Site of pyruvate formation and processing of mammalian S-adenosylmethionine decarboxylase proenzyme

Mammalian S-adenosylmethionine decarboxylase was expressed at a high level in an Escherichia coli mutant deficient in this enzyme. The proenzyme form of this enzyme was cleaved and processed to the mature decarboxylase which contains two pairs of nonidentical subunits, the larger of which contains a pyruvate prosthetic group. In order to determine the site of formation of the pyruvate, two approaches were used. First, the mammalian S-adenosylmethionine decarboxylase produced in E. coli was purified to homogeneity and the pyruvate converted to alanine by a reductive amination. The large subunit was then isolated by reversed phase high pressure liquid chromatography and the amino-terminal sequence determined and compared with the sequence of the proenzyme derived from its cDNA. These results indicated that the bond between glutamic acid 67 and serine 68 was the site of cleavage. Second, each of the serine residues in portion of the proenzyme likely to contain the cleavage site were altered by site-directed mutagenesis and the RNA produced from plasmids containing these mutations was translated in a reticulocyte lysate. The translation products were tested for processing and for S-adenosylmethionine decarboxylase activity. Altering the serine residues at positions 50, 66, and 69 to alanines had little effect but changing serine at position 68 to alanine completely prevented both processing and activity. These results indicate that the serine residue at position 68 of the proenzyme which is in the underlined position in the sequence -Leu-Ser-Glu-Ser-Ser-Met- is the residue which is converted to the pyruvate prosthetic group in human S-adenosylmethionine decarboxylase.     

Studies on the mechanism of formation of the pyruvate prosthetic group of phosphatidylserine decarboxylase from Escherichia coli

Phosphatidylserine decarboxylase from Escherichia coli uses a pyruvate group as the enzyme cofactor (Satre, M., and Kennedy, E. P. (1978) J. Biol. Chem. 253, 479-483). Comparison of the DNA sequence of the psd gene with the partial amino acid sequence of the mature gene product suggests that the two nonidentical subunits of the mature enzyme are formed by cleavage of a proenzyme resulting in the conversion of Ser-254 to an amino-terminal pyruvate residue (Li, Q.-X., and Dowhan, W. (1988) J. Biol. Chem. 263, 11516-11522). The cleavage of the wild-type proenzyme occurs rapidly with a half-time on the order of 2 min. When Ser-254 is changed to cysteine (S254C), threonine (S254T), or alanine (S254A) by site-directed mutagenesis, the rate of processing of the proenzyme and the production of the functional enzyme are drastically affected. Proenzymes with S254C or S254T are cleaved with a half-time of around 2-4 h while the S254A proenzyme does not undergo processing. The reduced processing rate for the mutant proenzymes is consistent with less of the functional enzyme being made. Mutants encoding the S254C and S254T protein produce 16 and 2%, respectively, of the activity of the wild-type allele but can still complement a temperature-sensitive mutant in the psd locus. There is no detectable activity or complementation observed with the S254A protein. These results are consistent with the hydroxyl group of Ser-254 playing a critical role in the cleavage of the peptide bond between Gly-253 and Ser-254 of the prophosphatidylserine decarboxylase and support the mechanism proposed by Snell and coworkers (Recsei and Snell (1984) Annul Rev. Biochem. 53, 357-387) for the formation of the prosthetic group of pyruvate-dependent decarboxylases.     

Mechanism of human S-adenosylmethionine decarboxylase proenzyme processing as revealed by the structure of the S68A mutant

