The structures of the unique sulfotransferase retinol dehydratase with product and inhibitors provide insight into enzyme mechanism and inhibition

The structure of retinol dehydratase (DHR) from Spodoptera frugiperda, a member of the sulfotransferase superfamily, in complexes with the inactive form of the cofactor PAP 3'-phosphoadenosine 5'-phosphate (PAP) and (1) the product of the reaction with retinol anhydroretinol (AR), (2) the retinoid inhibitor all-trans-4-oxoretinol (OR), and (3) the potent steroid inhibitor androsterone (AND) have been determined and compared to the enzyme complex with PAP and retinol. The structures show that the geometry of the active-site amino acids is largely preserved in the various complexes. However, the beta-ionone rings of the retinoids are oriented differently with respect to side chains that have been shown to be important for the enzymatic reaction. In addition, the DHR:PAP:AND complex reveals a novel mode for steroid binding that contrasts significantly with that for steroid binding in other sulfotransferases. The molecule is displaced and rotated approximately 180 degrees along its length so that there is no acceptor hydroxyl in close proximity to the site of sulfate transfer. This observation explains why steroids are potent inhibitors of retinol dehydratase activity, rather than substrates for sulfonation. Most of the steroid-protein contacts are provided by the alpha-helical cap that distinguishes this member of the superfamily. This observation suggests that in addition to providing a chemical environment that promotes the dehydration of a sulfonated intermediate, the cap may also serve to minimize a promiscuous sulfotransferases activity.     

Crystal structures of calpain-E64 and -leupeptin inhibitor complexes reveal mobile loops gating the active site

The endogenous calpain inhibitor, calpastatin, modulates some patho-physiological aspects of calpain signaling. Excess calpain can escape this inhibition and as well, many calpain isoforms and autolytically generated protease core fragments are not inhibited by calpastatin. There is a need, therefore, to develop specific, cell-permeable calpain inhibitors to block uncontrolled proteolysis and prevent tissue damage during brain and heart ischemia, spinal-cord injury and Alzheimer's diseases. Here, we report the first high-resolution crystal structures of rat mu-calpain protease core complexed with two traditional, low molecular mass inhibitors, leupeptin and E64. These structures show that access to a slightly deeper, but otherwise papain-like active site is gated by two flexible loops. These loops are divergent among the calpain isoforms giving a potential structural basis for substrate/inhibitor selectivity over other papain-like cysteine proteases and between members of the calpain family.     

Crystal structure of creatininase from Pseudomonas putida: a novel fold and a case of convergent evolution

Creatinine amidohydrolase (creatininase; EC 3.5.2.10) from Pseudomonas putida, a homohexameric enzyme with a molecular mass of 28.4 kDa per subunit, is a cyclic amidohydrolase catalysing the reversible conversion of creatinine to creatine. The enzyme plays a key role in the bacterial degradation of creatinine. The three-dimensional structure of creatininase from P.putida was determined and refined to 2.1A. The structure shows the six subunits arranged as a trimer of dimers and definitely disproves previous reports that the enzyme has an octameric quaternary structure. Each monomer consists of a central, four-stranded, parallel beta-sheet flanked by two alpha-helices on both sides of the beta-sheet. This topology is unique within the superfamily of amidohydrolases. Moreover, creatininase possesses a novel fold with no close structural relatives within the Protein Data Bank. Each creatininase monomer contains a binuclear zinc centre near the C termini of the beta-strands and the N termini of the main alpha-helices. These zinc ions indicate the location of the active site unambiguously. The active site is entirely buried and is not accessible from the solution without movement of parts of the protein. The two zinc ions are bridged by a water molecule and by an aspartate residue, which acts as a bidentate ligand. They differ from each other in the number and the spatial arrangement of their ligands. One of them is tetrahedrally and the other trigonal-bipyramidally ligated. Using two water molecules of the first coordination sphere as anchor points, a creatinine-water adduct resembling the transition state of the hydrolysation reaction was modelled into the active site. The resulting complex in combination with structural comparisons with other amidohydrolases enabled us to identify the most probable candidate for the catalytic base and to suggest a putative reaction mechanism. Surprisingly these structural comparisons revealed a similarity in the active-site arrangement between creatininase and the hydantoinase-like cyclic amidohydrolases that was unexpected, given the completely unrelated primary and tertiary structures. In particular, the zinc-bridging aspartate residue of creatininase is a spatially and functionally analogue to a carboxylated lysine residue found in dihydroorotase and the hydantoinases. Hence, creatininase and the hydantoinase-like cyclic amidohydrolases represent a further example of convergent evolution within the enzyme class of hydrolases.     

