Crystal structure of S-adenosylhomocysteine hydrolase from rat liver.

The crystal structure of rat liver S-adenosyl-L-homocysteine hydrolase (AdoHcyase, EC 3.3.1.1) which catalyzes the reversible hydrolysis of S-adenosylhomocysteine (AdoHcy) has been determined at 2.8 A resolution. AdoHcyase from rat liver is a tetrameric enzyme with 431 amino acid residues in each identical subunit. The subunit is composed of the catalytic domain, the NAD+-binding domain, and the small C-terminal domain. Both catalytic and NAD+-binding domains are folded into an ellipsoid with a typical alpha/beta twisted open sheet structure. The C-terminal section is far from the main body of the subunit and extends into the opposite subunit. An NAD+ molecule binds to the consensus NAD+-binding cleft of the NAD+-binding domain. The peptide folding pattern of the catalytic domain is quite similar to the patterns observed in many methyltransferases. Although the crystal structure does not contain AdoHcy or its analogue, there is a well-formed AdoHcy-binding crevice in the catalytic domain. Without introducing any major structural changes, an AdoHcy molecule can be placed in the catalytic domain. In the structure described here, the catalytic and NAD+-binding domains are quite far apart from each other. Thus, the enzyme appears to have an "open" conformation in the absence of substrate. It is likely that binding of AdoHcy induces a large conformational change so as to place the ribose moiety of AdoHcy in close proximity to the nicotinamide moiety of NAD+. A catalytic mechanism of AdoHcyase has been proposed on the basis of this crystal structure. Glu155 acts as a proton acceptor from the O3'-H when the proton of C3'-H is abstracted by NAD+. His54 or Asp130 acts as a general acid-base catalyst, while Cys194 modulates the oxidation state of the bound NAD+. The polypeptide folding pattern of the catalytic domain suggests that AdoHcy molecules can travel freely to and from AdoHcyase and methyltransferases to properly regulate methyltransferase activities. We believe that the crystal structure described here can provide insight into the molecular architecture of this important regulatory enzyme.     

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.     

Regulation of S-adenosylhomocysteine hydrolase in the halophilic cyanobacterium Aphanothece halophytica: a possible role in glycinebetaine biosynthesis

Aphanothece halophytica, a halophilic cyanobacterium capable of growing in saturated NaCl, accumulates high intracellular concentrations of glycinebetaine in response to increasing environmental NaCl. In this organism, intracellular levels of K⁺ rise dramatically with increasing external NaCl before an increase in glycinebetaine can be detected. Glycinebetaine synthesis requires three S-adenosylmethionine (AdoMet)-mediated transmethylations; each transmethylation reaction generates one molecule of the transmethylation inhibitor S-adenosylhomocysteine (AdoHcy). Thus, glycinebetaine synthesis should require continued removal of AdoHcy. In A. halophytica, catabolism of AdoHcy was shown to occur via the reversible reaction catalyzed by AdoHcy hydrolase (EC 3.3.1.1). Activity of AdoHcy hydrolase in the direction of synthesis of AdoHcy was inhibited by 0.4 M KCl in this organism. On the other hand, activity of AdoHcy hydrolase in the direction of AdoHcy hydrolysis was unaffected by 0.4 M KCl. Glycinebetaine increased synthesis of AdoHcy in the presence of 0.4 KCl, but had no effect on AdoHcy hydrolysis. Based upon these results, a mechanism is proposed for the regulation of glycinebetaine synthesis by K⁺ and glycinebetaine in A. halophytica. According to this mechanism, the regulatory response would be initiated by a K⁺-induced shift in the AdoMet/AdoHcy ratio.Abbreviations AdoMet S-adenosylmethionine - AdoHcy S-adenosyl homocysteine     

Structure of Escherichia coli 5'-methylthioadenosine/ S-adenosylhomocysteine nucleosidase inhibitor complexes provide insight into the conformational changes required for substrate binding and catalysis