S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl-dependent enzyme that catalyzes the formation of the aminopropyl group donor in the biosynthesis of the polyamines spermidine and spermine. The enzyme is synthesized as a protein precursor and is activated by an autocatalytic serinolysis reaction that creates the pyruvoyl group. The autoprocessing reaction proceeds via an N --> O acyl rearrangement, generating first an oxyoxazolidine anion intermediate followed by an ester intermediate. A similar strategy is utilized in self-catalyzed protein splicing reactions and in autoproteolytic activation of protein precursors. Mutation of Ser68 to alanine in human AdoMetDC prevents processing by removing the serine side chain necessary for nucleophilic attack at the adjacent carbonyl carbon atom. We have determined the X-ray structure of the S68A mutant and have constructed models of the proenzyme and the oxyoxazolidine intermediate. Formation of the oxyoxazolidine intermediate is promoted by a hydrogen bond from Cys82 and stabilized by a hydrogen bond from Ser229. These observations are consistent with mutagenesis studies, which show that the C82S and C82A mutants process slowly and that the S229A mutant does not process at all. Donation of a proton by His243 to the nitrogen atom of the oxyoxazolidine ring converts the oxyoxazolidine anion to the ester intermediate. The absence of a base to activate the hydroxyl group of Ser68 suggests that strain may play a role in the cleavage reaction. Comparison of AdoMetDC with other self-processing proteins shows no common structural features. Comparison to histidine decarboxylase and aspartate decarboxylase shows that these pyruvoyl-dependent enzymes evolved different catalytic strategies for forming the same cofactor.     

Histidine decarboxylase of Lactobacillus 30a. Hydroxylamine cleavage of the -seryl-seryl- bond at the activation site of prohistidine decarboxylase

Conversion of the pi subunit of prohistidine decarboxylase to the alpha beta subunits of the active enzyme proceeds by a nonhydrolytic, monovalent cation-dependent, serinolysis reaction in which the hydroxyl oxygen of serine 82 of the pi chain is incorporated into the carboxyl group at the COOH terminus (serine 81) of the beta chain. Serine-82 becomes the pyruvate residue at the NH2 terminus of the alpha chain (Recsei, P.A., Huynh, Q. K., and Snell, E.E. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 973-977). The unusual reactivity of this particular -Ser-Ser- bond is demonstrated by its sensitivity to 1 M hydroxylamine, which cleaves the native proenzyme under mild conditions (pH 8.0, 37 degrees C) to yield a modified beta chain with serine hydroxamate at the COOH terminus (Ser-81) and a modified alpha chain containing serine (Ser-82 of the proenzyme) rather than pyruvate at the NH2 terminus. Neither an -Asn-Gly- bond nor other -Ser-Ser- bonds in the proenzyme were cleaved under these conditions. The reaction also did not occur with the denatured enzyme or with model peptides, indicating that the enhanced reactivity is a result of the particular conformation at this position in the native protein. The reaction with the native proenzyme proceeded optimally at pH 7.5-8.0 with a half-time (30 min) substantially less than that (3.5-4.5 h) required for the activation reaction and was not increased in rate by addition of K+. Correspondingly, preincubation of the proenzyme at pH 8.0 in the absence of both hydroxylamine and K+ modestly increased the rate of activation when K+ was subsequently added. Although these findings do not exclude other mechanisms, they are all consistent with and most easily explained by rearrangement of the pi chain to form an internal ester intermediate prior to the beta-elimination that occurs during activation to yield the alpha and beta chains of the mature enzyme.     

Detection of proenzyme form of S-adenosylmethionine decarboxylase in extracts from rat prostate

Previous work in which the synthesis of S-adenosylmethionine decarboxylase was studied by translation of its mRNA indicated that it was formed as a proenzyme having a M.W. of about 37,000 that was cleaved to form the enzyme sub-unit of M.W. 32,000 in a putrescine-stimulated reaction. The extent to which the proenzyme accumulates in vivo and is affected by the putrescine concentration was studied by subjecting prostate extracts to Western immunoblotting procedures. The proenzyme form was readily detectable in control prostates (about 4% of the total) and this proportion was increased to 25% when the rats were pretreated for 3 days with the ornithine decarboxylase inhibitor, alpha-difluoromethylornithine. Conversely, it was decreased to almost undetectable levels after treatment with methylglyoxal bis(guanylhydrazone). These results indicate that the processing of the proenzyme form of S-adenosylmethionine decarboxylase is regulated by the cellular putrescine concentration. This conversion provides another step at which polyamine biosynthesis may be controlled.     