Crystal structures of Pasteurella multocida sialyltransferase complexes with acceptor and donor analogues reveal substrate binding sites and catalytic mechanism

Sialyltransferases are key enzymes involved in the biosynthesis of biologically and pathologically important sialic acid-containing molecules in nature. Binary X-ray crystal structures of a multifunctional Pasteurella multocida sialyltransferase (Delta24PmST1) with a donor analogue CMP-3F(a)Neu5Ac or CMP-3F(e)Neu5Ac were determined at 2.0 and 1.9 A resolutions, respectively. Ternary X-ray structures of the protein in complex with CMP or a donor analogue CMP-3F(a)Neu5Ac and an acceptor lactose have been determined at 2.0 and 2.27 A resolutions, respectively. This represents the first sialyltransferase structure and the first GT-B-type glycosyltransferase structure that is bound to both a donor analogue and an acceptor simultaneously. The four structures presented here reveal that binding of the nucleotide-activated donor sugar causes a buried tryptophan to flip out of the protein core to interact with the donor sugar and helps define the acceptor sugar binding site. Additionally, key amino acid residues involved in the catalysis have been identified. Structural and kinetic data support a direct displacement mechanism involving an oxocarbenium ion-like transition state assisted with Asp141 serving as a general base to activate the acceptor hydroxyl group.     

Crystal structures of Staphylococcus epidermidis mevalonate diphosphate decarboxylase bound to inhibitory analogs reveal new insight into substrate binding and catalysis

The polyisoprenoid compound undecaprenyl phosphate is required for biosynthesis of cell wall peptidoglycans in Gram-positive bacteria, including pathogenic Enterococcus, Streptococcus, and Staphylococcus spp. In these organisms, the mevalonate pathway is used to produce the precursor isoprenoid, isopentenyl 5-diphosphate. Mevalonate diphosphate decarboxylase (MDD) catalyzes formation of isopentenyl 5-diphosphate in an ATP-dependent irreversible reaction and is therefore an attractive target for inhibitor development that could lead to new antimicrobial agents. To facilitate exploration of this possibility, we report the crystal structure of Staphylococcus epidermidis MDD (1.85 Å resolution) and, to the best of our knowledge, the first structures of liganded MDD. These structures include MDD bound to the mevalonate 5-diphosphate analogs diphosphoglycolyl proline (2.05 Å resolution) and 6-fluoromevalonate diphosphate (FMVAPP; 2.2 Å resolution). Comparison of these structures provides a physical basis for the significant differences in K_i values observed for these inhibitors. Inspection of enzyme/inhibitor structures identified the side chain of invariant Ser¹⁹² as making potential contributions to catalysis. Significantly, Ser → Ala substitution of this side chain decreases k_cat by ∼10³-fold, even though binding interactions between FMVAPP and this mutant are similar to those observed with wild type MDD, as judged by the 2.1 Å cocrystal structure of S192A with FMVAPP. Comparison of microbial MDD structures with those of mammalian counterparts reveals potential targets at the active site periphery that may be exploited to selectively target the microbial enzymes. These studies provide a structural basis for previous observations regarding the MDD mechanism and inform future work toward rational inhibitor design.     

Crystal structures of methionine adenosyltransferase complexed with substrates and products reveal the methionine-ATP recognition and give insights into the catalytic mechanism

Methionine adenosyltransferases (MATs) are a family of enzymes in charge of synthesising S-adenosylmethionine (SAM), the most important methyl donor present in living organisms. These enzymes use methionine and ATP as reaction substrates, which react in a S(N)2 fashion where the sulphur atom from methionine attacks C5' from ATP while triphosphate chain is cleaved. A MAT liver specific isoenzyme has been detected, which exists in two distinct oligomeric forms, a dimer (MAT III) and a tetramer (MAT I). Our previously reported crystal structure of MAT I complexed with an inhibitor led to the identification of the methionine-binding site. We present here the results obtained from the complex of MAT I with a competitive inhibitor of methionine, (2S,4S)-amino-4,5-epoxypentanoic acid (AEP), which presents the same features at the methionine binding site reported before. We have also analysed several complexes of this enzyme with methionine and ATP and analogues of them, in order to characterise the interaction that is produced between both substrates. The crystal structures of the complexes reveal how the substrates recognise each other at the active site of the enzyme, and suggest a putative binding site for the product SAM. The residues involved in the interactions of substrates and products with MAT have been identified, and the results agree with all the previous data concerning mutagenesis experiments and crystallographic work. Moreover, all the information provided from the analysis of the complexes has allowed us to postulate a catalytic mechanism for this family of enzymes. In particular, we propose a key role for Lys182 in the correct positioning of the substrates, and Asp135(*), in stabilising the sulphonium group formed in the product (SAM).     

