Abietadiene synthase catalysis: conserved residues involved in protonation-initiated cyclization of geranylgeranyl diphosphate to (+)-copalyl diphosphate

Abietadiene synthase catalyzes two sequential, mechanistically distinct cyclization reactions in the formation of a mixture of abietadiene double bond isomers as the committed step in resin acid biosynthesis. Each reaction is carried out at a separate active site residing in a structurally distinct domain, and the reactions are kinetically separable. The first cyclization reaction is initiated by protonation of the terminal double bond of the universal diterpene precursor, geranylgeranyl diphosphate. The pH dependence of the overall reaction is consistent with an acid-base catalytic mechanism, and a divalent metal ion plays a role in this reaction probably by binding the diphosphate moiety to assist in positioning the substrate for catalysis. A putative active site for the protonation-initiated cyclization was defined by modeling abietadiene synthase and locating the DXDD motif previously shown to be involved in this reaction. A number of charged and aromatic residues, which are highly conserved in mechanistically related diterpene cyclases, line the putative active site. Alanine substitutions were made for each of these residues, as were asparagine and glutamate substitutions for the aspartates of the DXDD motif. Kinetic evaluation confirmed the involvement of most of the targeted residues in the reaction, and analysis of mutational effects on the pH-activity profile and affinity for a transition state analogue suggested specific roles for several of these residues in catalyzing the cyclization of geranylgeranyl diphosphate to (+)-copalyl diphosphate. A functional role was also suggested for the cryptic insertional element found in abietadiene synthase and other diterpene synthases that carry out similar protonation-initiated cyclizations.     

Biosynthesis of the earthy odorant geosmin by a bifunctional Streptomyces coelicolor enzyme

Geosmin is responsible for the characteristic odor of moist soil, as well as off-flavors in drinking water and foodstuffs. Geosmin is generated from farnesyl diphosphate (FPP, 2) by an enzyme that is encoded by the SCO6073 gene in the soil organism Streptomyces coelicolor A32 (ref. 3). We have now shown that the recombinant N-terminal half of this protein catalyzes the Mg2+-dependent cyclization of FPP to germacradienol and germacrene D, while the highly homologous C-terminal domain, previously thought to be catalytically silent, catalyzes the Mg2+-dependent conversion of germacradienol to geosmin. Site-directed mutagenesis confirmed that the N- and C-terminal domains each harbor a distinct, independently functioning active site. A mutation in the N-terminal domain of germacradienol-geosmin synthase of a catalytically essential serine to alanine results in the conversion of FPP to a mixture of sesquiterpenes that includes an aberrant product identified as isolepidozene, which was previously suggested to be an enzyme-bound intermediate in the cyclization of FPP to germacradienol.     

Abietadiene synthase from grand fir (Abies grandis): characterization and mechanism of action of the "pseudomature" recombinant enzyme

The oleoresin secreted by grand fir (Abies grandis) is composed of resin acids derived largely from the abietane family of diterpene olefins as precursors which undergo subsequent oxidation of the C18-methyl group to a carboxyl function, for example, in the conversion of abieta-7,13-diene to abietic acid. A cDNA encoding abietadiene synthase has been isolated from grand fir and the heterologously expressed bifunctional enzyme shown to catalyze both the protonation-initiated cyclization of geranylgeranyl diphosphate to the intermediate (+)-copalyl diphosphate and the ionization-dependent cyclization of (+)-copalyl diphosphate, via a pimarenyl intermediate, to the olefin end products. Abietadiene synthase is translated as a preprotein bearing an N-terminal plastidial targeting sequence, and this form of the recombinant protein expressed in Escherichia coli proved to be unsuitable for detailed structure-function studies. Since the transit peptide-mature protein cleavage site could not be determined directly, a truncation series was constructed to delete the targeting sequence and prepare a "pseudomature" form of the enzyme that resembled the native abietadiene synthase in kinetic properties. Both the native synthase and the pseudomature synthase having 84 residues deleted from the preprotein converted geranylgeranyl diphosphate and the intermediate (+)-copalyl diphosphate to a nearly equal mixture of abietadiene, levopimaradiene, and neoabietadiene, as well as to three minor products, indicating that this single enzyme accounts for production of all of the resin acid precursors of grand fir. Kinetic evaluation of abietadiene synthase with geranylgeranyl diphosphate and (+)-copalyl diphosphate provided evidence for two functionally distinct active sites, the first for the cyclization of geranylgeranyl diphosphate to (+)-copalyl diphosphate and the second for the cyclization of (+)-copalyl diphosphate to diterpene end products, and demonstrated that the rate-limiting step of the coupled reaction sequence resides in the second cyclization process. The structural implications of these findings are discussed in the context of primary sequence elements considered to be responsible for binding the substrate and intermediate and for initiating the respective cyclization steps.     