The structural basis for substrate specificity and inhibition of human S-adenosylmethionine decarboxylase

S-Adenosylmethionine decarboxylase belongs to a small class of amino acid decarboxylases that use a covalently bound pyruvate as a prosthetic group. It is an essential enzyme for polyamine biosynthesis and provides an important target for the design of anti-parasitic and cancer chemotherapeutic agents. We have determined the structures of S-adenosylmethionine decarboxylase complexed with the competitive inhibitors methylglyoxal bis(guanylhydrazone) and 4-amidinoindan-1-one-2'-amidinohydrazone as well as the irreversible inhibitors 5'-deoxy-5'-[N-methyl-N-[(2-aminooxy)ethyl]amino]adenosine, 5'-deoxy-5'-[N-methyl-N-(3-hydrazinopropyl)amino]adenosine, and the methyl ester analogue of S-adenosylmethionine. These structures elucidate residues important for substrate binding and show how those residues interact with both covalently and noncovalently bound inhibitors. S-Adenosylmethionine decarboxylase has a four-layer alphabeta betaalpha sandwich fold with residues from both beta-sheets contributing to substrate and inhibitor binding. The side chains of conserved residues Phe7, Phe223, and Glu247 and the backbone carbonyl of Leu65 play important roles in binding and positioning the ligands. The catalytically important residues Cys82, Ser229, and His243 are positioned near the methionyl group of the substrate. One molecule of putrescine per monomer is observed between the two beta-sheets but far away from the active site. The activating effects of putrescine may be due to conformational changes in the enzyme, to electrostatic effects, or both. The adenosyl moiety of the bound ligand is observed in the unusual syn conformation. The five structures reported here provide a framework for interpretation of S-adenosylmethionine decarboxylase inhibition data and suggest strategies for the development of more potent and more specific inhibitors of S-adenosylmethionine decarboxylase.     

The speEspeD operon of Escherichia coli. Formation and processing of a proenzyme form of S-adenosylmethionine decarboxylase

We have previously shown that the gene (speD) for S-adenosylmethionine decarboxylase is part of an operon that also contains the gene (speE) for spermidine synthase (Tabor, C. W., Tabor, H., and Xie, Q.-W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 6040-6044). We have now determined the nucleotide sequence of this operon and have found that speD codes for a polypeptide of Mr = 30,400, which is considerably greater than the subunit size of the purified enzyme. Our studies show that S-adenosylmethionine decarboxylase is first formed as a Mr = 30,400 polypeptide and that this proenzyme is then cleaved at the Lys111-Ser112 peptide bond to form a Mr = 12,400 subunit and a Mr = 18,000 subunit. The latter subunit contains the pyruvoyl moiety that we previously showed is required for enzymatic activity. Both subunits are present in the purified enzyme. These conclusions are based on (i) pulse-chase experiments with a strain containing a speD+ plasmid which showed a precursor-product relationship between the proenzyme and the enzyme subunits, (ii) the amino acid sequence of the proenzyme form of S-adenosylmethionine decarboxylase (derived from the nucleotide sequence of the speD gene), and (iii) comparison of this sequence of the proenzyme with the N-terminal amino acid sequences of the two subunits of the purified enzyme reported by Anton and Kutny (Anton, D. L., and Kutny, R. (1987) J. Biol. Chem. 262, 2817-2822).     

Pyruvoyl-dependent arginine decarboxylase from Methanococcus jannaschii: crystal structures of the self-cleaved and S53A proenzyme forms

The three-dimensional structure of pyruvoyl-dependent arginine decarboxylase from Methanococcus jannaschii was determined at 1.4 A resolution. The pyruvoyl group of arginine decarboxylase is generated by an autocatalytic internal serinolysis reaction at Ser53 in the proenzyme resulting in two polypeptide chains. The structure of the nonprocessing S53A mutant was also determined. The active site of the processed enzyme unexpectedly contained the reaction product agmatine. The crystal structure confirms that arginine decarboxylase is a homotrimer. The protomer fold is a four-layer alphabetabetaalpha sandwich with topology similar to pyruvoyl-dependent histidine decarboxylase. Highly conserved residues Asn47, Ser52, Ser53, Ile54, and Glu109 are proposed to play roles in the self-processing reaction. Agmatine binding residues include the C terminus of the beta chain (Ser52) from one protomer and the Asp35 side chain and the Gly44 and Val46 carbonyl oxygen atoms from an adjacent protomer. Glu109 is proposed to play a catalytic role in the decarboxylation reaction.     

