Structure-function relationships in the collagenase family member transin

We have developed a system for studying the proteinase activity of a collagenase family member, transin. Cos cells transfected with a vector designed to direct synthesis of a secretable fusion protein between staphylococcal protein A and transin secrete a latent proteinase, activable by 4-aminophenylmercuric acetate, which binds to IgG-Sepharose. Treatment with 4-aminophenylmercuric acetate leads to cleavage of the fusion protein and elution of the active proteinase transin. Based on results obtained with this system we propose that transin comprises an N-terminal proteinase domain and an independent C-terminal hemopexin-like domain. The latter domain is not required for binding of inhibitors or for maintenance of transin in its inactive form. The sequence PRCGVPDV is present in the proenzyme forms of collagenase family proteinases just upstream from the N termini of the active enzymes. We show that mutations within this sequence lead to transin variants with a much increased tendency to undergo spontaneous activation. Finally, we show that mutations within a region of transin having sequence similarity to the zinc-binding site of bacterial metalloproteinases inactivate the proteinase activity of transin, lending support to the notion that this region represents part of transin's active site.     

Purification and properties of extracellular matrix-degrading metallo-proteinase overproduced by Rous sarcoma virus-transformed rat liver cell line, and its identification as transin

Rous sarcoma virus-transformed rat liver cell line RSV-BRL secreted a neutral proteinase in a latent precursor form with a molecular weight (Mr) of 57,000 (57k) as a major secreted protein. This enzyme was a calcium-dependent metallo-proteinase. The proenzyme was purified from the serum-free conditioned medium of the transformed cells by affinity chromatographies on a zinc chelate Sepharose column and a reactive red agarose column. When activated by treatment with trypsin or p-aminophenylmercuric acetate (APMA) in the presence of Ca2+, the purified enzyme effectively hydrolyzed casein, fibronectin, and laminin. Type IV collagen was hydrolyzed at 37 degrees C but not at 30 degrees C by the enzyme, whereas type I and type III collagens were hardly hydrolyzed even at 37 degrees C. The treatment with trypsin or AMPA in the presence of Ca2+ converted this 57k proenzyme to an active and stable enzyme with Mr 42k. In the absence of Ca2+, however, APMA converted the proenzyme to an intermediate form with Mr 45k, while trypsin digested it to an inactive peptide with Mr 30k. These results demonstrate that calcium ion is essential for the activation, activity expression, and stabilization of this metallo-proteinase. Analysis of its partial amino acid sequence and amino acid composition showed that the 57k proenzyme was identical or closely related to the putative protein transin, a rat homologue of stromelysin.     

Identification of catalytically relevant amino acids of the extracellular Serratia marcescens endonuclease by alignment-guided mutagenesis.

By sequence alignment of the extracellular Serratia marcescens nuclease with three related nucleases we have identified seven charged amino acid residues which are conserved in all four sequences. Six of these residues together with four other partially conserved His or Asp residues were changed to alanine by site-directed PCR-mediated mutagenesis using a variant of the nuclease gene in which the coding sequence of the signal peptide was replaced by the coding sequence for an N-terminal affinity tag [Met(His)6GlySer]. Four of the mutant proteins showed almost no reduction in nuclease activity but five displayed a 10- to 1000-fold reduction in activity and one (His110Ala) was inactive. Based upon these results it is suggested that the S.marcescens nuclease employs a mechanism in which His110 acts in concert with a Mg2+ ion and three carboxylates (Asp107, Glu148 and Glu232) as well as one or two basic amino acid residues (Arg108, Arg152).     

