Pyruvoyl-dependent Enzyme Research Articles

Formation of the pyruvoyl-dependent proline reductase Prd from Clostridioides difficile requires the maturation enzyme PrdH.

Stickland fermentation, the coupled oxidation and reduction of amino acid pairs, is a major pathway for obtaining energy in the nosocomial bacterium Clostridioides difficile. D-proline is the preferred substrate for the reductive path, making it not only a key component of the general metabolism but also impacting on the expression of the clostridial toxins TcdA and TcdB. D-proline reduction is catalyzed by the proline reductase Prd, which belongs to the pyruvoyl-dependent enzymes. These enzymes are translated as inactive proenzymes and require subsequent processing to install the covalently bound pyruvate. Whereas pyruvoyl formation by intramolecular serinolysis has been studied in unrelated enzymes, details about pyruvoyl generation by cysteinolysis as in Prd are lacking. Here, we show that Prd maturation requires a small dimeric protein that we have named PrdH. PrdH (CD630_32430) is co-encoded with the PrdA and PrdB subunits of Prd and also found in species producing similar reductases. By producing stable variants of PrdA and PrdB, we demonstrate that PrdH-mediated cleavage and pyruvoyl formation in the PrdA subunit requires PrdB, which can be harnessed to produce active recombinant Prd for subsequent analyses. We further created PrdA- and PrdH-mutants to get insight into the interaction of the components and into the processing reaction itself. Finally, we show that deletion of prdH renders C. difficile insensitive to proline concentrations in culture media, suggesting that this processing factor is essential for proline utilization. Due to the link between Stickland fermentation and pathogenesis, we suggest PrdH may be an attractive target for drug development.

An activator for pyruvoyl‐dependent l‐aspartate α‐decarboxylase is conserved in a small group of the γ‐proteobacteria including Escherichia coli

In bacteria, β-alanine is formed via the action of l-aspartate α-decarboxylase (PanD) which is one of the small class of pyruvoyl-dependent enzymes. The pyruvoyl cofactor in these enzymes is formed via the intramolecular rearrangement of a serine residue in the peptide backbone leading to chain cleavage and formation of the covalently-bound cofactor from the serine residue. This reaction was previously thought to be uncatalysed. Here we show that in Escherichia coli, PanD is activated by the putative acetyltransferase YhhK, subsequently termed PanZ. Activation of PanD both in vivo and in vitro is PanZ-dependent. PanZ binds to PanD, and we demonstrate that a PanZ(N45A) site-directed mutant is unable to enhance cleavage of the proenzyme PanD despite retaining affinity for PanD. This suggests that the putative acetyltransferases domain of PanZ may be responsible for activation to enhance the processing of PanD. Although panD is conserved among most bacteria, the panZ gene is conserved only in E. coli-related enterobacterial species including Shigella, Salmonella, Klebsiella and Yersinia. These bacteria are found predominantly in the gut flora where pantothenate is abundant and regulation of PanD by PanZ allows these organisms to closely regulate production of β-alanine and hence pantothenate in response to metabolic demand.

Chemoinformatic Identification of Novel Inhibitors against Mycobacterium tuberculosis L-aspartate α-decarboxylase

L-Aspartate α-decarboxylase (ADC) belongs to a class of pyruvoyl dependent enzymes and catalyzes the conversion of aspartate to β-alanine in the pantothenate pathway, which is critical for the growth of several micro-organisms, including Mycobacterium tuberculosis (Mtb). Its presence only in micro-organisms, fungi and plants and its absence in animals, particularly human, make it a promising drug target. We have followed a chemoinformatics-based approach to identify potential drug-like inhibitors against Mycobacterium tuberculosis L-aspartate α-decarboxylase (MtbADC). The structure-based high throughput virtual screening (HTVS) mode of the Glide program was used to screen 333,761 molecules of the Maybridge, National Cancer Institute (NCI) and Food and Drug Administration (FDA) approved drugs databases. Ligands were rejected if they cross-reacted with S-adenosylmethionine (SAM) decarboxylase, a human pyruvoyl dependent enzyme. The lead molecules were further analyzed for physicochemical and pharmacokinetic parameters, based on Lipinski's rule of five, and ADMET (absorption, distribution, metabolism, excretion and toxicity) properties. This analysis resulted in eight small potential drug-like inhibitors that are in agreement with the binding poses of the crystallographic ADC:fumarate and ADC:isoasparagine complex structures and whose backbone scaffolds seem to be suitable for further experimental studies in therapeutic development against tuberculosis.