Structural basis for the inactivation of AdoMetDC K12R mutant

S-adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in the biosynthesis of the polyamines spermidine and spermine. Polyamines are ubiquitous organic cations that are absolutely required for normal cell proliferation and differentiation. AdoMetDC catalyzes decarboxylation of S-adenosylmethionine (AdoMet) which provides aminopropyl groups for spermidine and spermine synthesis. Mammalian AdoMetDC is produced as a proenzyme (38 kDa) which is cleaved to form the alpha (30.7 kDa) and beta (7.7 kDa) subunits of the mature enzyme. It is here shown that the catalytic activity of the enzyme was completely eliminated when lysine 12 was mutated to an arginine residue in the small subunit; however, the proenzyme processing was not affected. On the other hand, mutations of other lysine residues (Lys45-->Arg and Lys56-->Arg) did not affect either the enzyme activity or the proenzyme processing. Structure analysis using Swiss Deep Viewer v3.7 has indicated that Arg in place of Lys12 may eliminate AdoMetDC activity by restricting the mobility of Thr85 through hydrogen bonding. Sequence alignment of various AdoMetDC sequences indicated that Thr85 is in a highly conserved region, suggesting that Thr85 is critical for the decarboxylation reaction.     

Spermidine biosynthesis in Saccharomyces cerevisiae. Biosynthesis and processing of a proenzyme form of S-adenosylmethionine decarboxylase

We have cloned and sequenced the Saccharomyces cerevisiae gene for S-adenosylmethionine decarboxylase. This enzyme contains covalently bound pyruvate which is essential for enzymatic activity. We have shown that this enzyme is synthesized as a Mr 46,000 proenzyme which is then cleaved post-translationally to form two polypeptide chains: a beta subunit (Mr 10,000) from the amino-terminal portion and an alpha subunit (Mr 36,000) from the carboxyl-terminal portion. The protein was overexpressed in Escherichia coli and purified to homogeneity. The purified enzyme contains both the alpha and beta subunits. About half of the alpha subunits have pyruvate blocking the amino-terminal end; the remaining alpha subunits have alanine in this position. From a comparison of the amino acid sequence deduced from the nucleotide sequence with the amino acid sequence of the amino-terminal portion of each subunit (determined by Edman degradation), we have identified the cleavage site of the proenzyme as the peptide bond between glutamic acid 87 and serine 88. The pyruvate moiety, which is essential for activity, is generated from serine 88 during the cleavage. The amino acid sequence of the yeast enzyme has essentially no homology with S-adenosylmethionine decarboxylase of E. coli (Tabor, C. W., and Tabor, H. (1987) J. Biol. Chem. 262, 16037-16040) and only a moderate degree of homology with the human and rat enzymes (Pajunen, A., Crozat, A., J?nne, O. A., Ihalainen, R., Laitinen, P. H., Stanley, B., Madhubala, R., and Pegg, A. E. (1988) J. Biol. Chem. 263, 17040-17049); all of these enzymes are pyruvoyl-containing proteins. Despite this limited overall homology the cleavage site of the yeast proenzyme is identical to the cleavage sites in the human and rat proenzymes, and seven of the eight amino acids adjacent to the cleavage site are identical in the three eukaryote enzymes.     

Identification of the active site residues of Pseudomonas aeruginosa protease IV. Importance of enzyme activity in autoprocessing and activation

Protease IV is a lysine-specific endoprotease produced by Pseudomonas aeruginosa whose activity has been correlated with corneal virulence. Comparison of the protease IV amino acid sequence to other bacterial proteases suggested that amino acids His-72, Asp-122, and Ser-198 could form a catalytic triad that is critical for protease IV activity. To test this possibility, site-directed mutations by alanine substitution were introduced into six selected residues including the predicted triad and identical residues located close to the triad. Mutations at any of the amino acids of the predicted catalytic triad or Ser-197 caused a loss of enzymatic activity and absence of the mature form of protease IV. In contrast, mutations at His-116 or Ser-200 resulted in normal processing into the enzymatically active mature form. A purified proenzyme that accumulated in the His-72 mutant was shown in vitro to be susceptible to cleavage by protease IV purified from P. aeruginosa. Furthermore, similarities of protease IV to the lysine-specific endoprotease of Achromobacter lyticus suggested three possible disulfide bonds in protease IV. These results identify the catalytic triad of protease IV, demonstrate that autodigestion is essential for the processing of protease IV into a mature protease, and predict sites essential to enzyme conformation.     