Genomic organization of plant terpene synthases and molecular evolutionary implications

Terpenoids are the largest, most diverse class of plant natural products and they play numerous functional roles in primary metabolism and in ecological interactions. The first committed step in the formation of the various terpenoid classes is the transformation of the prenyl diphosphate precursors, geranyl diphosphate, farnesyl diphosphate, and geranylgeranyl diphosphate, to the parent structures of each type catalyzed by the respective monoterpene (C(10)), sesquiterpene (C(15)), and diterpene synthases (C(20)). Over 30 cDNAs encoding plant terpenoid synthases involved in primary and secondary metabolism have been cloned and characterized. Here we describe the isolation and analysis of six genomic clones encoding terpene synthases of conifers, [(-)-pinene (C(10)), (-)-limonene (C(10)), (E)-alpha-bisabolene (C(15)), delta-selinene (C(15)), and abietadiene synthase (C(20)) from Abies grandis and taxadiene synthase (C(20)) from Taxus brevifolia], all of which are involved in natural products biosynthesis. Genome organization (intron number, size, placement and phase, and exon size) of these gymnosperm terpene synthases was compared to eight previously characterized angiosperm terpene synthase genes and to six putative terpene synthase genomic sequences from Arabidopsis thaliana. Three distinct classes of terpene synthase genes were discerned, from which assumed patterns of sequential intron loss and the loss of an unusual internal sequence element suggest that the ancestral terpenoid synthase gene resembled a contemporary conifer diterpene synthase gene in containing at least 12 introns and 13 exons of conserved size. A model presented for the evolutionary history of plant terpene synthases suggests that this superfamily of genes responsible for natural products biosynthesis derived from terpene synthase genes involved in primary metabolism by duplication and divergence in structural and functional specialization. This novel molecular evolutionary approach focused on genes of secondary metabolism may have broad implications for the origins of natural products and for plant phylogenetics in general.     

Investigating conservation of the albaflavenone biosynthetic pathway and CYP170 bifunctionality in streptomycetes

Albaflavenone, a tricyclic sesquiterpene antibiotic, is biosynthesized in Streptomyces coelicolor A3(2) by enzymes encoded in a two-gene operon. Initially, sesquiterpene cyclase catalyzes the cyclization of farnesyl diphosphate to the terpenoid epi-isozizaene, which is oxidized to the final albaflavenone by cytochrome P450 (CYP)170A1. Additionally, this CYP is a bifunctional enzyme, being able to also generate farnesene isomers from farnesyl diphosphate, owing to a terpene synthase active site moonlighting on the CYP molecule. To explore the functionality of this operon in other streptomycetes, we have examined culture extracts by GC/MS and established the presence of albaflavenone in five Streptomyces species. Bioinformatics examination of the predicted CYP170 primary amino acid sequences revealed substitutions in the CYP terpene synthase active site. To examine whether the terpene synthase site was catalytically active in another CYP170, we characterized the least related CYP170 orthologue from Streptomyces albus (CYP170B1). Following expression and purification, CYP170B1 showed a normal reduced CO difference spectrum at 450 nm, in contrast to the unusual 440-nm peak observed for S. coelicolor A3(2) CYP170A1. CYP170B1 can catalyze the conversion of epi-isozizaene to albaflavenone, but was unable to catalyze the conversion of farnesyl diphosphate to farnesene. Molecular modeling with our crystal structure of CYP170A1 suggests that the absence of key amino acids for binding the essential terpene synthase cofactor Mg(2+) may be the explanation for the loss of CYP170B1 bifunctionality.     

Three-dimensional structure of the bifunctional enzyme phosphoribosylanthranilate isomerase: indoleglycerolphosphate synthase from Escherichia coli refined at 2.0 A resolution.