Expression of the creatininase gene from Pseudomonas putidaRS65 in Escherichia coli

The gene encoding creatininase from Pseudomonas putida RS65 was cloned, sequenced and expressed in Escherichia coli. One plasmid containing a 7.0-kb HindIII insert was selected by its ability to express creatininase activity. After deletion of the adjacent restriction fragments, a 1.1-kb SphI fragment, which contained the full length of the creatininase gene, was subcloned into a pUC18 vector and the nucleotide sequence of the creatininase gene was determined. The gene consists of 771 base pairs and encodes a protein of 257 amino acids. The constitutive creatininase productivity of E. coli DH5α (pCRN741) cultured in broth was about 8.5-fold higher than that of P. putida RS65 cultured in a creatinine-containing medium. The creatininase gene was expressed efficiently in E. coli from its own promoter. Journal of Industrial Microbiology & Biotechnology (2000) 24, 2–6. Received 02 April 1999/ Accepted in revised form 31 July 1999     

Crystal structures of substrate complexes of malic enzyme and insights into the catalytic mechanism

Malic enzymes catalyze the oxidative decarboxylation of L-malate to pyruvate and CO(2) with the reduction of the NAD(P)(+) cofactor in the presence of divalent cations. We report the crystal structures at up to 2.1 A resolution of human mitochondrial NAD(P)(+)-dependent malic enzyme in different pentary complexes with the natural substrate malate or pyruvate, the dinucleotide cofactor NAD(+) or NADH, the divalent cation Mn(2+), and the allosteric activator fumarate. Malate is bound deep in the active site, providing two ligands for the cation, and its C4 carboxylate group is out of plane with the C1-C2-C3 atoms, facilitating decarboxylation. The divalent cation is positioned optimally to catalyze the entire reaction. Lys183 is the general base for the oxidation step, extracting the proton from the C2 hydroxyl of malate. Tyr112-Lys183 functions as the general acid-base pair to catalyze the tautomerization of the enolpyruvate product from decarboxylation to pyruvate.     

Structures of S. aureus thymidylate kinase reveal an atypical active site configuration and an intermediate conformational state upon substrate binding

Methicillin-resistant Staphylococcus aureus (MRSA) poses a major threat to human health, particularly through hospital acquired infection. The spread of MRSA means that novel targets are required to develop potential inhibitors to combat infections caused by such drug-resistant bacteria. Thymidylate kinase (TMK) is attractive as an antibacterial target as it is essential for providing components for DNA synthesis. Here, we report crystal structures of unliganded and thymidylate-bound forms of S. aureus thymidylate kinase (SaTMK). His-tagged and untagged SaTMK crystallize with differing lattice packing and show variations in conformational states for unliganded and thymidylate (TMP) bound forms. In addition to open and closed forms of SaTMK, an intermediate conformation in TMP binding is observed, in which the site is partially closed. Analysis of these structures indicates a sequence of events upon TMP binding, with helix alpha3 shifting position initially, followed by movement of alpha2 to close the substrate site. In addition, we observe significant conformational differences in the TMP-binding site in SaTMK as compared to available TMK structures from other bacterial species, Escherichia coli and Mycobacterium tuberculosis as well as human TMK. In SaTMK, Arg 48 is situated at the base of the TMP-binding site, close to the thymine ring, whereas a cis-proline occupies the equivalent position in other TMKs. The observed TMK structural differences mean that design of compounds highly specific for the S. aureus enzyme looks possible; such inhibitors could minimize the transfer of drug resistance between different bacterial species.     