5'-Methylthioadenosine/S-adenosylhomocysteine (MTA/AdoHcy) nucleosidase is a key enzyme in a number of critical biological processes in many microbes. This nucleosidase catalyzes the irreversible hydrolysis of the N(9)-C(1') bond of MTA or AdoHcy to form adenine and the corresponding thioribose. The key role of the MTA/AdoHcy nucleosidase in biological methylation, polyamine biosynthesis, methionine recycling, and bacterial quorum sensing has made it an important antimicrobial drug target. The crystal structures of Escherichia coli MTA/AdoHcy nucleosidase complexed with the transition state analog, formycin A (FMA), and the nonhydrolyzable substrate analog, 5'-methylthiotubercidin (MTT) have been solved to 2.2- and 2.0-A resolution, respectively. These are the first MTA/AdoHcy nucleosidase structures to be solved in the presence of inhibitors. These structures clearly identify the residues involved in substrate binding and catalysis in the active site. Comparisons of the inhibitor complexes to the adenine-bound MTA/AdoHcy nucleosidase (Lee, J. E., Cornell, K. A., Riscoe, M. K., and Howell, P. L. (2001) Structure (Camb.) 9, 941-953) structure provide evidence for a ligand-induced conformational change in the active site and the substrate preference of the enzyme. The enzymatic mechanism has been re-examined.     

Molecular insights of SAH enzyme catalysis and implication for inhibitor design

Biological transmethylation reaction is a key step in the duplication of virus life cycle, in which S-adenosylmethionine plays as the methyl donor. The product of this reactions, S-adenosylhomocysteine (AdoHcy) inhibits the transmethylation process. AdoHcy is hydrolysed to adenosine and L-homocysteine by the action of S-adenosylhomocysteine hydrolase (SAH). Thus the virus life cycle should be cut off once the action of SAH is inhibited. Our study was focussed on the discovery of potential inhibitor against SAH. We performed a similarity search in Traditional Chinese Medicine Database and retrieved 17 hits with high similarity. After that we virtually docked the 17 compounds as well as the natural substrates to the hydrolase using Autodock 3.0.1 software. Then we discussed about the mechanism of the inhibition reaction, followed by proposing the potential inhibitors by comparing best docked solutions and possible modification for the best inhibitors.     

Inhibition of EcoRI DNA methylase with cofactor analogs

Four analogs of the natural cofactor S-adenosylmethionine (AdoMet) were tested for their ability to bind and inhibit the prokaryotic enzyme, EcoRI adenine DNA methylase. The EcoRI methylase transfers the methyl group from AdoMet to the second adenine in the double-stranded DNA sequence 5'GAATTC3'. Dissociation constants (KD) of the binary methylase-analog complexes obtained in the absence of DNA with S-adenosylhomocysteine (AdoHcy), sinefungin, N-methyl-AdoMet, and N-ethylAdoMet are 225, 43, greater than 1000, and greater than 1000 microM, respectively. In the presence of a DNA substrate, all four analogs show simple competitive inhibition with respect to AdoMet. The product of the enzymic reaction, AdoHcy, is a poor inhibitor of the enzyme (KI(AdoHcy) = 9 microM; KM(AdoMet) = 0.60 microM). Two synthetic analogs, N-methyl-AdoMet and N-ethyl-AdoMet, were also shown to be poor inhibitors with KI values of 50 and greater than 1000 microM, respectively. In contrast, the naturally occurring analog sinefungin was shown to be a highly potent inhibitor (KI = 10 nM). Gel retardation assays confirm that the methylase-DNA-sinefungin complex is sequence-specific. The ternary complex is the first sequence-specific complex detected for any DNA methylase. Potential applications to structural studies of methylase-DNA interactions are discussed.     

青岛文昌鱼S-腺苷高半胱氨酸水解酶基因的组织特异性表达

为了探索文昌鱼S-腺苷高半胱氨酸水解酶AdoHcyase基因在文昌鱼组织中的表达分布情况,利用组织原位杂交技术,以地高辛标记的反义RNA为探针,检测了文昌鱼AdoHcyase基因在组织中的表达分布特点.结果表明,AdoHcyase基因在雌性文昌鱼的卵巢、肝盲囊和后肠杂交信号十分强烈,在内柱、鳃等组织中也有微弱信号表达,...     