The three-dimensional structure of the monomeric bifunctional enzyme N-(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from Escherichia coli has been refined at 2.0 A resolution, using oscillation film data obtained from synchrotron radiation. The model includes the complete protein (452 residues), two phosphate ions and 628 water molecules. The final R-factor is 17.3% for all observed data between 15 and 2 A resolution. The root-mean-square deviations from ideal bond lengths and bond angles are 0.010 A and 3.2 degrees, respectively. The structure of N-(5'-phosphoribosyl)anthranilate isomerase: indole-3-glycerol-phosphate synthase from E. coli comprises two beta/alpha-barrel domains that superimpose with a root-mean-square deviation of 2.03 A for 138 C alpha-pairs. The C-terminal domain (residues 256 to 452) catalyses the PRAI reaction and the N-terminal domain (residues 1 to 255) catalyses the IGPS reaction, two sequential steps in tryptophan biosynthesis. The enzyme has the overall shape of a dumb-bell, resulting in a surface area that is considerably larger than normally observed for monomeric proteins of this size. The active sites of the PRAI and the IGPS domains, both located at the C-terminal side of the central beta-barrel, contain equivalent binding sites for the phosphate moieties of the substrates N-(5'-phosphoribosyl) anthranilate and 1-(o-carboxyphenylamino)-1-deoxyribulose-5-phosphate. These two phosphate binding sites are identical with respect to their positions within the tertiary structure of the beta/alpha-barrel, the conformation of the residues involved in phosphate binding and the hydrogen-bonding network between the phosphate ions and the protein. The active site cavities of both domains contain similar hydrophobic pockets that presumably bind the anthranilic acid moieties of the substrates. These similarities of the tertiary structures and the active sites of the two domains provide evidence that N-(5'-phosphoribosyl)anthranilate isomerase:indole-3-glycerol-phosphate synthase from E. coli results from a gene duplication event of a monomeric beta/alpha-barrel ancestor.     

Deletion mutagenesis of human cystathionine beta-synthase. Impact on activity,oligomeric status,and S-adenosylmethionine regulation

Cystathionine beta-synthase is a tetrameric hemeprotein that catalyzes the pyridoxal 5'-phosphate-dependent condensation of serine and homocysteine to cystathionine. We have used deletion mutagenesis of both the N and C termini to investigate the functional organization of the catalytic and regulatory regions of this enzyme. Western blot analysis of these mutants expressed in Escherichia coli indicated that residues 497-543 are involved in tetramer formation. Deletion of the 70 N-terminal residues resulted in a heme-free protein retaining 20% of wild type activity. Additional deletion of 151 C-terminal residues from this mutant resulted in an inactive enzyme. Expression of this double-deletion mutant as a glutathione S-transferase fusion protein generated catalytically active protein (15% of wild type activity) that was unaffected by subsequent removal of the fusion partner. The function of the N-terminal region appears to be primarily steric in nature and involved in the correct folding of the enzyme. The C-terminal region of human cystathionine beta-synthase contains two hydrophobic motifs designated "CBS domains." Partial deletion of the most C-terminal of these domains decreased activity and caused enzyme aggregation and instability. Removal of both of these domains resulted in stable constitutively activated enzyme. Deletion of as few as 8 C-terminal residues increased enzyme activity and abolished any further activation by S-adenosylmethionine indicating that the autoinhibitory role of the C-terminal region is not exclusively a function of the CBS domains.     

Crystal structure of FabG4 from Mycobacterium tuberculosis reveals the importance of C-terminal residues in ketoreductase activity

Rv0242c, also known as FabG4, is a beta-ketoacyl CoA reductase in Mycobacterium tuberculosis. The crystal structure of C-terminal truncated FabG4 is solved at 2.5? resolution which shows the presence of two distinct domains, domain I and II. Domain I partially resembles "flavodoxin type domain" and the domain II is a typical "ketoacyl CoA reductase (KAR) domain". The enzyme exhibits ketoacyl CoA reductase activity by reducing acetoacyl CoA to 3-hydroxyacyl CoA in presence of NADH. Conserved catalytic triad Ser347, Tyr360, and Lys364 constitute the active site residues of the KAR domain. Presence of the Tyr and the Lys residues in the triad in a particular orientation is imperative for effective catalytic mechanism. The importance of loop I and II and the role of the C-terminal residues of KAR domain are highlighted. Comparative structural analyses clearly demonstrate that loop II is stabilized by hydrophobic interaction with C-terminal residues to sustain the orientation of Tyr360. Loop I interacts with loop II via H-bonding network to restrict the active site residue Lys364 in a catalytically favorable orientation.     