Identification of active-site amino acid residues in the Chiba virus 3C-like protease

The 3C-like protease of the Chiba virus, a Norwalk-like virus, is one of the chymotrypsin-like proteases. To identify active-site amino acid residues in this protease, 37 charged amino acid residues and a putative nucleophile, Cys139, within the GDCG sequence were individually replaced with Ala in the 3BC precursor, followed by expression in Escherichia coli, where the active 3C-like protease would cleave 3BC into 3B (VPg) and 3C (protease). Among 38 Ala mutants, 7 mutants (R8A, H30A, K88A, R89A, D138A, C139A, and H157A) completely or nearly completely lost the proteolytic activity. Cys139 was replaceable only with Ser, suggesting that an SH or OH group in the less bulky side chain was required for the side chain of the residue at position 139. His30, Arg89, and Asp138 could not be replaced with any other amino acids. Although Arg8 was also not replaceable for the 3B/3C cleavage and the 3C/3D cleavage, the N-terminal truncated mutant devoid of Arg8 significantly cleaved 3CD into 3C and 3D (polymerase), indicating that Arg8 itself was not directly involved in the proteolytic cleavage. As for position 88, a positively charged residue was required because the Arg mutant showed significant activity. As deduced by the X-ray structure of the hepatitis A virus 3C protease, Arg8, Lys88, and Arg89 are far away from the active site, and the side chain of Asp138 is directed away from the active site. Therefore, these are not catalytic residues. On the other hand, all of the mutants of His157 in the S1 specificity pocket tended to retain very slight activity, suggesting a decreased level of substrate recognition. These results, together with a sequence alignment with the picornavirus 3C proteases, indicate that His30 and Cys139 are active-site residues, forming a catalytic dyad without a carboxylate directly participating in the proteolysis.     

Amino acids conserved in interleukin-1 receptors (IL-1Rs) and the Drosophila toll protein are essential for IL-1R signal transduction.

The cytoplasmic domain of the human T cell-type interleukin-1 receptor (hIL-1R) is not involved in the binding, internalization, or nuclear localization of interleukin-1 (IL-1), but is essential for signal transduction. We have previously localized a 50-amino acid region (residues 477-527) critical for IL-1-mediated activation of the interleukin-2 promoter in T cells. This region displays a striking degree of amino acid conservation in human, murine, and chicken IL-1Rs. Here we report the results of a site-directed mutational analysis of the cytoplasmic domain of the hIL-1R. We have introduced single-amino acid substitutions at positions conserved in all three receptors and at nonconserved positions and identified key amino acids for IL-1R function in signal transduction. Three basic (Arg431, Lys515, and Arg518) and 3 aromatic (Phe513, Trp514, and Tyr519) amino acids that are conserved in human, murine, and chicken IL-1Rs could not be replaced without abolishing IL-1R-mediated signal transduction. A substitution at another conserved position (Pro521) reduces significantly the ability of the IL-1R to transmit the IL-1 signal. Nonconserved residues could be replaced without affecting signal transduction. The cytoplasmic domain of the IL-1R is related to that of the Drosophila Toll protein, with a 26% identity and a 43% similarity in amino acid sequence. The amino acids shown to be essential for IL-1R function are conserved in the Toll protein. Our experimental data indicate that the amino acid sequence similarity between the IL-1R and the Drosophila toll protein reflects a functional homology between the two proteins.     

Inactivation of N-TIMP-1 by N-terminal acetylation when expressed in bacteria

The high-affinity binding of tissue inhibitors of metalloproteinases (TIMPs) to matrix metalloproteinases (MMPs) is essential for regulation of the turnover of the extracellular matrix during development, wound healing, and progression of inflammatory diseases, such as cancer, atherosclerosis, and arthritis. Bacterially expressed N-terminal inhibitory domains of TIMPs (N-TIMPs) have been used extensively for biochemical and biophysical study of interactions with MMPs. Titration of N-TIMP-1 expressed in E. coli indicates, however, that only about 42% of the protein is active as an MMP inhibitor. The separation of inactive from fully active N-TIMP-1 has been achieved both by MMP affinity and by high-resolution cation exchange chromatography at an appropriate pH, based on a slight difference of charge. Purification by cation exchange chromatography with a Mono S column enriches the active portion of N-TIMP-1 to >95%, with K(i) of 1.5 nM for MMP-12. Mass spectra reveal that the inactive form differs from active N-TIMP-1 in being N-terminally acetylated, underscoring the importance of the free alpha-NH(2) of Cys1 for MMP inhibition. N(alpha)-acetylation of the CTCVPP sequence broadens the N-terminal sequence motifs reported to be susceptible to alpha-amino acetylation by E. coli N-acetyl transferases. (c) 2008 Wiley Periodicals, Inc. Biopolymers 89: 960-968, 2008.This article was originally published online as an accepted preprint. The "Published Online" date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com.     