3-Fluoroaspartate and Pyruvoyl-Dependant Aspartate Decarboxylase: Exploiting the Unique Characteristics of Fluorine To Probe Reactivity and Binding

Fluorine-containing amino acids have been used with great success as mechanism-based inhibitors of pyridoxal phosphate (PLP)-dependent enzymes, and the influence of fluorine on the conformation of molecules has also been extensively studied and practically exploited. In this study, we sought to use these unique characteristics to probe the reactivity and binding of aspartate decarboxylase (ADC) enzymes, which are members of the small class of pyruvoyl-dependant decarboxylases. Since ADC activity has been shown to be essential to the virulence of Mycobacterium tuberculosis, information gained in this manner could be used for the development of inhibitors that selectively target pyruvoyl-dependent enzymes such as ADC, without affecting PLP-dependent enzymes in the host. For this purpose, we synthesized the L-erythro and L-threo isomers of 3-fluoroaspartate and tested their ability to act as substrates and/or inhibitors of the M. tuberculosis and Escherichia coli ADC enzymes. Trapping and MS-based binding analysis was additionally used to confirm that both isomers enter the enzymes' active sites. Our studies show that both isomers undergo single turnover decarboxylation and fluorine elimination reactions to give enamine products that can be trapped within the active site. Interestingly, the enamine/ADC complex that forms from the L-erythro (but not the L-threo) isomer is sufficiently stable that it can be observed even without any trapping. This finding suggests that the two 3-fluoroaspartates maintain different conformations within the ADC active site, which leads to the enamine products with configurations of different stabilities. Taken together, our results provide new insights for the development of cofactor-specific inhibitors, and confirm the utility of fluorine as a unique tool for probing reactivity and binding profiles within enzymes.

Complexes ofThermotoga maritimaS-adenosylmethionine decarboxylase provide insights into substrate specificity

The polyamines putrescine, spermidine and spermine are ubiquitous aliphatic cations and are essential for cellular growth and differentiation. S-Adenosylmethionine decarboxylase (AdoMetDC) is a critical pyruvoyl-dependent enzyme in the polyamine-biosynthetic pathway. The crystal structures of AdoMetDC from humans and plants and of the AdoMetDC proenzyme from Thermotoga maritima have been obtained previously. Here, the crystal structures of activated T. maritima AdoMetDC (TmAdoMetDC) and of its complexes with S-adenosylmethionine methyl ester and 5'-deoxy-5'-dimethylthioadenosine are reported. The results demonstrate for the first time that TmAdoMetDC autoprocesses without the need for additional factors and that the enzyme contains two complete active sites, both of which use residues from both chains of the homodimer. The complexes provide insights into the substrate specificity and ligand binding of AdoMetDC in prokaryotes. The conservation of the ligand-binding mode and the active-site residues between human and T. maritima AdoMetDC provides insight into the evolution of AdoMetDC.

Agmatine is essential for the cell growth ofThermococcus kodakaraensis

TK0149 (designated as Tk-PdaD) of a hyperthermophilic archaeon, Thermococcus kodakaraensis, was annotated as pyruvoyl-dependent arginine decarboxylase, which catalyzes agmatine formation by the decarboxylation of arginine as the first step of polyamine biosynthesis. In order to investigate its physiological roles, Tk-PdaD was purified as a recombinant form, and its substrate dependency was examined using the candidate compounds arginine, ornithine and lysine. Tk-PdaD, expressed in Escherichia coli, was cleaved into alpha and beta subunits, as other pyruvoyl-dependent enzymes, and the resulting subunits formed an (alphabeta)6 complex. The Tk-PdaD complex catalyzed the decarboxylation of arginine but not that of ornithine and lysine. A gene disruptant lacking Tk-pdaD was constructed, showing that it grew only in the medium in the presence of agmatine but not in the absence of agmatine. The obtained results indicate that Tk-pdaD encodes a pyruvoyl-dependent arginine decarboxylase and that agmatine is essential for the cell growth of T. kodakaraensis.

