Pyruvoyl-Dependent Enzymes

The genome sequence of the hyperthermophilic methanogen Methanococcus jannaschii contains homologs of most genes required for spermidine polyamine biosynthesis. Yet genomes from neither this organism nor any other euryarchaeon have orthologs of the pyridoxal 5-phosphate-dependent ornithine or arginine decarboxylase genes, required to produce putrescine. Instead, as shown here, these organisms have a new class of arginine decarboxylase (PvlArgDC) formed by the self-cleavage of a proenzyme into a 5-kDa subunit and a 12-kDa subunit that contains a reactive pyruvoyl group. Although this extremely thermostable enzyme has no significant sequence similarity to previously characterized proteins, conserved active site residues are similar to those of the pyruvoyl-dependent histidine decarboxylase enzyme, and its subunits form a similar (␣␤) 3 complex. Homologs of PvlArgDC are found in several bacterial genomes, including those of Chlamydia spp., which have no agmatine ureohydrolase enzyme to convert agmatine (decarboxylated arginine) into putrescine. In these intracellular pathogens, PvlArgDC may function analogously to pyruvoyl-dependent histidine decarboxylase; the cells are proposed to import arginine and export agmatine, increasing the pH and affecting the host cell's metabolism. Phylogenetic analysis of PvlArgDC proteins suggests that this gene has been recruited from the euryarchaeal polyamine biosynthetic pathway to function as a degradative enzyme in bacteria.

mentioning

confidence: 70%

Methanococcus jannaschii Uses a Pyruvoyl-dependent Arginine Decarboxylase in Polyamine Biosynthesis

Graham

White

2002

“…There is now formal evidence that these reactions exist in several protein maturation pathways, including protein splicing (36), hedgehog protein maturation (32), pyruvoyl enzymes (37), and N-terminal nucleophile hydrolases (29,38) activations. In these cases, the hydroxyl-or thiol-containing residues, serine, threonine, or cysteine, undergo a NϾO or a NϾS acyl-shift resulting in an ester or thioester bond much more reactive than the peptide bond.…”

Section: Discussionmentioning

confidence: 99%

Self-catalyzed Cleavage of the Yeast Nucleoporin Nup145p Precursor

Teixeira

Fabre

Dujon³

1999

Nup145p is a component of the nuclear pore complex of Saccharomyces cerevisiae and is essential for mRNA export. Nup145p and its apparent vertebrate homologue are the only known nucleoporins to be composed of two functionally independent peptide moieties resulting from the post-translational cleavage of a large precursor molecule. In this study, the proteolytic cleavage site of Nup145p has been mapped upstream of an evolutionary conserved serine residue. Cleavage occurs at the same site when a precursor is artificially expressed in Escherichia coli. A hydroxyl-containing residue is critical for the reaction, although a thiol-containing residue offers an acceptable replacement. In vitro kinetics experiments using a purified precursor molecule demonstrate that the cleavage is self-catalyzed and that the catalytic domain lies within the N-terminal moiety. Taken altogether, our data are consistent with a proteolytic mechanism involving an N>O acyl rearrangement and a subsequent ester intermediate uncovered in other selfprocessing proteins.

“…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).…”

mentioning

confidence: 99%

“…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)(8)(9)(10)(11). Three distinct classes of AdoMetDC have been described, with little sequence similarity among them.…”

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confidence: 99%

“…The human and potato enzymes have been characterized by crystallographic and functional studies, which have revealed much about the protein structure and active site, as well as the allosteric nature of putrescine activation (13)(14)(15)(16)(17)(18). A second class of AdoMet decarboxylase with a distinct sequence is typified by the Mg 2ϩ -dependent Escherichia coli enzyme that is composed of 12.4-and 18-kDa subunits in an (␣␤) 4 arrangement (7, 15, 19 -23). Less is known about the structure or mechanism of this protein; in particular, the role of the Mg 2ϩ has not been elucidated.…”

mentioning

confidence: 99%

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

Markham

2004

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...