Structure of E. coli 5'-methylthioadenosine/S-adenosylhomocysteine nucleosidase reveals similarity to the purine nucleoside phosphorylases

BACKGROUND: 5'-methylthioadenosine/S-adenosyl-homocysteine (MTA/AdoHcy) nucleosidase catalyzes the irreversible cleavage of 5'-methylthioadenosine and S-adenosylhomocysteine to adenine and the corresponding thioribose, 5'-methylthioribose and S-ribosylhomocysteine, respectively. While this enzyme is crucial for the metabolism of AdoHcy and MTA nucleosides in many prokaryotic and lower eukaryotic organisms, it is absent in mammalian cells. This metabolic difference represents an exploitable target for rational drug design. RESULTS: The crystal structure of E. coli MTA/AdoHcy nucleosidase was determined at 1.90 A resolution with the multiwavelength anomalous diffraction (MAD) technique. Each monomer of the MTA/AdoHcy nucleosidase dimer consists of a mixed alpha/beta domain with a nine-stranded mixed beta sheet, flanked by six alpha helices and a small 3(10) helix. Intersubunit contacts between the two monomers present in the asymmetric unit are mediated primarily by helix-helix and helix-loop hydrophobic interactions. The unexpected presence of an adenine molecule in the active site of the enzyme has allowed the identification of both substrate binding and potential catalytic amino acid residues. CONCLUSIONS: Although the sequence of E. coli MTA/AdoHcy nucleosidase has almost no identity with any known enzyme, its tertiary structure is similar to both the mammalian (trimeric) and prokaryotic (hexameric) purine nucleoside phosphorylases. The structure provides evidence that this protein is functional as a dimer and that the dual specificity for MTA and AdoHcy results from the truncation of a helix. The structure of MTA/AdoHcy nucleosidase is the first structure of a prokaryotic nucleoside N-ribohydrolase specific for 6-aminopurines.     

S-adenosylhomocysteine metabolism in different cell lines: effect of hypoxia and cell density.

BACKGROUND/AIMS: The methylation potential (MP) is defined as the ratio of S-adenosylmethionine (AdoMet) to S-adenosylhomocysteine (AdoHcy). It was shown recently that hypoxia increases AdoMet/AdoHcy ratio in HepG2 cells (Hermes et al., Exp Cell Res 294: 325-334, 2004). In the present study, we compared AdoMet/AdoHcy ratio and energy metabolism in HepG2, HEK-293, HeLa, MCF-7 and SK-HEP-1 cell lines under normoxia and hypoxia. METHODS: Metabolite concentrations were measured by HPLC. In addition, AdoHcy hydrolase (AdoHcyase) activity was determined photometrically. RESULTS: Under normoxia HepG2 cells show the highest AdoMet/AdoHcy ratio of 53.4 +/- 3.3 followed by MCF-7 and SK-HEP-1 cells with a AdoMet/AdoHcy ratio of 14.4 +/- 1.1 and 21.1 +/- 1.3, respectively. The lowest AdoMet/AdoHcy ratios are exhibited by HeLa and HEK-293 cells (6.6 +/- 0.7 and 7.1 +/- 0.3). Hypoxia does not significantly change the MP in MCF-7 and HeLa cells, but alters the MP in HepG2, HEK-293 and SK-HEP-1 cells. These alterations are dependent on the cell density. Under normoxia HepG2 cells exhibit AdoHcyase activity of 2.5 +/- 0.2 nmol min(-1) mg(-1) protein. All other cell lines show 3-5 times lower enzyme activity. Interestingly, hypoxia affects AdoHcyase activity only in HepG2 cells. CONCLUSIONS: Our data clearly show that the cell lines are characterized by different MP and different behavior under hypoxia. That implies that a lower MP is not necessarily associated with impaired transmethylation activity and cellular function.     

Structural snapshots of MTA/AdoHcy nucleosidase along the reaction coordinate provide insights into enzyme and nucleoside flexibility during catalysis

MTA/AdoHcy nucleosidase (MTAN) irreversibly hydrolyzes the N9-C1' bond in the nucleosides, 5'-methylthioadenosine (MTA) and S-adenosylhomocysteine (AdoHcy) to form adenine and the corresponding thioribose. MTAN plays a vital role in metabolic pathways involving methionine recycling, biological methylation, polyamine biosynthesis, and quorum sensing. Crystal structures of a wild-type (WT) MTAN complexed with glycerol, and mutant-enzyme and mutant-product complexes have been determined at 2.0A, 2.0A, and 2.1A resolution, respectively. The WT MTAN-glycerol structure provides a purine-free model and in combination with the previously solved thioribose-free MTAN-ADE structure, we now have separate apo structures for both MTAN binding subsites. The purine and thioribose-free states reveal an extensive enzyme-immobilized water network in their respective binding subsites. The Asp197Asn MTAN-MTA and Glu12Gln MTAN-MTR.ADE structures are the first enzyme-substrate and enzyme-product complexes reported for MTAN, respectively. These structures provide representative snapshots along the reaction coordinate and allow insight into the conformational changes of the enzyme and the nucleoside substrate. A "catalytic movie" detailing substrate binding, catalysis, and product release is presented.     