Structural basis for light activation of a chloroplast enzyme: the structure of sorghum NADP-malate dehydrogenase in its oxidized form

Some key chloroplast enzymes are activated by light via a ferredoxin-thioredoxin reduction system which reduces disulfide bridges in the enzymes. We describe for the first time the structural basis for the redox activation of a chloroplast enzyme, the NADP-dependent malate dehydrogenase (MDH) from Sorghum vulgare whose structure has been determined and refined at 2.4 A resolution. In addition to the normal structural components of MDHs, the enzyme exhibits extensions at both the N- and C-termini, each of which contains a regulatory disulfide bridge which must be reduced for activation. The N-terminal disulfide motif is inserted in a cleft between the two subunits of the dimer, thereby locking the domains in each subunit. The C-terminal disulfide keeps the C-terminal residues tight to the enzyme surface and blocks access to the active site. Reduction of the N-terminal disulfide would release the stopper between the domains and give the enzyme the necessary flexibility. Simultaneous reduction of the C-terminal disulfide would free the C-terminal residues from binding to the enzyme and make the active site accessible.     

Split target specificity of ResT: a design for protein delivery,site selectivity and regulation of enzyme activity?

The ResT telomere resolvase is responsible for maintaining the hairpin telomeres that cap the linear chromosome and minichromosomes of Borrelia burgdorferi. This enzyme acts at the tandem telomere junctions present within circular dimers resulting from DNA replication. ResT mediates the transesterification steps of resolution using a constellation of active site residues similar to that found in tyrosine recombinases and type IB topoisomerases. By combining this reaction mechanism with a hairpin binding module in its N-terminal domain, ResT reduces a fused telomere dimer into two hairpin monomers. ResT displays a split DNA binding specificity, with the N- and C-terminal domains targeting distinct regions of the telomere. This bi-specificity in binding is likely to be important in protein delivery, substrate selection and regulation of enzyme activity.     

Crystal Structure and Functional Analysis of Homocitrate Synthase, an Essential Enzyme in Lysine Biosynthesis

Homocitrate synthase (HCS) catalyzes the first and committed step in lysine biosynthesis in many fungi and certain Archaea and is a potential target for antifungal drugs. Here we report the crystal structure of the HCS apoenzyme from Schizosaccharomyces pombe and two distinct structures of the enzyme in complex with the substrate 2-oxoglutarate (2-OG). The structures reveal that HCS forms an intertwined homodimer stabilized by domain-swapping between the N- and C-terminal domains of each monomer. The N-terminal catalytic domain is composed of a TIM barrel fold in which 2-OG binds via hydrogen bonds and coordination to the active site divalent metal ion, whereas the C-terminal domain is composed of mixed α/β topology. In the structures of the HCS apoenzyme and one of the 2-OG binary complexes, a lid motif from the C-terminal domain occludes the entrance to the active site of the neighboring monomer, whereas in the second 2-OG complex the lid is disordered, suggesting that it regulates substrate access to the active site through its apparent flexibility. Mutations of the active site residues involved in 2-OG binding or implicated in acid-base catalysis impair or abolish activity in vitro and in vivo. Together, these results yield new insights into the structure and catalytic mechanism of HCSs and furnish a platform for developing HCS-selective inhibitors.     

Crystal Structure of Albaflavenone Monooxygenase Containing a Moonlighting Terpene Synthase Active Site

Albaflavenone synthase (CYP170A1) is a monooxygenase catalyzing the final two steps in the biosynthesis of this antibiotic in the soil bacterium, Streptomyces coelicolor A3(2). Interestingly, CYP170A1 shows no stereo selection forming equal amounts of two albaflavenol epimers, each of which is oxidized in turn to albaflavenone. To explore the structural basis of the reaction mechanism, we have studied the crystal structures of both ligand-free CYP170A1 (2.6 Å) and complex of endogenous substrate (epi-isozizaene) with CYP170A1 (3.3 Å). The structure of the complex suggests that the proximal epi-isozizaene molecules may bind to the heme iron in two orientations. In addition, much to our surprise, we have found that albaflavenone synthase also has a second, completely distinct catalytic activity corresponding to the synthesis of farnesene isomers from farnesyl diphosphate. Within the cytochrome P450 α-helical domain both the primary sequence and x-ray structure indicate the presence of a novel terpene synthase active site that is moonlighting on the P450 structure. This includes signature sequences for divalent cation binding and an α-helical barrel. This barrel is unusual because it consists of only four helices rather than six found in all other terpene synthases. Mutagenesis establishes that this barrel is essential for the terpene synthase activity of CYP170A1 but not for the monooxygenase activity. This is the first bifunctional P450 discovered to have another active site moonlighting on it and the first time a terpene synthase active site is found moonlighting on another protein.     