Bioinformatic comparison of structures and homology-models of matrix metalloproteinases

The entire family of human matrix metalloproteinases (MMPs) was investigated using phylogenetic trees and homology modeling. The phylogenetic analysis indicates that individual domains of each MMP have evolved in a correlated manner. Despite their high sequence similarity, the phylogenetic tree of the catalytic domains already allows functional (e.g., linked to regulation and substrate recognition) homologies between different MMPs to be identified. The same pattern of functional homologies is confirmed by the phylogenetic analysis of the mature proteins. Structural models were built for the catalytic domains of the entire MMP family, for twelve hemopexin domains and for twelve mature proteins. The surface properties around the active site cleft of the modeled and experimental structures are quite conserved, whereas the hemopexin domains are more differentiated, possibly indicating a role in determining substrate specificity. The analysis of mature MMPs showed that the area of the interface between the catalytic and hemopexin domains is essentially conserved, with both hydrophobic and hydrophilic amino acids at the interface. The absence of specific conserved interdomain contacts suggests that the interface is tolerant to amino acid replacements, and that there may be a certain degree of plasticity with respect to the reciprocal orientation of the two domains.     

Characteristics of a 95-kDa matrix metalloproteinase produced by mammary carcinoma cells

A Mr 95,000 matrix metalloproteinase (MMP) produced by rat mammary carcinoma cells has been isolated and characterized. The MMP was secreted in a proteolytically inactive form that was free from bound tissue inhibitor of metalloproteinases. The enzyme was highly glycosylated as evident from an apparent drop of Mr from 95,000 to 83,000 after treatment with N-glycanase. Rotary shadowing electron micrographs of purified proenzyme preparations revealed a uniform set of ellipsoidal molecules. Treatment of the proenzyme with 1% SDS resulted in generation of catalytic activity and exposed a cryptic unpaired Cys residue. The latent proenzyme may be activated in at least three additional ways: either spontaneously upon storage, by treatment with organomercurials, or by limited proteolysis by trypsin. Each mode of activation yielded a distinct pattern of cleavage of the enzyme. The activated enzyme cleaved gelatin (denatured type I collagen) and native type IV and V collagen at 30-37 degrees C. Noncollagenous proteins including alpha 1-proteinase inhibitor, casein, and fibrinogen also were cleaved. The rat mammary carcinoma cell line that produces the Mr 95,000 MMP is composed of two distinct (epithelial- and myoepithelial-like) cell types. The enzyme is expressed constitutively by the epithelial cells. This suggests that expression of the Mr 95,000 MMP is regulated differently from that of interstitial collagenase, which is produced by the epithelial cells only in response to specific inductive factor(s) from the myoepithelial-like cells. Monoclonal antibodies raised against the purified latent Mr 95,000 form of the enzyme bind specifically to the Mr 95,000 MMP and have been used to localize the enzyme to the Golgi region and cytoplasmic granules of the epithelial cells.     

Acyl-coenzyme A: isopenicillin N acyltransferase from Penicillium chrysogenum: effect of amino acid substitutions at Ser227, Ser230 and Ser309 on proenzyme cleavage and activity

