Structure and Mechanism of Ferulic Acid Decarboxylase (FDC1) from Saccharomyces cerevisiae

The nonoxidative decarboxylation of aromatic acids occurs in a range of microbes and is of interest for bioprocessing and metabolic engineering. Although phenolic acid decarboxylases provide useful tools for bioindustrial applications, the molecular bases for how these enzymes function are only beginning to be examined. Here we present the 2.35-Å-resolution X-ray crystal structure of the ferulic acid decarboxylase (FDC1; UbiD) from Saccharomyces cerevisiae. FDC1 shares structural similarity with the UbiD family of enzymes that are involved in ubiquinone biosynthesis. The position of 4-vinylphenol, the product of p-coumaric acid decarboxylation, in the structure identifies a large hydrophobic cavity as the active site. Differences in the β2e-α5 loop of chains in the crystal structure suggest that the conformational flexibility of this loop allows access to the active site. The structure also implicates Glu285 as the general base in the nonoxidative decarboxylation reaction catalyzed by FDC1. Biochemical analysis showed a loss of enzymatic activity in the E285A mutant. Modeling of 3-methoxy-4-hydroxy-5-decaprenylbenzoate, a partial structure of the physiological UbiD substrate, in the binding site suggests that an ∼30-Å-long pocket adjacent to the catalytic site may accommodate the isoprenoid tail of the substrate needed for ubiquinone biosynthesis in yeast. The three-dimensional structure of yeast FDC1 provides a template for guiding protein engineering studies aimed at optimizing the efficiency of aromatic acid decarboxylation reactions in bioindustrial applications.     

Purification and characterization of a ferulic acid decarboxylase from Pseudomonas fluorescens.

A ferulic acid decarboxylase enzyme which catalyzes the decarboxylation of ferulic acid to 4-hydroxy-3-methoxystyrene was purified from Pseudomonas fluorescens UI 670. The enzyme requires no cofactors and contains no prosthetic groups. Gel filtration estimated an apparent molecular mass of 40.4 (+/- 6%) kDa, whereas sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a molecular mass of 20.4 kDa, indicating that ferulic acid decarboxylase is a homodimer in solution. The purified enzyme displayed an optimum temperature range of 27 to 30 degrees C, exhibited an optimum pH of 7.3 in potassium phosphate buffer, and had a Km of 7.9 mM for ferulic acid. This enzyme also decarboxylated 4-hydroxycinnamic acid but not 2- or 3-hydroxycinnamic acid, indicating that a hydroxy group para to the carboxylic acid-containing side chain is required for the enzymatic reaction. The enzyme was inactivated by Hg2+, Cu2+, p-chloromercuribenzoic acid, and N-ethylmaleimide, suggesting that sulfhydryl groups are necessary for enzyme activity. Diethyl pyrocarbonate, a histidine-specific inhibitor, did not affect enzyme activity.     

Evolution of Substrate Specificity within a Diverse Family of β/α-Barrel-fold Basic Amino Acid Decarboxylases: X-RAY STRUCTURE DETERMINATION OF ENZYMES WITH SPECIFICITY FOR l-ARGININE AND CARBOXYNORSPERMIDINE*

Pyridoxal 5′-phosphate (PLP)-dependent basic amino acid decarboxylases from the β/α-barrel-fold class (group IV) exist in most organisms and catalyze the decarboxylation of diverse substrates, essential for polyamine and lysine biosynthesis. Herein we describe the first x-ray structure determination of bacterial biosynthetic arginine decarboxylase (ADC) and carboxynorspermidine decarboxylase (CANSDC) to 2.3- and 2.0-Å resolution, solved as product complexes with agmatine and norspermidine. Despite low overall sequence identity, the monomeric and dimeric structures are similar to other enzymes in the family, with the active sites formed between the β/α-barrel domain of one subunit and the β-barrel of the other. ADC contains both a unique interdomain insertion (4-helical bundle) and a C-terminal extension (3-helical bundle) and it packs as a tetramer in the asymmetric unit with the insertions forming part of the dimer and tetramer interfaces. Analytical ultracentrifugation studies confirmed that the ADC solution structure is a tetramer. Specificity for different basic amino acids appears to arise primarily from changes in the position of, and amino acid replacements in, a helix in the β-barrel domain we refer to as the “specificity helix.” Additionally, in CANSDC a key acidic residue that interacts with the distal amino group of other substrates is replaced by Leu³¹⁴, which interacts with the aliphatic portion of norspermidine. Neither product, agmatine in ADC nor norspermidine in CANSDC, form a Schiff base to pyridoxal 5′-phosphate, suggesting that the product complexes may promote product release by slowing the back reaction. These studies provide insight into the structural basis for the evolution of novel function within a common structural-fold.     