Domain structure of pleiotrophin required for transformation

The pleiotrophin (PTN) gene (Ptn) is a potent proto-oncogene that is highly expressed in many primary human tumors and constitutively expressed in cell lines derived from these tumors. The product of the Ptn gene is a secreted 136-amino acid heparin binding cytokine with distinct lysine-rich clusters within both the N- and C-terminal domains. To seek domains of PTN functionally important in neoplastic transformation, we constructed a series of mutants and tested their transforming potential by four independent criteria. Our data establish that a domain within PTN residues 41 to 64 and either but not both the N- or C-terminal domains are required for transformation; deletion of both the N and C termini abolishes the transformation potential of PTN. Furthermore, deletion of two internal 5-amino acid residue repeats enhances the transformation potency of PTN 2-fold. Our data indicate that PTN residues 41-64 contain an essential domain for transformation and suggest the hypothesis that this domain requires an additional interaction of the highly basic clusters of the N or C terminus of PTN with a negatively charged "docking" site to enable the transforming domain itself to engage and initiate PTN signaling through its cognate receptor.     

Functional analysis of the two interacting cyclase domains in ent-kaurene synthase from the fungus Phaeosphaeria sp. L487 and a comparison with cyclases from higher plants

We report here kinetic analysis and identification of the two cyclase domains in a bifunctional diterpene cyclase, Phaeosphaeria ent-kaurene synthase (FCPS/KS). Kinetics of a recombinant FCPS/KS protein indicated that the affinity for copalyl diphosphate is higher than that for geranylgeranyl diphosphate (GGDP). ent-Kaurene production from GGDP by FCPS/KS was enhanced by the addition of a plant ent-kaurene synthase (KS) but not by plant CDP synthase (CPS), suggesting that the rate of ent-kaurene production of FCPS/KS may be limited by the KS activity. Site-directed mutagenesis of aspartate-rich motifs in FCPS/KS indicated that the (318)DVDD motif near the N terminus and the (656)DEFFE motif near the C terminus may be part of the active site for the CPS and KS reactions, respectively. The other aspartate-rich (132)DDVLD motif near the N terminus is thought to be involved in both reactions. Functional analysis of the N- and C-terminal truncated mutants revealed that a N-terminal 59-kDa polypeptide catalyzed the CPS reaction and a C-terminal 66-kDa polypeptide showed KS activity. A 101-kDa polypeptide lacking the first 43 amino acids of the N terminus reduced KS activity severely without CPS activity. These results indicate that there are two separate interacting domains in the 106-kDa polypeptide of FCPS/KS.     

Interdomain compactization in human tyrosyl-tRNA synthetase studied by the hierarchical rotations technique

Aminoacyl-tRNA synthetases are key enzymes of protein biosynthesis which usually possess multidomain structures. Mammalian tyrosyl-tRNA synthetase is composed of two structural modules: N-terminal catalytic core and an EMAPII-like C-terminal domain separated by long flexible linker. The structure of full-length human cytoplasmic tyrosyl-tRNA synthetase is still unknown. The structures of isolated N-terminal and C-terminal domains of the protein are resolved, but their compact packing in a functional enzyme is a subject of debates. In this work we studied putative compactization of the N- and C-terminal modules of human tyrosyl-tRNA synthetase by the coarse-grained hierarchical rotations technique (HIEROT). The large number of distinct types of binding interfaces between N- and C-terminal modules is revealed in the absence of enzyme substrates. The binding propensities of different residues are computed and several binding "hot spots" are observed on the surfaces of N and C modules. These results could be used to govern atomistic molecular dynamics simulations, which will sample preferable binding interfaces effectively.     

Crystal structure of mycothiol synthase (Rv0819) from Mycobacterium tuberculosis shows structural homology to the GNAT family of N-acetyltransferases

Mycothiol is the predominant low-molecular weight thiol produced by actinomycetes, including Mycobacterium tuberculosis. The last reaction in the biosynthetic pathway for mycothiol is catalyzed by mycothiol synthase (MshD), which acetylates the cysteinyl amine of cysteine-glucosamine-inositol (Cys-GlcN-Ins). The crystal structure of MshD was determined in the presence of coenzyme A and acetyl-CoA. MshD consists of two tandem-repeated domains, each exhibiting the Gcn5-related N-acetyltransferase (GNAT) fold. These two domains superimpose with a root-mean-square deviation of 1.7 A over 88 residues, and each was found to bind one molecule of coenzyme, although the binding sites are quite different. The C-terminal domain has a similar active site to many GNAT members in which the acetyl group of the coenzyme is presented to an open active site slot. However, acetyl-CoA bound to the N-terminal domain is buried, and is apparently not positioned to promote acetyl transfer. A modeled substrate complex indicates that Cys-GlcN-Ins would only fill a portion of a negatively charged channel located between the two domains. This is the first structure determined for an enzyme involved in the biosynthesis of mycothiol.     