Abstract Using a high level Escherichia coli expression system for the Penicillium chrysogenum penDE gene, we have produced acyl-coenzyme A: isopenicillin N acyltransferase (AT) enzymes containing amino acid substitutions at three conserved Ser residues. Chosen for study based on amino acid sequence homologies to other proteins, Ser²²⁷, Ser²³⁰ and Ser³⁰⁹ were changed to Cys or Ala to assess amino acid side chain involvement in proenzyme cleavage and AT enzyme mechanism. Substitutions at Ser²³⁰ had no effect on proenzyme cleavage, acyl-coenzyme A: IPN acyltransferase (IAT) or acyl-coenzyme A: 6-aminopenicillanic acid acyltransferase (AAT) activities. While Ser²²⁷→Cys had no effect, Ser²²⁷→Ala produced uncleaved proenzyme lacking both AAT and IAT activities, suggesting that the presence of a nucleophilic side chain at this residue is required for proenzyme cleavage and AT activity. Substitution of Ser³⁰⁹→Cys did not appreciably prevent proenzyme cleavage, IAT or AAT activity. Recombinant AT (recAT) proenzyme containing Ser³⁰⁹→Ala was cleaved; however, IAT and AAT activities were not observed. This separation of proenzyme cleavage from IAT and AAT activities has not been previously observed, and suggests that Ser³⁰⁹ is involved in substrate acylation.     

Unconventional activation mechanisms of MMP-26, a human matrix metalloproteinase with a unique PHCGXXD cysteine-switch motif

ProMMP-26 has the unique Pro-His(81)-Cys-Gly-Xaa-Xaa-Asp cysteine-switch motif that discriminates this protease from all other matrix metalloproteinases (MMPs) known so far. The conserved, free cysteine residue of the conventional PRCXXPD sequence interacts with the zinc ion of the catalytic domain and provides the fourth coordination site for the catalytic zinc, thereby preventing latent proMMPs from becoming active. MMPs become functionally active when proteolytic cleavage releases the prodomain and the PRCXXPD sequence and exposes the zinc atom. Here, we report that the Pro-His(81)-Cys-Gly-Xaa-Xaa-Asp motif is not functional in proMMP-26 and consequently is not involved in the activation mechanisms. Organomercurial treatment failed to activate proMMP-26. The autolytic Lys-Lys-Gln(59) downward arrow Gln(60)-Phe-His cleavage upstream of the Pro-His(81)-Cys-Gly-Xaa-Xaa-Asp motif induced the proteolytic activity of recombinant proMMP-26 whereas any further cleavage inactivated the enzyme. The His(81) --> Arg(81) mutation restored the conventional cysteine-switch sequence in the prodomain but failed to induce the cysteine-switch activation mechanism. These data and computer modeling studies allowed us to hypothesize that the presence of His(81) significantly modified the fold of proMMP-26, abolished the functionality of the cysteine-switch motif, and stimulated an alternative intramolecular activation pathway of the proenzyme.     

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 mutations in the T-protein gene causing typical and atypical nonketotic hyperglycinemia

We have investigated the molecular lesions of T-protein deficiency causing typical or atypical nonketotic hyperglycinemia (NKH) in two unrelated pedigrees. A patient with typical NKH was identified as being homozygous for a missense mutation in the T-protein gene, a G-to-A transition leading to a Gly-to-Asp substitution at amino acid 269 (G269D). Sibling patients of a second family with atypical NKH had two different missense mutations in the T-protein gene (compound heterozygote), a G-to-A transition leading to a Gly-to-Arg substitution at amino acid 47 (G47R) in one allele, and a G-to-A transition leading to an Arg-to-His substitution at amino acid 320 (R320H) in the other allele. Gly 269 is conserved in T-proteins of various species, even in E. coli, whereas Gly 47 and Arg 320 are replaced by Ala and Leu, respectively, in E. coli. The mutation occurring in more conservative amino acid residues thus results in more deleterious damage to the T-protein, and gives the severe clinical phenotype, viz., typical NKH.     

Potential ligands to the [2Fe-2S] Rieske cluster of the cytochrome bc1 complex of Rhodobacter capsulatus probed by site-directed mutagenesis.

