Catalytic Properties of the Archaeal S-Adenosylmethionine Decarboxylase from Methanococcus jannaschii

Abstract

S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl cofactor-dependent enzyme that participates in polyamine biosynthesis. AdoMetDC from the Archaea Methanococcus jannaschii is a prototype for a recently discovered class that is not homologous to the eucaryotic enzymes or to a distinct group of microbial enzymes. M. jannaschii AdoMetDC has a K m of 95 M and the turnover number (k cat ) of 0.0075 s ؊1 at pH 7.5 and 22°C. The turnover number increased ϳ38-fold at a more physiological temperature of 80°C. AdoMetDC was inactivated by treatment with the imine reductant NaCNBH 3 only in the presence of substrate. Mass spectrometry of the inactivated protein showed modification solely of the pyruvoyl-containing subunit, with a mass increase corresponding to reduction of a Schiff base adduct with decarboxylated AdoMet. The presteady state time course of the AdoMetDC reaction revealed a burst of product formation; thus, a step after CO 2 formation is rate-limiting in turnover. Comparable D 2 O kinetic isotope effects of were seen on the first turnover (1.9) and on k cat /K m (1.6); there was not a significant D 2 O isotope effect on k cat , suggesting that product release is rate-limiting in turnover. The pH dependence of the steady state rate showed participation of acid and basic groups with pK values of 5.3 and 8.2 for k cat and 6.5 and 8.3 for k cat /K m , respectively. The competitive inhibitor methylglyoxal bis(guanylhydrazone) binds at a single site per (␣␤) heterodimer. UV spectroscopic studies show that methylglyoxal bis(guanylhydrazone) binds as the dication with a 23 M dissociation constant. Studies with substrate analogs show a high specificity for AdoMet.The decarboxylation of S-adenosylmethionine (AdoMet) 1 occurs at a metabolic branch point at which AdoMet becomes committed to the synthesis of the ubiquitous polyamines rather than partaking in methylation or its many other roles (1-3). S-Adenosylmethionine decarboxylase (AdoMetDC) is one of a small group of decarboxylases that use a covalently attached pyruvoyl group rather than pyridoxal phosphate to form a Schiff base with the substrate during catalysis (4, 5). The pyruvoyl-containing enzymes are initially synthesized as proenzymes that self-cleave at an internal serine residue to yield two polypeptides, one of which (designated the ␣ subunit) has the serine transmogrified into a pyruvoyl group at the new N terminus, a reaction in which the formation of an ester intermediate is analogous to intein processing (6).A remarkable feature of AdoMetDC is that the enzyme from all studied eucarya, bacteria, and Archaea contains the pyruvoyl cofactor, whereas for other decarboxylases for which pyruvoyl-containing enzymes are known there are often pyridoxaldependent enzymes with the same function (4, 7-11). Three distinct classes of AdoMetDC have been described, with little sequence similarity among them. The enzymes from eucarya are well conserved within this kingdom and are typically composed of ϳ8-kDa (␤) and 32-kDa (␣) polypeptides; in some organisms th...

Evolution and Multifarious Horizontal Transfer of an Alternative Biosynthetic Pathway for the Alternative Polyamine sym-Homospermidine

“…In eukaryotes, the triamine spermidine is synthesized from the precursor diamine putrescine by the addition of an aminopropyl group donated by decarboxylated S-adenosylmethionine. The enzymes involved in eukaryotic spermidine biosynthesis, S-adenosylmethionine decarboxylase and spermidine synthase, are also present in many bacteria and archaea and have been extensively characterized (13)(14)(15)(16)(17)(18)(19). The decarboxylated S-adenosylmethionine pathway is also involved in norspermidine and norspermine biosynthesis in thermophilic Crenarchaeota and Clostridia (20,21) and probably in other bacterial species.…”

Metal Ion Activation of S-Adenosylmethionine Decarboxylase Reflects Cation Charge Density

“…After the samples were shaken for 30 min at room temperature, the 14 CO 2 absorbed on the filters was quantified by scintillation counting (18). Kinetic analyses of AdoMetDC activity with other metal ions also measured the production of 14 CO 2 from [carboxy-14 C]AdoMet; protein concentrations were adjusted so that 14 CO 2 formation was linear with time (28). Determination of the pH variation in V max was conducted in the presence of 10 mM MgCl 2 or 10 µM TbCl 3 , as described previously (28).…”

“…Kinetic analyses of AdoMetDC activity with other metal ions also measured the production of 14 CO 2 from [carboxy-14 C]AdoMet; protein concentrations were adjusted so that 14 CO 2 formation was linear with time (28). Determination of the pH variation in V max was conducted in the presence of 10 mM MgCl 2 or 10 µM TbCl 3 , as described previously (28). Kinetic data were fit to the Michaelis-Menten or Hill equations.…”

Discovery of novel inhibitors of human S-adenosylmethionine decarboxylase based on in silico high-throughput screening and a non-radioactive enzymatic assay

“…This limitation becomes a burden especially when it comes to experimental HTP screening of AdoMetDC inhibitors813141516. Although the high-performance liquid chromatography (HPLC) analysis of the other product, dcAdoMet, is an effective alternative method8121415171833, it is also quite complicated and not suitable for HTP screening. Thus,, the lack of an easy-to-use enzymatic assays has largely hampered the development of novel AdoMetDC inhibitors.…”