Mechanisms of Allosteric Regulation of Trypanosoma cruzi S-Adenosylmethionine Decarboxylase

S-Adenosylmethionine decarboxylase (AdoMetDC) is a pyruvoyl-dependent enzyme that catalyzes an essential step in polyamine biosynthesis. The polyamines are required for cell growth, and the biosynthetic enzymes are targets for antiproliferative drugs. The function of AdoMetDC is regulated by the polyamine-precursor putrescine in a species-specific manner. AdoMetDC from the protozoal parasite Trypanosoma cruzi requires putrescine for maximal enzyme activity, but not for processing to generate the pyruvoyl cofactor. The putrescine-binding site is distant from the active site, suggesting a mechanism of allosteric regulation. To probe the structural basis by which putrescine stimulates T. cruzi AdoMetDC we generated mutations in both the putrescine-binding site and the enzyme active site. The catalytic efficiency of the mutant enzymes, and the binding of the diamidine inhibitors, CGP 48664A and CGP 40215, were analyzed. Putrescine stimulates the k(cat)/K(m) for wild-type T. cruzi AdoMetDC by 27-fold, and it stimulates the binding of both inhibitors (IC(50)s decrease 10-20-fold with putrescine). Unexpectedly CGP 48664A activated the T. cruzi enzyme at low concentrations (0.1-10 microM), while at higher concentrations (>100 microM), or in the presence of putrescine, inhibition was observed. Analysis of the mutant data suggests that this inhibitor binds both the putrescine-binding site and the active site, providing evidence that the putrescine-binding site of the T. cruzi enzyme has broad ligand specificity. Mutagenesis of the active site identified residues that are important for putrescine stimulation of activity (F7 and T245), while none of the active site mutations altered the apparent putrescine-binding constant. Mutations of residues in the putrescine-binding site that resulted in reduced (S111R) and enhanced (F285H) catalytic efficiency were both identified. These data provide evidence for coupling between residues in the putrescine-binding site and the active site, consistent with a mechanism of allosteric regulation.

Expression of Bacterial l-aspartate-α-decarboxylase in Tobacco Increases β-Alanine and Pantothenate Levels and Improves Thermotolerance

L- Aspartate-alpha-decarboxylase catalyzes the decarboxylation of L -aspartate to generate Beta-alanine and carbon dioxide. This is an unusual pyruvoyl-dependent enzyme unique to prokaryotes that undergoes limited self-processing. The Escherichia coli pan D gene encoding L- aspartate-alpha-decarboxylase was expressed under a constitutive promoter in transgenic tobacco. Transgene expression was verified by assays based on RNA blots, immunoblots and enzyme activity in vitro. The pan D lines had increased levels of leaf Beta-alanine (1.2- to 4-fold), pantothenate (3.2- to 4.1-fold) and total free amino acids (up to 3.7-fold) compared to wild-type and vector controls. Growth of homozygous lines expressing E. coli L- aspartate-alpha-decarboxylase was less affected than that of the control lines when the plants were stressed for 1 week at 35 degrees C. When transferred from 35 to 30 degrees C for 3 weeks, the Pan D transgenic lines recovered significantly (P <or= 0.001) better than the controls: Pan D lines had on an average 54% and 84% greater fresh and dry weights respectively, compared to the controls. Homozygous lines expressing E. coli L- aspartate-alpha-decarboxylase had significantly greater thermotolerance (P<or=0.05) during germination. At 42 degrees C, 95% of two T3 Pan D transgenic line seeds germinated after 12 days compared to 73% for the wild-type seeds. Our results indicated that E. coli L-aspartate-alpha-decarboxylase was correctly processed and active in the transgenic eukaryotic host and its expression resulted in increased thermotolerance in tobacco.