The Rieske protein of the ubiquinol-cytochrome c oxidoreductase (bc1 complex or b6f complex) contains a [2Fe-2S] cluster which is thought to be bound to the protein via two nitrogen and two sulfur ligands [Britt, R. D., Sauer, K., Klein, M. P., Knaff, D. B., Kriauciunas, A., Yu, C.-A., Yu, L., & Malkin, R. (1991) Biochemistry 30, 1892-1901; Gurbiel, R. J., Ohnishi, T., Robertson, D. E., Daldal, F., & Hoffman, B. M. (1991) Biochemistry 30, 11579-11584]. All available Rieske amino acid sequences have carboxyl termini featuring two conserved regions containing four cysteine (Cys) and two or three histidine (His) residues. Site-directed mutagenesis was applied to the Rieske protein of the photosynthetic bacterium Rhodobacter capsulatus, and the mutants obtained were studied biochemically in order to identify which of these conserved residues are the ligands of the [2Fe-2S] cluster. It was found that His159 (in the R. capsulatus numbering) is not a ligand and that the presence of the Rieske protein in the intracytoplasmic membrane is greatly decreased by alteration of any of the remaining six His or Cys residues. Among these mutations, only the substitution Cys155 to Ser resulted in the synthesis of Rieske protein (in a small amount) which contained a [2Fe-2S] cluster with altered biophysical properties. This finding suggested that Cys155 is not a ligand to the cluster. A comparison of the conserved regions of the Rieske proteins with bacterial aromatic dioxygenases (which contain a spectrally and electrochemically similar [2Fe-2S] cluster) indicated that Cys133, His135, Cys153, and His156 are conserved in both groups of enzymes, possibly as ligands to their [2Fe-2S] clusters. These findings led to the proposal that Cys138 and Cys155, which are not conserved in bacterial dioxygenases, may form an internal disulfide bond which is important for the structure of the Rieske protein and the conformation of the quinol oxidation (Qo) site of the bc1 complex.     

Proximity between Glu126 and Arg144 in the lactose permease of Escherichia coli.

Evidence has been presented [Venkatesan, P., and Kaback, H. R. (1998) Proc. Natl. Acad. Sci. U.S.A. 95, 9802-9807] that Glu126 (helix IV) and Arg144 (helix V) which are critical for substrate binding in the lactose permease of Escherichia coli are charge paired and therefore in close proximity. To test this conclusion more directly, three different site-directed spectroscopic techniques were applied to permease mutants in which Glu126 and/or Arg144 were replaced with either His or Cys residues. (1) Glu126-->His/Arg144-->His permease containing a biotin acceptor domain was purified by monomeric avidin affinity chromatography, and Mn(II) binding was assessed by electron paramagnetic resonance spectroscopy. The mutant protein binds Mn(II) with a KD of about 40 microM at pH 7.5, while no binding is observed at pH 5.5. In addition, no binding is detected with Glu126-->His or Arg144-->His permease. (2) Permease with Glu126-->Cys/Arg144-->Cys and a biotin acceptor domain was purified, labeled with a thiol-specific nitroxide spin-label, and shown to exhibit spin-spin interactions in the frozen state after reconstitution into proteoliposomes. (3) Glu126-->Cys/Arg144-->Cys permease with a biotin acceptor domain was purified and labeled with a thiol-specific pyrene derivative, and fluorescence spectra were obtained after reconstitution into lipid bilayers. An excimer band is observed with the reconstituted E126C/R144C mutant, but not with either single-Cys mutant or when the single-Cys mutants are mixed prior to reconstitution. The results provide strong support for the conclusion that Glu126 (helix IV) and Arg144 (helix V) are in close physical proximity.     

Characterization of Bacillus caldotenax anthranilate synthase I produced in Escherichia coli and identification of its essential arginine residue by site-directed mutagenesis.

Anthranilate synthase I (ASI) of Bacillus caldotenax, a thermophilic bacterium, was purified from a plasmid-bearing Escherichia coli and characterized. The molecular weight determination under native and denaturing conditions revealed that it was a monomeric enzyme of M(r) = 54,000. The N-terminal amino acid sequence is the same as expected from DNA sequence of trpE except that the N-terminal methionine is lacking. All four cysteines in the molecule could be titrated with 5,5'-dithiobis (2-nitrobenzoic acid) in more than 8 M urea. The purified enzyme retained its full enzymatic activity after being heated at 60 degrees C. Six mutated genes for the ASI with histidine in place of each conserved arginine, Arg321, Arg353, Arg358, Arg416, Arg429, and Arg452, were prepared by site-directed mutagenesis. All the mutated genes except one, the gene encoding an ASI mutant with histidine in place of Arg452 (R452H ASI) complemented an E. coli (trpE). The mutated ASIs were purified and compared with the wild type ASI. No distinctive differences in enzymatic properties were found between the wild type and the enzymatically active mutated ASIs. R452H ASI was enzymatically inactive, though its conformation seemed to be unchanged after the substitution based on CD spectra and the SH titration curve.     