Determinants of substrate specificity in KdcA, a thiamin diphosphate-dependent decarboxylase

Thiamin diphosphate-dependent decarboxylases catalyze the non-oxidative decarboxylation of 2-keto carboxylic acids. Although they display relatively low sequence similarity, and broadly different range of substrates, these enzymes show a common homotetrameric structure. Here we describe a kinetic characterization of the substrate spectrum of a recently identified member of this class, the branched chain 2-keto acid decarboxylase (KdcA) from Lactococcus lactis. In order to understand the structural basis for KdcA substrate recognition we developed a homology model of its structure. Ser286, Phe381, Val461 and Met358 were identified as residues that appeared to shape the substrate binding pocket. Subsequently, site-directed mutagenesis was carried out on these residues with a view to converting KdcA into a pyruvate decarboxylase. The results show that the mutations all lowered the Km value for pyruvate and both the S286Y and F381W variants also had greatly increased values of k(cat) with pyruvate as a substrate.     

Molecular basis of phosphatidylinositol 4-phosphate and ARF1 GTPase recognition by the FAPP1 pleckstrin homology (PH) domain

Four-phosphate-adaptor protein 1 (FAPP1) regulates secretory transport from the trans-Golgi network (TGN) to the plasma membrane. FAPP1 is recruited to the Golgi through binding of its pleckstrin homology (PH) domain to phosphatidylinositol 4-phosphate (PtdIns(4)P) and a small GTPase ADP-ribosylation factor 1 (ARF1). Despite the critical role of FAPP1 in membrane trafficking, the molecular basis of its dual function remains unclear. Here, we report a 1.9 Å resolution crystal structure of the FAPP1 PH domain and detail the molecular mechanisms of the PtdIns(4)P and ARF1 recognition. The FAPP1 PH domain folds into a seven-stranded β-barrel capped by an α-helix at one edge, whereas the opposite edge is flanked by three loops and the β4 and β7 strands that form a lipid-binding pocket within the β-barrel. The ARF1-binding site is located on the outer side of the β-barrel as determined by NMR resonance perturbation analysis, mutagenesis, and measurements of binding affinities. The two binding sites have little overlap, allowing FAPP1 PH to associate with both ligands simultaneously and independently. Binding to PtdIns(4)P is enhanced in an acidic environment and is required for membrane penetration and tubulation activity of FAPP1, whereas the GTP-bound conformation of the GTPase is necessary for the interaction with ARF1. Together, these findings provide structural and biochemical insight into the multivalent membrane anchoring by the PH domain that may augment affinity and selectivity of FAPP1 toward the TGN membranes enriched in both PtdIns(4)P and GTP-bound ARF1.     

Metal-free Superoxide Dismutase-1 and Three Different Amyotrophic Lateral Sclerosis Variants Share a Similar Partially Unfolded ��-Barrel at Physiological Temperature

The structure and unfolding of metal-free (apo) human wild-type SOD1 and three pathogenic variants of SOD1 (A4V, G93R, and H48Q) that cause familial amyotrophic lateral sclerosis have been studied with amide hydrogen/deuterium exchange and mass spectrometry. The results indicate that a significant proportion of each of these proteins exists in solution in a conformation in which some strands of the β-barrel (i.e. β2) are well protected from exchange at physiological temperature (37 °C), whereas other strands (i.e. β3 and β4) appear to be unprotected from hydrogen/deuterium exchange. Moreover, the thermal unfolding of these proteins does not result in the uniform incorporation of deuterium throughout the polypeptide but involves the local unfolding of different residues at different temperatures. Some regions of the proteins (i.e. the “Greek key” loop, residues 104–116) unfold at a significantly higher temperature than other regions (i.e. β3 and β4, residues 21–53). Together, these results show that human wild-type apo-SOD1 and variants have a partially unfolded β-barrel at physiological temperature and unfold non-cooperatively.     

解淀粉芽孢杆菌Q-426酚酸脱羧酶的克隆表达及酶学性质鉴定*

目的:生物法脱羧制备4-乙烯基衍生物具有诸多优势和良好的发展前景,研究解淀粉芽孢杆菌Q-426酚酸脱羧酶(BaPAD-Q-426)的酶学性质,为其进一步应用提供理论基础。方法:从解淀粉芽孢杆菌中克隆酚酸脱羧酶基因;以pET-28a(+)为载体,将重组质粒转化至E. coli BL21(DE3)中,实现酚酸脱羧酶BaPAD-Q-426的高效表达,利用Ni-NTA亲和层析进行纯化,并进行酶学性质鉴定。结果:酚酸脱羧酶BaPAD-Q-426在pH 7.0~9.0范围内保持良好的pH稳定性,最适pH为8.0;在25~40℃范围内保持着较高的酶活性,最适温度为35℃,在4℃时保持30 min后该酶依然保持95%以上的酶活性;K⁺对BaPAD-Q-426的酶活具有明显促进作用,酶活力提高60%;该酶在石油醚中具有良好的耐受能力,在40%石油醚存在下,仍保留50%以上的酶活力;BaPAD-Q-426的最适底物为阿魏酸,酶活力达到19.5 IU/mL。结论:与其他来源的酚酸脱羧酶相比,BaPAD-Q-426在低温时具有更好的稳定性,在弱碱性环境下对阿魏酸的催化脱羧能力最强。     

Cloning, sequencing, and expression in Escherichia coli of the Bacillus pumilus gene for ferulic acid decarboxylase.