Site-directed alteration of serine 82 causes nonproductive chain cleavage in prohistidine decarboxylase

Prohistidine decarboxylase from Lactobacillus 30a normally autoactivates by cleavage of the Ser-81-Ser-82 peptide bond, converting Ser-82 to a pyruvoyl moiety which serves as the enzymatic cofactor. We have used site-directed methods to make two conservative mutations, converting Ser-82 to cysteine (S82C) and threonine (S82T). Both mutant proteins autoactivate, although dramatically (20- to 80-fold), more slowly than wild type. Roughly 55% of the mutant protein in each case undergoes a nonproductive chain cleavage which does not result in cofactor production. This finding suggests that an important feature in the enzyme's evolution has been development of an activation scheme which minimizes nonproductive side reactions. Catalytic constants are also affected by the mutations, particularly kcat which drops 8-fold in S82C and 450-fold in S82T. In addition, the S82T protein activates to produce a novel alpha-ketobutyroyl cofactor.     

Structure of a human S-adenosylmethionine decarboxylase self-processing ester intermediate and mechanism of putrescine stimulation of processing as revealed by the H243A mutant

S-Adenosylmethionine decarboxylase (AdoMetDC) is synthesized as a proenzyme that cleaves itself in a putrescine-stimulated reaction via an N-->O acyl shift and beta-elimination to produce an active enzyme with a catalytically essential pyruvoyl residue at the new N-terminus. N-->O acyl shifts initiate the self-processing of other proteins such as inteins and amidohydrolases, but their mechanisms in such proteins are not well understood. We have solved the crystal structure of the H243A mutant of AdoMetDC to 1.5 A resolution. The mutant protein is trapped in the ester form, providing clear evidence for the structure of the ester intermediate in the processing of pyruvoyl enzymes. In addition, a putrescine molecule is bound in a charged region within the beta-sandwich, and cross-links the two beta-sheets through hydrogen bonds to several acidic residues and ordered water molecules. The high-resolution structure provides insight into the mechanism for the self-processing reaction and provides evidence for the mechanism for simulation of the self-processing reaction by putrescine. Studies of the effects of putrescine or 4-aminobutanol on the processing of mutant AdoMetDC proenzymes are consistent with a model in which a single activator molecule interacts with buried Asp174, Glu178, and Glu256, leading to an alteration in the position of Glu11, resulting in stimulation of self-processing.     

In vitro processing of the proproteins GrdE of protein B of glycine reductase and PrdA of D-proline reductase from Clostridium sticklandii: formation of a pyruvoyl group from a cysteine residue.

GrdE and PrdA of Clostridium sticklandii are subunits of glycine reductase and D-proline reductase, respectively, that are processed post-translationally to form a catalytic active pyruvoyl group. The cleavage occurred on the N-terminal side of a cysteine residue, which is thus the precursor of a pyruvoyl moiety. Both proproteins could be over-expressed in Escherichia coli and conditions were developed for in vitro processing. GrdE could be expressed as full-size protein, whereas PrdA had to be truncated N-terminally to achieve successful over-expression. Both proproteins were cleaved at the in vivo observed cleavage site after addition of 200 mM NaBH4 in Tris buffer (pH 7.6) at room temperature as analysed by SDS/PAGE and MS. Cleavage of GrdE was observed with a half-time of approximately 30 min. Cys242, as the precursor of the pyruvoyl group in GrdE, was changed to alanine, serine, or threonine by site-directed mutagenesis. The Cys242-->Ser and Cys242-->Thr mutant proteins were also cleaved under similar conditions with extended half-times. However, the Cys242-->Ala mutant protein was not cleaved indicating a pivotal role of the thiol group of cysteine or hydroxyl group of serine and threonine during the processing of pyruvoyl group-dependent reductases.     