Mutational analysis of white spruce (Picea glauca) ent-kaurene synthase (PgKS) reveals common and distinct mechanisms of conifer diterpene synthases of general and specialized metabolism

Conifer diterpene synthases (diTPSs) catalyze the multi-step cycloisomerization of geranylgeranyl diphosphate, or copalyl diphosphate, to a variety of diterpenes in general (i.e., primary) and specialized (i.e., secondary) metabolism. Despite their functional diversity, the known conifer diTPSs are structurally closely related, with variations in three conserved domains, α, β and γ. The catalytic specificity of conifer class I and class I/II diTPSs is predominantly determined by the protein environment of the C-terminal class I active site through stabilization of common and unique carbocation intermediates. Using the crystal structure of Taxus brevifolia taxadiene synthase as template, comparative modeling and mutagenesis of the class I diTPS ent-kaurene synthase from Picea glauca (PgKS) was performed to elucidate the catalytic specificity of PgKS relative to spruce diTPSs of specialized metabolism. N-terminal truncations demonstrated a role for the βγ domain in class I enzyme activity for PgKS, facilitating the closure of the class I active site upon substrate binding. Based on position, Arg476 and Asp736 in the C-terminal α domain of PgKS may contribute to this conformational transition and appear critical for catalysis. Consistent with the mechanism of other diTPSs, the subsequent ionization of a copalyl diphosphate substrate and coordination of the diphosphate group is controlled by strictly conserved residues in the DDxxD and NDIQGCKRE motif of PgKS, such as Asn656 and Arg653. Furthermore, Lys478, Trp502, Met588, Ala615 and Ile619 control the enzymatic activity and specificity of PgKS via carbocation stabilization en route to ent-kaurene. These positions show a high level of amino acid variation, consistent with functional plasticity among conifer diTPSs of different functions in general or specialized metabolism.     

The crystal structure of yeast protein disulfide isomerase suggests cooperativity between its active sites

Protein disulfide isomerase plays a key role in catalyzing the folding of secretory proteins. It features two catalytically inactive thioredoxin domains inserted between two catalytically active thioredoxin domains and an acidic C-terminal tail. The crystal structure of yeast PDI reveals that the four thioredoxin domains are arranged in the shape of a twisted "U" with the active sites facing each other across the long sides of the "U." The inside surface of the "U" is enriched in hydrophobic residues, thereby facilitating interactions with misfolded proteins. The domain arrangement, active site location, and surface features strikingly resemble the Escherichia coli DsbC and DsbG protein disulfide isomerases. Biochemical studies demonstrate that all domains of PDI, including the C-terminal tail, are required for full catalytic activity. The structure defines a framework for rationalizing the differences between the two active sites and their respective roles in catalyzing the formation and rearrangement of disulfide bonds.     

Crystal structure of human spermine synthase: implications of substrate binding and catalytic mechanism

The crystal structures of two ternary complexes of human spermine synthase (EC 2.5.1.22), one with 5'-methylthioadenosine and spermidine and the other with 5'-methylthioadenosine and spermine, have been solved. They show that the enzyme is a dimer of two identical subunits. Each monomer has three domains: a C-terminal domain, which contains the active site and is similar in structure to spermidine synthase; a central domain made up of four beta-strands; and an N-terminal domain with remarkable structural similarity to S-adenosylmethionine decarboxylase, the enzyme that forms the aminopropyl donor substrate. Dimerization occurs mainly through interactions between the N-terminal domains. Deletion of the N-terminal domain led to a complete loss of spermine synthase activity, suggesting that dimerization may be required for activity. The structures provide an outline of the active site and a plausible model for catalysis. The active site is similar to those of spermidine synthases but has a larger substrate-binding pocket able to accommodate longer substrates. Two residues (Asp(201) and Asp(276)) that are conserved in aminopropyltransferases appear to play a key part in the catalytic mechanism, and this role was supported by the results of site-directed mutagenesis. The spermine synthase.5'-methylthioadenosine structure provides a plausible explanation for the potent inhibition of the reaction by this product and the stronger inhibition of spermine synthase compared with spermidine synthase. An analysis to trace possible evolutionary origins of spermine synthase is also described.