Affinity-chromatographic purification of <i>S</i>-adenosyl-<scp>l</scp>-homocysteine hydrolase. Some properties of the enzyme from rat liver

Kajander, E. Olavi; Raina, Aarne

doi:10.1042/bj1930503

Cited by 44 publications

(3 citation statements)

References 31 publications

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“…[a-35S]dATP ( 1168Ci/mmol), [a-35S]dCTP (1039 Ci/mmol), GeneScreen Plus nylon membranes, and Colony/Plaque screen hybridization membranes were purchased from DuPont NEN Research Products (Boston, MA). The coupling of 8-[(3-aminopropyl)amino] adenosine to CH-Sepharose 4B to give Ado-Sepharose was carried out as described (Trayer et al, 1974;Kajander & Raina, 1981). [1-3H]FPP (16.7 and 71.7 MCi/jmiol) was synthesized as previously reported (Cane & Ha, 1988).…”

Section: Methodsmentioning

confidence: 99%

Pentalenene Synthase. Purification, Molecular Cloning, Sequencing, and High-Level Expression in Escherichia coli of a Terpenoid Cyclase from Streptomyces UC5319

Cane

Sohng²,

Lamberson³

et al. 1994

Biochemistry

149

118

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Pentalenene synthase, which catalyzes the cyclization of farnesyl diphosphate (1) to the tricyclic sesquiterpene hydrocarbon pentalenene (2), was purified from Streptomyces UC5319. A 450-bp hybridization probe, generated by PCR amplification of genomic DNA using primers based on N-terminal and internal tryptic peptide sequence data for pentalenene synthase, was used to screen both plasmid and phage DNA libraries of Streptomyces genomic DNA, resulting in the isolation and sequencing of the complete pentalenene synthase gene. PCR was used to insert the pentalenene synthase gene into the T7 expression vector pLM1. Cloning of the resulting construct in the expression host Escherichia coli BL21 (DE3) gave transformants that expressed pentalenene synthase as greater than 10% of soluble protein. The recombinant enzyme has been purified, and initial physical and kinetic characterization has been performed. The recombinant enzyme appears to be identical in every respect with the native Streptomyces synthase and exhibits the following steady-state kinetic parameters: Km = 0.31 +/- 0.05 microM, kcat = 0.32 +/- s-1, KI(PPi) = 3.2 +/- 0.6 microM. Both enzymes have an absolute requirement of Mg2+ for catalysis and an optimum pH of 8.2-8.4. Both proteins have M(r) values of 41-42 kDa, as determined by SDS-PAGE.

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Section: Methodsmentioning

confidence: 99%

Pentalenene Synthase. Purification, Molecular Cloning, Sequencing, and High-Level Expression in Escherichia coli of a Terpenoid Cyclase from Streptomyces UC5319

Cane

Sohng²,

Lamberson³

et al. 1994

Biochemistry

149

118

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“…AdoHcyase has been purified from a number of organisms and tissues (1,(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35)(36)(37). The enzymes from all sources are oligomeric proteins with subunits with M r values of 45000-55000 and isoelectric points of 5.3-6.0.…”

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confidence: 99%

Crystal Structure ofS-Adenosylhomocysteine Hydrolase from Rat Liver^,

et al. 1999

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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.

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“…Frozen male Wistar rat livers were homogenized in 2 volumes (w/v) of 10 mM acetic acid, 1 mM DTT, and 1 mM EDTA and centrifuged 15 min at 28000g, and the supernatant fluid was filtered through glass wool. All subsequent steps were performed as described by Kajander and Raina (1981). Agadenosine type 3 (agarosehexane-adenosine) was used for the affinity chromatography procedure.…”

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confidence: 99%

S-Adenosylhomocysteinase: mechanism of reversible and irreversible inactivation by ATP, cAMP, and 2'-deoxyadenosine

Haba¹,

Agostini²,

Bozzi³

et al. 1986

Biochemistry

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Homogeneous S-adenosylhomocysteinase (AdoHcyase) from rat liver is a tetrameric enzyme that contains four molecules of tightly bound NAD per mole of enzyme. We report here that incubation of the rat liver enzyme with ATP, Mg2+, and KCl leads to conversion of the active enzyme to an inactive form with release of all enzyme-bound NAD which can be recovered quantitatively by gel filtration. At various concentrations of ATP, the release of NAD corresponds closely with the degree of inactivation, suggesting that the four subunits are equivalent. Hydrolysis of ATP is not required for the inactivation process since nonhydrolyzable ATP analogues can replace ATP in the inactivation process. The ATP-dependent inactivation is fully reversible upon incubation of the inactivated enzyme with NAD. The ATP-dependent inactivation of the enzyme appears to be analogues to the cAMP-dependent inactivation of the enzyme from Dictyostelium discoideum described earlier by Hohman et al. (1985) [Hohman, R. J., Guitton, M. C., & Veron, M. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 4578-4581; Hohman, R. J., Veron, M., & Guitton, M. C. (1985) Curr. Top. Cell. Regul. 26, 233-245] but differs from the irreversible inactivation studied earlier by Abeles et al. (1982) [Abeles, R. H., Fish, S., & Lapinskas, B. (1982) Biochemistry 21, 5557-5562]. These authors have ascribed the time-dependent inactivation that results from incubation of the enzyme with 2'-deoxyadenosine at the C-3' and concluded that AdoHcyase "probably consists of two nonequivalent pairs of subunits".(ABSTRACT TRUNCATED AT 250 WORDS)

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Affinity-chromatographic purification of S-adenosyl-l-homocysteine hydrolase. Some properties of the enzyme from rat liver

Cited by 44 publications

References 31 publications

Pentalenene Synthase. Purification, Molecular Cloning, Sequencing, and High-Level Expression in Escherichia coli of a Terpenoid Cyclase from Streptomyces UC5319

Pentalenene Synthase. Purification, Molecular Cloning, Sequencing, and High-Level Expression in Escherichia coli of a Terpenoid Cyclase from Streptomyces UC5319

Crystal Structure ofS-Adenosylhomocysteine Hydrolase from Rat Liver^,

S-Adenosylhomocysteinase: mechanism of reversible and irreversible inactivation by ATP, cAMP, and 2'-deoxyadenosine

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Affinity-chromatographic purification of S-adenosyl-l-homocysteine hydrolase. Some properties of the enzyme from rat liver

Cited by 44 publications

References 31 publications

Pentalenene Synthase. Purification, Molecular Cloning, Sequencing, and High-Level Expression in Escherichia coli of a Terpenoid Cyclase from Streptomyces UC5319

Pentalenene Synthase. Purification, Molecular Cloning, Sequencing, and High-Level Expression in Escherichia coli of a Terpenoid Cyclase from Streptomyces UC5319

Crystal Structure ofS-Adenosylhomocysteine Hydrolase from Rat Liver,

S-Adenosylhomocysteinase: mechanism of reversible and irreversible inactivation by ATP, cAMP, and 2'-deoxyadenosine

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Crystal Structure ofS-Adenosylhomocysteine Hydrolase from Rat Liver^,