The Bacillus pumilus gene encoding a ferulic acid decarboxylase (fdc) was identified and isolated by its ability to promote ferulic acid decarboxylation in Escherichia coli DH5 alpha. The DNA sequence of the fdc gene was determined, and the recombinant enzyme produced in E. coli was purified and characterized.     

Oxalate decarboxylase uses electron hole hopping for catalysis

The hexameric low-pH stress response enzyme oxalate decarboxylase catalyzes the decarboxylation of the oxalate mono-anion in the soil bacterium Bacillus subtilis. A single protein subunit contains two Mn-binding cupin domains, and catalysis depends on Mn(III) at the N-terminal site. The present study suggests a mechanistic function for the C-terminal Mn as an electron hole donor for the N-terminal Mn. The resulting spatial separation of the radical intermediates directs the chemistry toward decarboxylation of the substrate. A π-stacked tryptophan pair (W96/W274) links two neighboring protein subunits together, thus reducing the Mn-to-Mn distance from 25.9 Å (intrasubunit) to 21.5 Å (intersubunit). Here, we used theoretical analysis of electron hole-hopping paths through redox-active sites in the enzyme combined with site-directed mutagenesis and X-ray crystallography to demonstrate that this tryptophan pair supports effective electron hole hopping between the C-terminal Mn of one subunit and the N-terminal Mn of the other subunit through two short hops of ∼8.5 Å. Replacement of W96, W274, or both with phenylalanine led to a large reduction in catalytic efficiency, whereas replacement with tyrosine led to recovery of most of this activity. W96F and W96Y mutants share the wildtype tertiary structure. Two additional hole-hopping networks were identified leading from the Mn ions to the protein surface, potentially protecting the enzyme from high Mn oxidation states during turnover. Our findings strongly suggest that multistep hole-hopping transport between the two Mn ions is required for enzymatic function, adding to the growing examples of proteins that employ aromatic residues as hopping stations.     

Structure and Catalytic Mechanism of 3-Ketosteroid-{Delta}4-(5α)-dehydrogenase from Rhodococcus jostii RHA1 Genome

3-Ketosteroid Δ4-(5α)-dehydrogenases (Δ4-(5α)-KSTDs) are enzymes that introduce a double bond between the C4 and C5 atoms of 3-keto-(5α)-steroids. Here we show that the ro05698 gene from Rhodococcus jostii RHA1 codes for a flavoprotein with Δ4-(5α)-KSTD activity. The 1.6 Å resolution crystal structure of the enzyme revealed three conserved residues (Tyr-319, Tyr-466, and Ser-468) in a pocket near the isoalloxazine ring system of the FAD co-factor. Site-directed mutagenesis of these residues confirmed that they are absolutely essential for catalytic activity. A crystal structure with bound product 4-androstene-3,17-dione showed that Ser-468 is in a position in which it can serve as the base abstracting the 4β-proton from the C4 atom of the substrate. Ser-468 is assisted by Tyr-319, which possibly is involved in shuttling the proton to the solvent. Tyr-466 is at hydrogen bonding distance to the C3 oxygen atom of the substrate and can stabilize the keto-enol intermediate occurring during the reaction. Finally, the FAD N5 atom is in a position to be able to abstract the 5α-hydrogen of the substrate as a hydride ion. These features fully explain the reaction catalyzed by Δ4-(5α)-KSTDs.     

Crystallographic snapshots of oxalyl-CoA decarboxylase give insights into catalysis by nonoxidative ThDP-dependent decarboxylases

Despite more than five decades of extensive studies of thiamin diphosphate (ThDP) enzymes, there remain many uncertainties as to how these enzymes achieve their rate enhancements. Here, we present a clear picture of catalysis for the simple nonoxidative decarboxylase, oxalyl-coenzyme A (CoA) decarboxylase, based on crystallographic snapshots along the catalytic cycle and kinetic data on active site mutants. First, we provide crystallographic evidence that, upon binding of oxalyl-CoA, the C-terminal 13 residues fold over the substrate, aligning the substrate alpha-carbon for attack by the ThDP-C2 atom. The second structure presented shows a covalent reaction intermediate after decarboxylation, interpreted as being nonplanar. Finally, the structure of a product complex is presented. In accordance with mutagenesis data, no side chains of the enzyme are implied to directly participate in proton transfer except the glutamic acid (Glu-56), which promotes formation of the 1',4'-iminopyrimidine tautomer of ThDP needed for activation.     