Crystal structures of Toxoplasma gondii adenosine kinase reveal a novel catalytic mechanism and prodrug binding

Adenosine kinase (AK) is a key purine metabolic enzyme from the opportunistic parasitic protozoan Toxoplasma gondii and belongs to the family of carbohydrate kinases that includes ribokinase. To understand the catalytic mechanism of AK, we determined the structures of the T. gondii apo AK, AK:adenosine complex and the AK:adenosine:AMP-PCP complex to 2.55 A, 2.50 A and 1.71 A resolution, respectively. These structures reveal a novel catalytic mechanism that involves an adenosine-induced domain rotation of 30 degrees and a newly described anion hole (DTXGAGD), requiring a helix-to-coil conformational change that is induced by ATP binding. Nucleotide binding also evokes a coil-to-helix transition that completes the formation of the ATP binding pocket. A conserved dipeptide, Gly68-Gly69, which is located at the bottom of the adenosine-binding site, functions as the switch for domain rotation. The synergistic structural changes that occur upon substrate binding sequester the adenosine and the ATP gamma phosphate from solvent and optimally position the substrates for catalysis. Finally, the 1.84 A resolution structure of an AK:7-iodotubercidin:AMP-PCP complex reveals the basis for the higher affinity binding of this prodrug over adenosine and thus provides a scaffold for the design of new inhibitors and subversive substrates that target the T. gondii AK.     

Crystal structures of Toxoplasma gondii adenosine kinase reveal a novel catalytic mechanism and prodrug binding

Adenosine kinase (AK) is a key purine metabolic enzyme from the opportunistic parasitic protozoan Toxoplasma gondii and belongs to the family of carbohydrate kinases that includes ribokinase. To understand the catalytic mechanism of AK, we determined the structures of the T. gondii apo AK, AK:adenosine complex and the AK:adenosine:AMP-PCP complex to 2.55 A, 2.50 A and 1.71 A resolution, respectively. These structures reveal a novel catalytic mechanism that involves an adenosine-induced domain rotation of 30 degrees and a newly described anion hole (DTXGAGD), requiring a helix-to-coil conformational change that is induced by ATP binding. Nucleotide binding also evokes a coil-to-helix transition that completes the formation of the ATP binding pocket. A conserved dipeptide, Gly68-Gly69, which is located at the bottom of the adenosine-binding site, functions as the switch for domain rotation. The synergistic structural changes that occur upon substrate binding sequester the adenosine and the ATP gi phosphate from solvent and optimally position the substrates for catalysis. Finally, the 1.84 A resolution structure of an AK:7-iodotubercidin:AMP-PCP complex reveals the basis for the higher affinity binding of this prodrug over adenosine and thus provides a scaffold for the design of new inhibitors and subversive substrates that target the T. gondii AK.     

Crystal structure of pullulanase: evidence for parallel binding of oligosaccharides in the active site

The crystal structures of Klebsiella pneumoniae pullulanase and its complex with glucose (G1), maltose (G2), isomaltose (isoG2), maltotriose (G3), or maltotetraose (G4), have been refined at around 1.7-1.9A resolution by using a synchrotron radiation source at SPring-8. The refined models contained 920-1052 amino acid residues, 942-1212 water molecules, four or five calcium ions, and the bound sugar moieties. The enzyme is composed of five domains (N1, N2, N3, A, and C). The N1 domain was clearly visible only in the structure of the complex with G3 or G4. The N1 and N2 domains are characteristic of pullulanase, while the N3, A, and C domains have weak similarity with those of Pseudomonas isoamylase. The N1 domain was found to be a new type of carbohydrate-binding domain with one calcium site (CBM41). One G1 bound at subsite -2, while two G2 bound at -1 approximately -2 and +2 approximately +1, two G3, -1 approximately -3 and +2 approximately 0', and two G4, -1 approximately -4 and +2 approximately -1'. The two bound G3 and G4 molecules in the active cleft are almost parallel and interact with each other. The subsites -1 approximately -4 and +1 approximately +2, including catalytic residues Glu706 and Asp677, are conserved between pullulanase and alpha-amylase, indicating that pullulanase strongly recognizes branched point and branched sugar residues, while subsites 0' and -1', which recognize the non-reducing end of main-chain alpha-1,4 glucan, are specific to pullulanase and isoamylase. The comparison suggested that the conformational difference around the active cleft, together with the domain organization, determines the different substrate specificities between pullulanase and isoamylase.     