Effects of site-directed mutagenesis on structure and function of recombinant rat liver S-adenosylhomocysteine hydrolase. Crystal structure of D244E mutant enzyme

A site-directed mutagenesis, D244E, of S-adenosylhomocysteine hydrolase (AdoHcyase) changes drastically the nature of the protein, especially the NAD(+) binding affinity. The mutant enzyme contained NADH rather than NAD(+) (Gomi, T., Takata, Y., Date, T., Fujioka, M., Aksamit, R. R., Backlund, P. S., and Cantoni, G. L. (1990) J. Biol. Chem. 265, 16102-16107). In contrast to the site-directed mutagenesis study, the crystal structures of human and rat AdoHcyase recently determined have shown that the carboxyl group of Asp-244 points in a direction opposite to the bound NAD molecule and does not participate in any hydrogen bonds with the NAD molecule. To explain the discrepancy between the mutagenesis study and the x-ray studies, we have determined the crystal structure of the recombinant rat-liver D244E mutant enzyme to 2.8-A resolution. The D244E mutation changes the enzyme structure from the open to the closed conformation by means of a approximately 17 degrees rotation of the individual catalytic domains around the molecular hinge sections. The D244E mutation shifts the catalytic reaction from a reversible to an irreversible fashion. The large affinity difference between NAD(+) and NADH is mainly due to the enzyme conformation, but not to the binding-site geometry; an NAD(+) in the open conformation is readily released from the enzyme, whereas an NADH in the closed conformation is trapped and cannot leave the enzyme. A catalytic mechanism of AdoHcyase has been proposed on the basis of the crystal structures of the wild-type and D244E enzymes.     

Structural comparison of MTA phosphorylase and MTA/AdoHcy nucleosidase explains substrate preferences and identifies regions exploitable for inhibitor design

The development of new and effective antiprotozoal drugs has been a difficult challenge because of the close similarity of the metabolic pathways between microbial and mammalian systems. 5'-Methylthioadenosine/S-adenosylhomocysteine (MTA/AdoHcy) nucleosidase is thought to be an ideal target for therapeutic drug design as the enzyme is present in many microbes but not in mammals. MTA/AdoHcy nucleosidase (MTAN) irreversibly depurinates MTA or AdoHcy to form adenine and the corresponding thioribose. The inhibition of MTAN leads to a buildup of toxic byproducts that affect various microbial pathways such as quorum sensing, biological methylation, polyamine biosynthesis, and methionine recycling. The design of nucleosidase-specific inhibitors is complicated by its structural similarity to the human MTA phosphorylase (MTAP). The crystal structures of human MTAP complexed with formycin A and 5'-methylthiotubercidin have been solved to 2.0 and 2.1 A resolution, respectively. Comparisons of the MTAP and MTAN inhibitor complexes reveal size and electrostatic potential differences in the purine, ribose, and 5'-alkylthio binding sites, which account for the substrate specificity and reactions catalyzed. In addition, the differences between the two enzymes have allowed the identification of exploitable regions that can be targeted for the development of high-affinity nucleosidase-specific inhibitors. Sequence alignments of Escherichia coli MTAN, human MTAP, and plant MTA nucleosidases also reveal potential structural changes to the 5'-alkylthio binding site that account for the substrate preference of plant MTA nucleosidases.     