Tools for metabolic engineering in Escherichia coli: inactivation of panD by a point mutation

l-Aspartate-α-decarboxylase (PanD) catalyzes the decarboxylation of aspartate to produce β-alanine, a precursor of Coenzyme A (CoA). The pyruvoyl-dependent enzyme from Escherichia coli is activated by self-cleavage at serine 25 to generate a 102-residue α subunit with the pyruvoyl group at its N terminus and a 24-residue β subunit with a hydroxy at its C terminus. A mutant form of the panD gene from E. coli in which serine 25 was replaced with an alanine (S25A) was constructed. Assays conducted in vitro and in vivo confirmed that the mutant version was completely inactive and was incapable of undergoing self-cleavage to generate the active form of the enzyme. The S25A panD mutant was used to replace the chromosomal copy of panD in BAP1, a strain of E. coli modified for polyketide production. Comparison of this strain with panD2 mutant strains derived from E. coli SJ16 showed an equivalent dependence on exogenous β-alanine for growth in liquid medium. Unlike the undefined and leaky panD2 mutation, the panD S25A mutation is defined and tight. The panD S25A E. coli strain enables analysis of intracellular acyl-CoA pools in both defined and complex media and is a useful tool in metabolic engineering studies that require the manipulation of acyl-CoA pools for the heterologous production of polyketides.

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.

Pyrroloquinoline quinone (PQQ) from methanol dehydrogenase and tryptophan tryptophylquinone (TTQ) from methylamine dehydrogenase

This chapter focuses on two well-characterized representative members of each class of these enzymes, the Pyrroloquinoline quinine (PQQ)-dependent methanol dehydrogenase (MEDH) and the tryptophan tryptophylquinone (TTQ)-dependent methylamine dehydrogenase (MADH). The chemical reaction mechanisms for catalysis by PQQ- and TTQ dependent enzymes are very similar to those used by other enzymes that possess carbonyl cofactors, such as pyridoxal phosphate-, topaquinone-, and pyruvoyl-dependent enzymes. The studies on these two classes of enzymes are facilitating a great deal of learning about mechanisms of carbonyl catalysis by enzymes, mechanisms of longrange interprotein electron transfer, and mechanisms of protein biosynthesis and posttranslational modification. PQQ and TTQ enzymes have an interesting common mechanistic feature of coupling active-site chemistry to surface-mediated electron transfer reactions. With the recent determinations of high-resolution crystal structures of more enzymes, and the development of systems for expression and site-directed mutagenesis of these enzymes, further insight into these fundamental biochemical processes is anticipated.

Escherichia coli L-aspartate-alpha-decarboxylase: preprotein processing and observation of reaction intermediates by electrospray mass spectrometry.

The Escherichia coli panD gene, encoding l-aspartate-alpha-decarboxylase, was cloned by PCR, and shown to complement a panD mutant defective in beta-alanine biosynthesis. Aspartate decarboxylase is a pyruvoyl-dependent enzyme, and is synthesized initially as an inactive proenzyme (the pi-protein), which is proteolytically cleaved at a specific X-Ser bond to produce a beta-subunit with XOH at its C-terminus and an alpha-subunit with a pyruvoyl group at its N-terminus, derived from the serine. The recombinant enzyme, as purified, is a tetramer, and comprises principally the unprocessed pi-subunit (of 13.8 kDa), with a small proportion of the alpha- and beta-subunits (11 kDa and 2.8 kDa respectively). Incubation of the purified enzyme at elevated temperatures for several hours results in further processing. Using fluorescein thiosemicarbazide, the completely processed enzyme was shown to contain three pyruvoyl groups per tetrameric enzyme. The presence of unchanged serine at the N-terminus of some of the alpha-subunits was confirmed by electrospray mass spectrometry (ESMS) and N-terminal amino acid sequencing. A novel HPLC assay for aspartate decarboxylase was established and used to determine the Km and kcat for l-aspartate as 151+/-16 microM and 0.57 s-1 respectively. ESMS was also used to observe substrate and product adducts trapped on the pyruvoyl group by sodium cyanoborohydride treatment.