Crystal structures of the T4 phage beta-glucosyltransferase and the D100A mutant in complex with UDP-glucose: glucose binding and identification of the catalytic base for a direct displacement mechanism

T4 phage beta-glucosyltransferase (BGT) is an inverting glycosyltransferase (GT) that transfers glucose from uridine diphospho-glucose (UDP-glucose) to an acceptor modified DNA. BGT belongs to the GT-B structural superfamily, represented, so far, by five different inverting or retaining GT families. Here, we report three high-resolution X-ray structures of BGT and a point mutant solved in the presence of UDP-glucose. The two co-crystal structures of the D100A mutant show that, unlike the wild-type enzyme, this mutation prevents glucose hydrolysis. This strongly indicates that Asp100 is the catalytic base. We obtained the wild-type BGT-UDP-glucose complex by soaking substrate-free BGT crystals. Comparison with a previous structure of BGT solved in the presence of the donor product UDP and an acceptor analogue provides the first model of an inverting GT-B enzyme in which both the donor and acceptor substrates are bound to the active site. The structural analyses support the in-line displacement reaction mechanism previously proposed, locate residues involved in donor substrate specificity and identify the catalytic base.     

The first structure of dipeptidyl-peptidase III provides insight into the catalytic mechanism and mode of substrate binding

Dipeptidyl-peptidases III (DPP III) are zinc-dependent enzymes that specifically cleave the first two amino acids from the N terminus of different length peptides. In mammals, DPP III is associated with important physiological functions and is a potential biomarker for certain types of cancer. Here, we present the 1.95-A crystal structure of yeast DPP III representing the prototype for the M49 family of metallopeptidases. It shows a novel fold with two domains forming a wide cleft containing the catalytic metal ion. DPP III exhibits no overall similarity to other metallopeptidases, such as thermolysin and neprilysin, but zinc coordination and catalytically important residues are structurally conserved. Substrate recognition is accomplished by a binding site for the N terminus of the peptide at an appropriate distance from the metal center and by a series of conserved arginine residues anchoring the C termini of different length substrates.     

Crystal structures and kinetic studies of human Kappa class glutathione transferase provide insights into the catalytic mechanism

GSTs (glutathione transferases) are a family of enzymes that primarily catalyse nucleophilic addition of the thiol of GSH (reduced glutathione) to a variety of hydrophobic electrophiles in the cellular detoxification of cytotoxic and genotoxic compounds. GSTks (Kappa class GSTs) are a distinct class because of their unique cellular localization, function and structure. In the present paper we report the crystal structures of hGSTk (human GSTk) in apo-form and in complex with GTX (S-hexylglutathione) and steady-state kinetic studies, revealing insights into the catalytic mechanism of hGSTk and other GSTks. Substrate binding induces a conformational change of the active site from an 'open' conformation in the apo-form to a 'closed' conformation in the GTX-bound complex, facilitating formations of the G site (GSH-binding site) and the H site (hydrophobic substrate-binding site). The conserved Ser(16) at the G site functions as the catalytic residue in the deprotonation of the thiol group and the conserved Asp(69), Ser(200), Asp(201) and Arg(202) form a network of interactions with γ-glutamyl carboxylate to stabilize the thiolate anion. The H site is a large hydrophobic pocket with conformational flexibility to allow the binding of different hydrophobic substrates. The kinetic mechanism of hGSTk conforms to a rapid equilibrium random sequential Bi Bi model.     

Crystal structures of the lytic transglycosylase MltA from N.gonorrhoeae and E.coli: insights into interdomain movements and substrate binding

MltA is a lytic transglycosylase of Gram-negative bacteria that cleaves the beta-1,4 glycosidic linkages between N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc) in peptidoglycan. We have determined the crystal structures of MltA from Neisseria gonorrhoeae and Escherichia coli (NgMltA and EcMltA), which have only 21.5% sequence identity. Both proteins have two main domains separated by a deep groove. Domain 1 shows structural similarity with the so-called double-psi barrel family of proteins. Comparison of the two structures reveals substantial differences in the relative positions of domains 1 and 2 such that the active site groove in NgMltA is much wider and appears more able to accommodate peptidoglycan substrate than EcMltA, suggesting that domain closure occurs after substrate binding. Docking of a peptidoglycan molecule into the structure of NgMltA reveals a number of conserved residues that are likely involved in substrate binding, including a potential binding pocket for the peptidyl moieties. This structure supports the assignment of Asp405 as the acid catalyst responsible for cleavage of the glycosidic bond. In EcMltA, the equivalent residue is Asp328, which has been identified previously. The structures also suggest a catalytic role for Asp393 (Asp317 in EcMltA) in activating the C6 hydroxyl group during formation of the 1,6-anhydro linkage. Finally, in comparison to EcMltA, NgMltA contains a unique third domain that is an insertion within domain 2. The domain is beta in structure and may mediate protein-protein interactions that are specific to peptidoglycan metabolism in N.gonorrhoeae.     