Improved purification protocol for wild-type and mutant human foamy virus proteases

Wild-type and an active site mutant (S25T) human foamy virus (HFV) proteases were expressed in fusion with maltose binding protein in Escherichia coli. The mutant enzyme contained a Ser to Thr mutation in the -Asp-Ser-Gly- active site triplet of the enzyme, which forms the "fireman's grip" between the two subunits of the homodimeric enzyme. The fusion proteins were purified by affinity chromatography on amylose resin, cleaved with factor Xa, and the processed enzymes were purified by gel filtration under denaturing condition. Refolding after purification resulted in active enzymes with comparable yields. Furthermore, both enzymes showed similar catalytic activities in an oligopeptide substrate representing an HFV Gag cleavage site. However, the S25T mutant showed increased stability in urea unfolding experiment, in a good agreement with the suggested role of the Thr residue of fireman's grip.     

Pyridoxal 5'-phosphate dependent histidine decarboxylase: overproduction, purification, biosynthesis of soluble site-directed mutant proteins, and replacement of conserved residues

The hdc gene coding for the pyridoxal 5'-phosphate dependent histidine decarboxylase from Morganella morganii has been expressed in Escherichia coli under control of the lac promoter. The enzyme accumulates to 7-8% of total cell protein and is purified to homogeneity by passage through three columns. Fourteen site-directed mutant enzymes were constructed to explore the roles of residues of interest, especially those in the sequence Ser229-X230-His231-N epsilon-(phosphopyridoxylidene)Lys232, since identical sequences also appear in several other decarboxylases. Most of the overproduced mutant proteins were aggregated into inclusion bodies, but when the late log phase cultures were cooled from 37 to 25 degrees C before induction, the mutant proteins were obtained as soluble products. Ala or Cys in place of Ser-229 yielded mutant enzymes about 7% as active as wild-type, indicating that this serine residue is not essential for catalysis but contributes to activity through conformational or other effects. Of the replacements made for His-231 (Asn, Gln, Phe, and Arg), only Gln and Asn gave partially active enzymes (about 12% and 0.2% of wild-type, respectively). The side-chain amide of Gln may act by mimicking the positionally equivalent tau-nitrogen on the imidazole ring of histidine to provide an interaction (e.g., a hydrogen bond) required for efficient catalysis. The Lys-232 residue that interacts with pyridoxal 5'-phosphate appears central to catalytic efficiency since replacing it with Ala yields a mutant protein that is virtually inactive but retains the ability to bind both pyridoxal 5'-phosphate and histidine efficiently.(ABSTRACT TRUNCATED AT 250 WORDS)     

Processing and assembly in vitro of engineered soybean beta-conglycinin subunits with the asparagine-glycine proteolytic cleavage site of 11S globulins

A short interdomain sequence between the N- and C-terminal domains of beta-conglycinin, the major 7S seed storage protein of soybean, was selected as a target for insertion of amino acid residues specifically cleaved by an asparaginyl endopeptidase that processes globulins into acidic and basic chains. Modified beta-conglycinin subunits containing the proteolytic cleavage site self-assembled into trimers in vitro at an efficiency similar to that of the unmodified subunit. In contrast to the absence of cleavage of the unmodified subunits, however, the modified beta-conglycinin trimers were processed by purified soybean asparaginyl endopeptidase into two polypeptides, each the size expected for the beta-conglycinin N- and C-terminal domains, respectively. The cleavage did not alter the assembly of mutant beta-conglycinins and the cleaved mutant trimers remained stable to further proteolytic attack. To examine the possibility of coassembly between the cleaved 11S and 7S subunits, in vitro processed mutant beta-conglycinin subunits were mixed with native dissociated 11S globulin preparations. Reassembly at a high ionic condition did not induce the 7S subunits to interact with 11S subunits to form hexameric complexes. Thus, cleavage of 7S globulin subunits into acidic and basic domains may not be sufficient for hexamer assembly to occur. Biotechnological implications of the engineered proteins are discussed.