10] Phosphatidylserine decarboxylases: Pyruvoyl-dependent enzymes from bacteria to mammals

Phosphatidylserine decarboxylases have been identified both genetically and biochemically in Escherichia coli (E. coli), Saccharomyces, Chinese hamster ovary (CHO) cells, and humans. In yeast phosphatidylserine and possibly in all eukaryotic cells there are two nonhomologous decarboxylases. In all cases these enzymes are integral membrane proteins requiring the use of detergents for solubilization, purification, and assay. The gene encoding each respective decarboxylase has been isolated and sequenced, revealing the sequence of each enzyme. The E. coli enzyme has been overproduced using a plasmid-borne copy of its gene and purified to homogeneity; the enzyme from rat liver mitochondria has been partially purified. The α-amino-acid decarboxylases including mammalian and some bacterial histidine decarboxylases utilize pyridoxal 5'-phosphate as their prosthetic group and in some cases also require pyrroloquinolinequinone. This chapter reviews E. coli phosphatidylserine decarboxylase and the histidine decarboxylase of Lactobacillus 30a. In other well-characterized enzymes of this class, the pyruvoyl residue is covalently bound to the amino terminus of one of the two nonidentical subunits of the E. coli phosphatidylserine decarboxylase. The enzyme is synthesized as a single subunit proenzyme form, which undergoes posttranslational processing in which an internal serine residue becomes the covalently bound prosthetic group of one of the two resulting subunits.

Purification and characterization of an oxygen-sensitive, reversible 3,4-dihydroxybenzoate decarboxylase from Clostridium hydroxybenzoicum

A 3,4-dihydroxybenzoate decarboxylase (EC 4.1.1.63) from Clostridium hydroxybenzoicum JW/Z-1T was purified and partially characterized. The estimated molecular mass of the enzyme was 270 kDa. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave a single band of 57 kDa, suggesting that the enzyme consists of five identical subunits. The temperature and pH optima were 50 degrees C and pH 7.0, respectively. The Arrhenius energy for decarboxylation of 3,4-dihydroxybenzoate was 32.5 kJ . mol(-1) for the temperature range from 22 to 50 degrees C. The Km and kcat for 3,4-dihydroxybenzoate were 0.6 mM and 5.4 x 10(3) min(-1), respectively, at pH 7.0 and 25 degrees C. The enzyme optimally catalyzed the reverse reaction, that is, the carboxylation of catechol to 3,4-dihydroxybenzoate, at pH 7.0. The enzyme did not decarboxylate 2-hydroxybenzoate, 3-hydroxybenzoate, 4-hydroxybenzoate, 2,3-dihydroxybenzoate, 2,4-dihydroxybenzoate, 2,5-dihydroxybenzoate, 2,3,4-trihydroxybenzoate, 3,4,5-trihydroxybenzoate, 3-F-4-hydroxybenzoate, or vanillate. The decarboxylase activity was inhibited by 25 and 20%, respectively, by 2,3,4- and 3,4,5-trihydroxybenzoate. Thiamine PPi and pyridoxal 5'-phosphate did not stimulate and hydroxylamine and sodium borohydride did not inhibit the enzyme activity, indicating that the 3,4-dihydroxybenzoate decarboxylase is not a thiamine PPi-, pyridoxal 5'-phosphate-, or pyruvoyl-dependent enzyme.