A single mutation at Tyr143 of human S-adenosylhomocysteine hydrolase renders the enzyme thermosensitive and affects the oxidation state of bound cofactor nicotinamide-adenine dinucleotide

Recently, we have described the first human case of AdoHcyase (S-adenosylhomocysteine hydrolase) deficiency. Two point mutations in the AdoHcyase gene, the missense mutation p.Y143C (AdoHcyase in which Tyr143 is replaced by cysteine) and the truncation mutation p.W112stop (AdoHcyase in which Trp112 is replaced by opal stop codon) were identified [Bari?, Fumi?, Glenn, Cuk, Schulze, Finkelstein, James, Mejaski-Bosnjak, Pazanin, Pogribny et al. (2004) Proc. Natl. Acad. Sci. U.S.A. 101, 4234-4239]. To elucidate the molecular and catalytic properties of AdoHcyase, we have made recombinant wild-type and mutant p.Y143C (AdoHcyase in which Tyr143 is replaced by cysteine) enzymes for a comparative analysis. The catalytic rates of p.Y143C protein in the directions of S-adenosylhomocysteine synthesis or hydrolysis are decreased from 65% to 75%. Further, the oxidation states of coenzyme NAD differ between mutant and wild-type protein, with an increased NADH accumulation in the mutant p.Y143C enzyme of 88% NADH (wild-type contains 18% NADH). Quantitative binding of NAD is not affected. Native polyacrylamide gel electrophoresis showed, that mutant p.Y143C subunits are able to form the tetrameric complex as is the wild-type enzyme. CD analysis showed that the p.Y143C mutation renders the recombinant protein thermosensitive, with an unfolding temperature significantly reduced by 7 degrees C compared with wild-type protein. Change of Glu115 to lysine in wild-type protein causes a change in thermosensitivity almost identical with that found in the p.Y143C enzyme, indicating that the thermosensitivity is due to a missing hydrogen bond between Tyr143 and Glu115. We emphasize involvement of this particular hydrogen bond for subunit folding and/or holoenyzme stability. In summary, a single mutation in the AdoHcyase affecting both the oxidation state of bound co-factor NAD and enzyme stability is present in a human with AdoHcyase deficiency.     

Effect of limited proteolysis on the stability and enzymatic activity of human placental S-adenosylhomocysteine hydrolase.

Human placental S-adenosylhomocysteine (AdoHcy) hydrolase was subjected to limited papain digestion. The multiple cleavage sites in the enzyme were identified to be Lys94-Ala95, Tyr100-Ala101, Glu243-Ile244, Met367-Ala368, Gln369-Ile370, and Gly382-Val383. Despite multiple cleavage sites in the backbone of the protein, the digested enzyme was able to maintain its quaternary structure and retain its full catalytic activity. The enzyme activity of the partially digested AdoHcy hydrolase was essentially identical to that of the native enzyme at several pH values. The thermal stabilities of the native and partially digested enzymes were only slightly different at all temperatures tested. The stability of both native and partially digested enzymes were examined in guanidine hydrochloride and equilibrium unfolding transitions were monitored by CD spectroscopy and tryptophan fluorescence spectroscopy. The results of these experiments can be summarized as follows: (1) CD spectroscopic analysis showed that the overall secondary and tertiary structures of the partially digested enzyme are essentially identical with those of the native enzyme; and (2) tryptophan fluorescence spectroscopic analysis indicated that there are small differences in the environments of surface-exposed tryptophan residues between the partially digested enzyme and the native enzyme under unfolding conditions. The differences in the free energy of unfolding, delta(delta Gu) [delta Gu(native)-delta Gu(digested)], is approximately 1.3 kcal/mol. When NAD+ was removed from the partially digested enzyme, the secondary and tertiary structures of the apo form of the digested AdoHcy hydrolase were completely lost and the enzymatic activity could not be recovered by incubation with excess NAD+. These results suggest that AdoHcy hydrolase exists as a very compact enzyme with extensive intramolecular bonding, which contributes significantly to the overall global protein stabilization. Identification of the surface-exposed peptide bonds, which are susceptible to papain digestion, has provided some constraints on the spatial orientations of subunits of the enzyme. This information, in turn, has provided supplemental data for X-ray crystallographic studies currently ongoing in our laboratories.     