Cloning of three Caenorhabditis elegans genes potentially encoding novel matrix metalloproteinases

Three genes potentially encoding novel matrix metalloproteinases (MMPs) were identified by sequence similarity searching of Caenorhabditis elegans genome database, and cDNAs for these MMPs were cloned. The predicted gene products (MMP-C31,-H19 and -Y19) display a similar domain organization to human MMPs. MMP-H19 and -Y19 are unique in that they have an RXKR motif between the propeptide and catalytic domains that is a furin-like cleavage site, and conserved only in stromelysin-3 and membrane-type MMPs. The amino acid sequence homology with MMP-1/human interstitial collagenase at the catalytic domain is 45%, 34% and 23% for MMP-C31, -H19 and -Y19, respectively. Recombinant proteins of C. elegans MMPs cleaved an MMP peptide substrate with efficiency proportional to their amino acid homology with human MMPs. Digestion of gelatin was observed only with MMP-C31. Enzyme activity of MMP-C31 and -H19 was inhibited by human tissue inhibitor of MMPs (TIMP)-1, TIMP-2 and synthetic MMP inhibitors, BB94 and CT543, indicating that the catalytic sites of these C. elegans MMPs are structurally closely related with those of mammalian MMPs.     

The amino acid sequence of human chorionic gonadotropin. The alpha subunit and beta subunit.

The amino acid sequences of both the alpha and beta subunits of human chorionic gonadotropin have been determined. The amino acid sequence of the alpha subunit is: Ala - Asp - Val - Gln - Asp - Cys - Pro - Glu - Cys-10 - Thr - Leu - Gln - Asp - Pro - Phe - Ser - Gln-20 - Pro - Gly - Ala - Pro - Ile - Leu - Gln - Cys - Met - Gly-30 - Cys - Cys - Phe - Ser - Arg - Ala - Tyr - Pro - Thr - Pro-40 - Leu - Arg - Ser - Lys - Lys - Thr - Met - Leu - Val - Gln-50 - Lys - Asn - Val - Thr - Ser - Glu - Ser - Thr - Cys - Cys-60 - Val - Ala - Lys - Ser - Thr - Asn - Arg - Val - Thr - Val-70 - Met - Gly - Gly - Phe - Lys - Val - Glu - Asn - His - Thr-80 - Ala - Cys - His - Cys - Ser - Thr - Cys - Tyr - Tyr - His-90 - Lys - Ser. Oligosaccharide side chains are attached at residues 52 and 78. In the preparations studied approximately 10 and 30% of the chains lack the initial 2 and 3 NH2-terminal residues, respectively. This sequence is almost identical with that of human luteinizing hormone (Sairam, M. R., Papkoff, H., and Li, C. H. (1972) Biochem. Biophys. Res. Commun. 48, 530-537). The amino acid sequence of the beta subunit is: Ser - Lys - Glu - Pro - Leu - Arg - Pro - Arg - Cys - Arg-10 - Pro - Ile - Asn - Ala - Thr - Leu - Ala - Val - Glu - Lys-20 - Glu - Gly - Cys - Pro - Val - Cys - Ile - Thr - Val - Asn-30 - Thr - Thr - Ile - Cys - Ala - Gly - Tyr - Cys - Pro - Thr-40 - Met - Thr - Arg - Val - Leu - Gln - Gly - Val - Leu - Pro-50 - Ala - Leu - Pro - Gin - Val - Val - Cys - Asn - Tyr - Arg-60 - Asp - Val - Arg - Phe - Glu - Ser - Ile - Arg - Leu - Pro-70 - Gly - Cys - Pro - Arg - Gly - Val - Asn - Pro - Val - Val-80 - Ser - Tyr - Ala - Val - Ala - Leu - Ser - Cys - Gln - Cys-90 - Ala - Leu - Cys - Arg - Arg - Ser - Thr - Thr - Asp - Cys-100 - Gly - Gly - Pro - Lys - Asp - His - Pro - Leu - Thr - Cys-110 - Asp - Asp - Pro - Arg - Phe - Gln - Asp - Ser - Ser - Ser - Ser - Lys - Ala - Pro - Pro - Pro - Ser - Leu - Pro - Ser-130 - Pro - Ser - Arg - Leu - Pro - Gly - Pro - Ser - Asp - Thr-140 - Pro - Ile - Leu - Pro - Gln. Oligosaccharide side chains are found at residues 13, 30, 121, 127, 132, and 138. The proteolytic enzyme, thrombin, which appears to cleave a limited number of arginyl bonds, proved helpful in the determination of the beta sequence.     