CDNAs for S-Adenosyl-L-Methionine Decarboxylase from Catharanthus Roseus, Heterologous Expression, Identification of the Proenzyme-Processing Site, Evidence for the Presence of Both Subunits in the Active Enzyme, and a Conserved Region in the 5' mRNA Leader

S-Adenosyl-L-methionine decarboxylases (AdoMetDC) are pyruvoyl-dependent enzymes producing the aminopropyl group for spermidine biosynthesis, and this reaction is the rate-limiting step in polyamine biosynthesis. We characterized cDNAs from Catharanthus roseus (Madagascar periwinkle) and investigated the enzyme after heterologous expression. The largest cDNA (1842 bp) had an 5' leader of 469 bp and encoded a protein of 357 residues and 30-35% identity with mammalian AdoMetDC. The proenzyme expressed in Escherichia coli was processed into active enzyme, and the processing site was identified by site-directed mutagenesis as the second Ser in the sequence Leu-Ser-Glu-Ser-Ser. The analysis of affinity-purified proteins indicated that the active enzyme contained both subunits. The Km for S-adenosyl-L-methionine was 35-40 microM, and the enzyme activity was not stimulated by putrescine. The 5' leader of the mRNA contained start and stop codons for a polypeptide of 51 amino acids, and this region was conserved in the 5' leaders of other plant AdoMetDC mRNAs. The putative polypeptide had no similarity with the hexapeptide responsible for modulation of AdoMetDC mRNA translation in mammals. The possibility is discussed that plants evolved a different type of translational regulation.

Structural characterization of Escherichia coli phosphatidylserine decarboxylase.

Phosphatidylserine decarboxylase of Escherichia coli is one of a small group of pyruvoyl-dependent enzymes (Satre, M., and Kennedy, E.P. (1978) J. Biol. Chem. 253, 479-483). The DNA sequence of the structural gene (psd) and partial protein sequence studies demonstrate that the enzyme contains two nonidentical subunits, alpha (Mr = 7,332) and beta (Mr = 28,579), which are derived from a single proenzyme. These two subunits are blocked at their respective amino termini. Reduction of the enzyme with NaCNBH3 in the presence of radiolabeled phosphatidylserine resulted in association of the label with the alpha subunit. Similar reduction in the presence of ammonium ions exposed a new amino terminus for the alpha subunit beginning with alanine. Therefore, the pyruvate prosthetic group is in amide linkage to the amino terminus of the alpha subunit. The amino terminus of the beta subunit was determined to be formylmethionine. The carboxyl terminus of the beta subunit was determined to be glycine as predicted by the DNA sequence. Comparison of the DNA sequence and protein sequence information revealed that the decarboxylase is made as a proenzyme (Mr = 35,893), and the predicted amino acid at the position of the pyruvate within the open reading frame of the proenzyme is serine. Therefore, as with other pyruvoyl-dependent decarboxylases, the prosthetic group is derived from serine through a post-translational cleavage of a proenzyme.

Structure determination of histidine decarboxylase from Lactobacillus 30a at 3.0 Å resolution

The crystal structure of histidine decarboxylase from Lactobacillus 30a has been determined by X-ray diffraction methods to a resolution of 3.0 Å. This protein is a pyruvoyl-dependent enzyme that is formed by an unusual self-activation process. The structure was determined from an electron density map calculated using multiple isomorphous replacement phases from two heavy-atom derivatives and included contributions from anomalous scattering measurements. The final mean figure of merit was 0.79, based on 28,805 independent reflections. The molecule has an ( αβ) 6 subunit composition and crystallizes in the space group I4 122 with a = b = 221.7 A ̊ and c = 107.1 A ̊ . There is one ( αβ) 3 half molecule per asymmetric unit. The ( αβ) 6 particle is dumbbell-shaped, with each ( αβ) 3 unit being approximately spherical, with a diameter of about 65 Å. There is a large central cavity approximately 30 Å deep around the molecular 3-fold axis of the ( αβ) 3 unit. The 3-fold related active site pockets are located around the bottom of this cavity and are separated from each other by a distance of approximately 23 Å. The inner portion of each (αβ) unit, which lies near the interface between the two ( αβ) 3 particles, consists mainly of random coil with several small helical and sheet regions. The outer region of each (αβ) unit has an unusual structure consisting of two overlapping, predominantly antiparallel β-pleated sheets, lined on each side by an α-helix. The walls of the central cavity are formed by the 3-fold repeat of two strands from this β-sandwich structure and one of the helices.