An enzyme-coupled continuous spectrophotometric assay for S-adenosylmethionine-dependent methyltransferases

Modification of small molecules and proteins by methyltransferases affects a wide range of biological processes. Here, we report an enzyme-coupled continuous spectrophotometric assay to quantitatively characterize S-adenosyl-L-methionine (AdoMet/SAM)-dependent methyltransferase activity. In this assay, S-adenosyl-L-homocysteine (AdoHcy/SAH), the transmethylation product of AdoMet-dependent methyltransferases, is hydrolyzed to S-ribosylhomocysteine and adenine by recombinant S-adenosylhomocysteine/5'-methylthioadenosine nucleosidase (SAHN/MTAN, EC 3.2.2.9). Subsequently, adenine generated from AdoHcy is further hydrolyzed to hypoxanthine and ammonia by recombinant adenine deaminase (EC 3.5.4.2). This deamination is associated with a decrease in absorbance at 265 nm that can be monitored continuously. Coupling enzymes are recombinant and easily purified. The utility of this assay was shown using recombinant rat protein arginine N-methyltransferase 1 (PRMT1, EC 2.1.1.125), which catalyzes the mono- and dimethylation of guanidino nitrogens of arginine residues in select proteins. Using this assay, the kinetic parameters of PRMT1 with three synthetic peptides were determined. An advantage of this assay is the destruction of AdoHcy by AdoHcy nucleosidase, which alleviates AdoHcy product feedback inhibition of S-adenosylmethionine-dependent methyltransferases. Finally, this method may be used to assay other enzymes that produce AdoHcy, 5'-methylthioadenosine, or compounds that can be cleaved by AdoHcy nucleosidase.     

Effects of ligand binding and oxidation on hinge-bending motions in S-adenosyl-L-homocysteine hydrolase

Domain motions of S-adenosyl-l-homocysteine (AdoHcy) hydrolase have been detected by time-resolved fluorescence anisotropy measurements. Time constants for reorientational motions in the native enzyme were compared with those for enzymes where key residues were altered by site-directed mutation. Mutations M351P, H353A, and P354A were selected in a hinge region for motion between the open and closed forms of the enzyme, as identified in a previous normal-mode study [Wang et al. (2005) Domain motions and the open-to-closed conformational transition of an enzyme: A normal-mode analysis of S-adenosyl-l-homocysteine hydrolase, Biochemistry 44, 7228-7239]. In wild-type, substrate-free AdoHcy hydrolase (NAD(+) cofactor in each subunit), reorientational motions were detected on time scales of 10-20 and 80-90 ns. The faster motion is attributed to the domain motion, and the slower motion is attributed to the tumbling of the enzyme. The domain motion was also detected for the enzyme complexes E(NADH/3'-keto-adenosine) and E(NAD(+)/3'-deoxyadenosine) but was absent for the complex E(NADH/3'-keto-neplanocin A). The results indicate that AdoHcy hydrolase exists in equilibrium of open and closed structures, with the equilibrium shifted toward the more mobile open form for the substrate-free enzyme, E(NAD(+)), and for intermediates formed early in the catalytic cycle after substrate binding or formed late prior to product release, E(NAD(+)/ligand). However, the strong inhibitor neplanocin A upon binding undergoes oxidation, forming the complex E(NADH/3'-keto-neplanocin). For this complex, which is analogous to the enzyme complex with the central catalytic intermediate, the equilibrium was shifted toward the more rigid closed form. A similar pattern was observed for M351P and P354A mutants. In contrast, the domain motion could not be detected, either in the absence or presence of ligands or with the cofactor in either the oxidized or reduced state, for the H353A protein, suggesting that this mutation changes the hinge-bending dynamics of the enzyme.     

Inhibition of phosphoinositide metabolism in human polymorphonuclear leukocytes by S-adenosylhomocysteine