Evolutionary links as revealed by the structure of Thermotoga maritima S-adenosylmethionine decarboxylase

S-adenosylmethionine decarboxylase (AdoMetDC) is a critical regulatory enzyme of the polyamine biosynthetic pathway and belongs to a small class of pyruvoyl-dependent amino acid decarboxylases. Structural elucidation of the prokaryotic AdoMetDC is of substantial interest in order to determine the relationship between the eukaryotic and prokaryotic forms of the enzyme. Although both forms utilize pyruvoyl groups, there is no detectable sequence similarity except at the site of pyruvoyl group formation. The x-ray structure of the Thermatoga maritima AdoMetDC proenzyme reveals a dimeric protein fold that is remarkably similar to the eukaryotic AdoMetDC protomer, suggesting an evolutionary link between the two forms of the enzyme. Three key active site residues (Ser55, His68, and Cys83) involved in substrate binding, catalysis or proenzyme processing that were identified in the human and potato AdoMet-DCs are structurally conserved in the T. maritima AdoMetDC despite very limited primary sequence identity. The role of Ser55, His68, and Cys83 in the self-processing reaction was investigated through site-directed mutagenesis. A homology model for the Escherichia coli AdoMetDC was generated based on the structures of the T. maritima and human AdoMetDCs.     

Alkylation of an active-site cysteinyl residue during substrate-dependent inactivation of Escherichia coli S-adenosylmethionine decarboxylase

S-Adenosylmethionine decarboxylase from Escherichia coli is a member of a small class of enzymes that uses a pyruvoyl prosthetic group. The pyruvoyl group is proposed to form a Schiff base with the substrate and then act as an electron sink facilitating decarboxylation. We have previously shown that once every 6000-7000 turnovers the enzyme undergoes an inactivation that results in a transaminated pyruvoyl group and the formation of an acrolein-like species from the methionine moiety. The acrolein then covalently alkylates the enzyme [Anton, D. L., & Kutny, R. (1987) Biochemistry 26, 6444]. After reduction of the alkylated enzyme with NaBH4, a tryptic peptide with the sequence Ala-Asp-Ile-Glu-Val-Ser-Thr-[S-(3-hydroxypropyl)Cys]-Gly-Val-Ile-Ser-Pro - Leu-Lys was isolated. This corresponds to acrolein alkylation of a cysteine residue in the second tryptic peptide from the NH2 terminal of the alpha-subunit [Anton, D. L., & Kutny, R. (1987) J. Biol. Chem. 262, 2817-2822]. The modified residue derived is from Cys-140 of the proenzyme [Tabor, C. W., & Tabor, H. (1987) J. Biol. Chem. 262, 16037-16040] and lies in the only sequence conserved between rat liver and E. coli S-adenosylmethionine decarboxylase [Pajunen et al. (1988) J. Biol. Chem. 263, 17040-17049]. We suggest that the alkylated Cys residue could have a role in the catalytic mechanism.