Structural studies of neuropilin/antibody complexes provide insights into semaphorin and VEGF binding

Neuropilins (Nrps) are co-receptors for class 3 semaphorins and vascular endothelial growth factors and important for the development of the nervous system and the vasculature. The extracellular portion of Nrp is composed of two domains that are essential for semaphorin binding (a1a2), two domains necessary for VEGF binding (b1b2), and one domain critical for receptor dimerization (c). We report several crystal structures of Nrp1 and Nrp2 fragments alone and in complex with antibodies that selectively block either semaphorin or vascular endothelial growth factor (VEGF) binding. In these structures, Nrps adopt an unexpected domain arrangement in which the a2, b1, and b2 domains form a tightly packed core that is only loosely connected to the a1 domain. The locations of the antibody epitopes together with in vitro experiments indicate that VEGF and semaphorin do not directly compete for Nrp binding. Based upon our structural and functional data, we propose possible models for ligand binding to neuropilins.     

Crystal structures of a psychrophilic metalloprotease reveal new insights into catalysis by cold-adapted proteases

Enzymes from psychrophilic organisms differ from their mesophilic counterparts in having a lower thermostability and a higher specific activity at low and moderate temperatures. It is in general accepted that psychrophilic enzymes are more flexible to allow easy accommodation and transformation of the substrates at low energy costs. Here, we report the structures of two crystal forms of the alkaline protease from an Antarctic Pseudomonas species (PAP), solved to 2.1- and 1.96-A resolution, respectively. Comparative studies of PAP structures with mesophilic counterparts show that the overall structures are similar but that the conformation of the substrate-free active site in PAP resembles that of the substrate-bound region of the mesophilic homolog, with both an active-site tyrosine and a substrate-binding loop displaying a conformation as in the substrate-bound form of the mesophilic proteases. Further, a region in the catalytic domain of PAP undergoes a conformational change with a loop movement as large as 13 A, induced by the binding of an extra calcium ion. Finally, the active site is more accessible due to deletions occurring in surrounding loop regions.     

Cryo-EM structures of human fucosidase FucA1 reveal insight into substrate recognition and catalysis

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Crystal structure of alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase: insight into the active site and catalytic mechanism of a novel decarboxylation reaction

Alpha-amino-beta-carboxymuconate-epsilon-semialdehyde decarboxylase (ACMSD) is a widespread enzyme found in many bacterial species and all currently sequenced eukaryotic organisms. It occupies a key position at the branching point of two metabolic pathways: the tryptophan to quinolinate pathway and the bacterial 2-nitrobenzoic acid degradation pathway. The activity of ACMSD determines whether the metabolites in both pathways are converted to quinolinic acid for NAD biosynthesis or to acetyl-CoA for the citric acid cycle. Here we report the first high-resolution crystal structure of ACMSD from Pseudomonas fluorescens which validates our previous predictions that this enzyme is a member of the metal-dependent amidohydrolase superfamily of the (beta/alpha)(8) TIM barrel fold. The structure of the enzyme in its native form, determined at 1.65 A resolution, reveals the precise spatial arrangement of the active site metal center and identifies a potential substrate-binding pocket. The identity of the native active site metal was determined to be Zn. Also determined was the structure of the enzyme complexed with cobalt at 2.50 A resolution. The hydrogen bonding network around the metal center suggests that Arg51 and His228 may play important roles in catalysis. The metal center configuration of PfACMSD is very similar to that of Zn-dependent adenosine deaminase and Fe-dependent cytosine deaminase, suggesting that ACMSD may share certain similarities in its catalytic mechanism with these enzymes. These data enable us to propose possible catalytic mechanisms for ACMSD which appear to be unprecedented among all currently characterized decarboxylases.