., and Takusagawa, F. (2000) J. Biol. Chem. 275, 32147-32156). To confirm the proposed catalytic mechanism, we have made the D130N, K185N, D189N, and N190S mutated enzymes and measured the catalytic activities. The catalytic rates (k(cat)) of D130N, K185N, D189N, and N190S mutated enzymes are reduced to 0.7%, 0.5%, 0.1%, and 0.5%, respectively, in comparison with the wild type enzyme, indicating that Asp-130, Lys-185, Asp-189, and Asn-190 are involved in the catalytic reaction. K(m) values of the mutated enzymes are increased significantly, except for the N190S mutation, suggesting that Asp-130, Lys-185, and Asp-189 participate in the substrate binding. To interpret the kinetic data, the oxidation states of the bound NAD molecules of the wild type and mutated enzymes were measured during the catalytic reaction by monitoring the absorbance at 340 nm. The crystal structures of the WT and D244E.Ado*, containing four subunits in the crystallographic asymmetric unit, were re-refined to have the same subunit structures. A detailed catalytic mechanism of AdoHcyase has been revealed based on the oxidation states of the bound NAD and the re-refined crystal structures of WT and D244E.Ado*. Lys-185 and Asp-130 abstract hydrogen atoms from 3'-OH and 4'-CH, respectively. Asp-189 removes a proton from Lys-185 and produces the neutral N zeta (-NH(2)), and Asn-190 facilitates formation of the neutral Lys-185. His-54 and His-300 hold and polarize a water molecule, which nucleophilically attacks the C5'- of 3'-keto-4',5'-dehydroadenosine to produce 3'-keto-Ado.

Evidence for a dimeric structure of rat liver serine dehydratase.

Rat liver serine dehydratase (SDH) is known to be involved in gluconeogenesis. It has long been believed to be a dimeric protein with the subunit molecular weight (M(r)) of 34,000. Recently, sheep liver SDH was reported to be a monomer with a M(r) of 38,000. The native M(r) of rat SDH was only determined by the ultracentrifugation method more than three decades ago, and that of sheep SDH was done by the method of gel chromatography. The primary to quaternary structures of a given enzyme in a specific mammalian organ are usually conserved among various species. The aim of the present investigation is to clarify the structural differences between rat and sheep SDHs. First, we found that the amino acid composition reported for sheep SDH was statistically similar to that of rat SDH. Second, immunoblot analysis using anti-rat SDH IgG as the probe showed the size of sheep SDH to be a M(r) of 30,500, whereas that of SDH was about M(r) of 35,000. On the other hand, the native size of rat SDH was assessed by two methods: (1) the laser light scattering method demonstrated that rat SDH had a M(r) of 66,800, consistent with the previous value (M(r)=64,000); (2) cross-linking experiments of the purified rat SDH with dimethyl suberimidate revealed the existence of a dimeric form by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The present results clearly confirm that rat SDH is a dimer, and suggest that sheep SDH is similar to rat SDH immunologically, but with a molecular weight 7500 smaller than reported previously.

Inhibition of S-adenosylhomocysteine hydrolase by acyclic sugar adenosine analogue D-eritadenine. Crystal structure of S-adenosylhomocysteine hydrolase complexed with D-eritadenine.

D-eritadenine (DEA) is a potent inhibitor (IC(50) = 7 nm) of S-adenosyl-l-homocysteine hydrolase (AdoHcyase). Unlike cyclic sugar Ado analogue inhibitors, including mechanism-based inhibitors, DEA is an acyclic sugar Ado analogue, and the C2' and C3' have opposite chirality to those of the cyclic sugar Ado inhibitors. Crystal structures of DEA alone and in complex with AdoHcyase have been determined to elucidate the DEA binding scheme to AdoHcyase. The DEA-complexed structure has been analyzed by comparing it with two structures of AdoHcyase complexed with cyclic sugar Ado analogues. The DEA-complexed structure has a closed conformation, and the DEA is located near the bound NAD(+). However, a UV absorption measurement shows that DEA is not oxidized by the bound NAD(+), indicating that the open-closed conformational change of AdoHcyase is due to the substrate/inhibitor binding, not the oxidation state of the bound NAD. The adenine ring of DEA is recognized by four essential hydrogen bonds as observed in the cyclic sugar Ado complexes. The hydrogen bond network around the acyclic sugar moiety indicates that DEA is more tightly connected to the protein than the cyclic sugar Ado analogues. The C3'-H of DEA is pointed toward C4 of the bound NAD(+) (C3'...C4 = 3.7 A), suggesting some interaction between DEA and NAD(+). By placing DEA into the active site of the open structure, the major forces to stabilize the closed conformation of AdoHcyase are identified as the hydrogen bonds between the backbone of His-352 and the adenine ring, and the C3'-H...C4 interaction. DEA has been believed to be an inactivator of AdoHcyase, but this study indicates that DEA is a reversible inhibitor. On the basis of the complexed structure, selective inhibitors of AdoHcyase have been designed.