Transmembrane signaling by chemoattractants in leukocytes appears to require activation of phosphoinositide metabolism with subsequent generation of the second messenger substances, inositol(1,4,5)trisphosphate and diacylglycerol. In addition, previous studies have shown that conditions which lead to an intracellular increase in S-adenosylhomocysteine (AdoHcy), a by-product and competitive inhibitor of S-adenosylmethionine-mediated methylation reactions, inhibit all chemoattractant-mediated functions of leukocytes, suggesting that AdoHcy also interferes with chemoattractant transmembrane signaling. In the present study, we determined whether AdoHcy altered the metabolism of phosphoinositides in human polymorphonuclear leukocytes. Treatment of 32P-labeled polymorphonuclear leukocytes with the adenosine deaminase inhibitor, erythro-9-(2-hydroxy-3-nonyl)adenine, plus exogenous adenosine and L-homocysteine thiolactone, conditions which cause an increase in AdoHcy, produced as much as a 37% decrease in the amount of [32P]phosphatidylinositol 4-monophosphate associated with the cells. The formation of inositol bisphosphate was inhibited by as much as 45% by erythro-9-(2-hydroxy-3-nonyl)adenine, adenosine, and L-homocysteine thiolactone suggesting decreased availability of phosphatidylinositol 4-monophosphate. In support of this, AdoHcy, in concentrations ranging from 0.01 to 0.1 mM, inhibited the transfer of gamma-32P from gamma-[32P] ATP to phosphatidylinositol (PtdIns). The inhibition of PtdIns kinase was competitive with an apparent Ki for AdoHcy of 43 microM. Increased intracellular AdoHcy reduced chemoattractant-mediated increases in inositol(1,4,5)trisphosphate formation suggesting abrogation of transmembrane signaling. These findings for the first time demonstrate that AdoHcy is a competitive inhibitor of PtdIns kinase and thus a regulator of the phosphoinositide pathway.     

Cofactor regeneration in phototrophic cyanobacteria applied for asymmetric reduction of ketones

The obligate photoautotrophic cyanobacterium Synechococcus PCC7942 and the photoheterotrophic heterocystous cyanobacterium Noctoc muscorum are able to reduce prochiral ketones asymmetrically to optical pure chiral alcohols without light. An example is the synthesis of S-pentafluoro(phenyl-)ethanol with an enantiomeric excess >99% if 2′-3′-4′-5′-6′-pentafluoroacetophenone is used as substrate. If no light is available for regeneration of the cofactor nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH), glucose is used as cosubstrate. Membrane disintegration during asymmetric reduction promotes cytosolic energy generating metabolic pathways. Observed regulatory effects depicted by an adenosine triphosphate (ATP) to nicotinamide adenine dinucleotide phosphate (oxidized form) (NADP⁺) ratio of 3:1 for efficient cofactor recycling indicate a metabolization via glycolisis. The stoichiometric formation of the by-product acetate (1 mol acetate/1 mol chiral alcohol) indicates homoacetic acid fermentation for cofactor regeneration including the obligate photoautotrophic cyanobacterium Synechococcus PCC7942.     

Kinetic and catalytic mechanism of HhaI methyltransferase

Kinetic and catalytic properties of the DNA (cytosine-5)-methyltransferase HhaI are described. With poly(dG-dC) as substrate, the reaction proceeds by an equilibrium (or processive) ordered Bi-Bi mechanism in which DNA binds to the enzyme first, followed by S-adenosylmethionine (AdoMet). After methyl transfer, S-adenosylhomocysteine (AdoHcy) dissociates followed by methylated DNA. AdoHcy is a potent competitive inhibitor with respect to AdoMet (Ki = 2.0 microM) and its generation during reactions results in non-linear kinetics. AdoMet and AdoHcy significantly interact with only the substrate enzyme-DNA complex; they do not bind to free enzyme and bind poorly to the methylated enzyme-DNA complex. In the absence of AdoMet, HhaI methylase catalyzes exchange of the 5-H of substrate cytosines for protons of water at about 7-fold the rate of methylation. The 5-H exchange reaction is inhibited by AdoMet or AdoHcy. In the enzyme-DNA-AdoHcy complex, AdoHcy also suppresses dissociation of DNA and reassociation of the enzyme with other substrate sequences. Our studies reveal that the catalytic mechanism of DNA (cytosine-5)-methyltransferases involves attack of the C6 of substrate cytosines by an enzyme nucleophile and formation of a transient covalent adduct. Based on precedents of other enzymes which catalyze similar reactions and the susceptibility of HhaI to inactivation by N-ethylmaleimide, we propose that the sulfhydryl group of a cysteine residue is the nucleophilic catalyst. Furthermore, we propose that Cys-81 is the active-site catalyst in HhaI. This residue is found in a Pro-Cys doublet which is conserved in all DNA (cytosine-5)-methyltransferases whose sequences have been determined to date and is found in related enzymes. Finally, we discuss the possibility that covalent adducts between C6 of pyrimidines and nucleophiles of proteins may be important general components of protein-nucleic acid interactions.