Nondisjunctional Segregations in Drosophila Female Meiosis I Are Preceded by Homolog Malorientation at Metaphase Arrest

The essential outer membrane β-barrel protein BamA forms a complex with four lipoprotein partners BamBCDE that assembles β-barrel proteins into the outer membrane of Escherichia coli. Detailed genetic studies have shown that BamA cycles through multiple conformations during substrate assembly, suggesting that a complex network of residues may be involved in coordinating conformational changes and lipoprotein partner function. While genetic analysis of BamA has been informative, it has also been slow in the absence of a straightforward selection for mutants. Here we take a bioinformatic approach to identify candidate residues for mutagenesis using direct coupling analysis. Starting with the BamA paralog FhaC, we show that direct coupling analysis works well for large β-barrel proteins, identifying pairs of residues in close proximity in tertiary structure with a true positive rate of 0.64 over the top 50 predictions. To reduce the effects of noise, we designed and incorporated a novel structured prior into the empirical correlation matrix, dramatically increasing the FhaC true positive rate from 0.64 to 0.88 over the top 50 predictions. Our direct coupling analysis of BamA implicates residues R661 and D740 in a functional interaction. We find that the substitutions R661G and D740G each confer OM permeability defects and destabilize the BamA β-barrel. We also identify synthetic phenotypes and cross-suppressors that suggest R661 and D740 function in a similar process and may interact directly. We expect that the direct coupling analysis approach to informed mutagenesis will be particularly useful in systems lacking adequate selections and for dynamic proteins with multiple conformations.     

The mechanism of orotidine 5'-monophosphate decarboxylase: catalysis by destabilization of the substrate

The mechanism of orotidine 5'-monophosphate decarboxylase (OMP decarboxylase, ODCase) was studied using the decarboxylation of orotic acid analogues as a model system. The rate of decarboxylation of 1,3-dimethylorotic acid and its analogues as well as the stability of their corresponding carbanion intermediates was determined. The results have shown that the stability of the carbanion intermediate is not a critical factor in the rate of decarboxylation. On the other hand, the reaction rate is largely dependent on the equilibrium constant for the formation of a zwitterion. Based on these results, we have proposed a new mechanism in which ODCase catalyzes the decarboxylation of OMP by binding the substrate in a zwitterionic form and providing a destabilizing environment for the carboxylate group of OMP.     

Three-dimensional Model of Human Platelet Integrin ��IIb��3 in Solution Obtained by Small Angle Neutron Scattering

Integrin αIIbβ3 is the major membrane protein and adhesion receptor at the surface of blood platelets, which after activation plays a key role in platelet plug formation in hemostasis and thrombosis. Small angle neutron scattering (SANS) and shape reconstruction algorithms allowed formation of a low resolution three-dimensional model of whole αIIbβ3 in Ca²⁺/detergent solutions. Model projections after 90° rotation along its long axis show an elongated and “arched” form (135°) not observed before and a “handgun” form. This 20-nm-long structure is well defined, despite αIIbβ3 multidomain nature and expected segmental flexibility, with the largest region at the top, followed by two narrower and smaller regions at the bottom. Docking of this SANS envelope into the high resolution structure of αIIbβ3, reconstructed from crystallographic and NMR data, shows that the solution structure is less constrained, allows tentative assignment of the disposition of the αIIb and β3 subunits and their domains within the model, and points out the structural analogies and differences of the SANS model with the crystallographic models of the recombinant ectodomains of αIIbβ3 and αVβ3 and with the cryo-electron microscopy model of whole αIIbβ3. The ectodomain is in the bent configuration at the top of the model, where αIIb and β3 occupy the concave and convex sides, respectively, at the arched projection, with their bent knees at its apex. It follows the narrower transmembrane region and the cytoplasmic domains at the bottom end. αIIbβ3 aggregated in Mn²⁺/detergent solutions, which impeded to get its SANS model.     

Functional Roles of the Dimer-Interface Residues in Human Ornithine Decarboxylase

Ornithine decarboxylase (ODC) catalyzes the decarboxylation of ornithine to putrescine and is the rate-limiting enzyme in the polyamine biosynthesis pathway. ODC is a dimeric enzyme, and the active sites of this enzyme reside at the dimer interface. Once the enzyme dissociates, the enzyme activity is lost. In this paper, we investigated the roles of amino acid residues at the dimer interface regarding the dimerization, protein stability and/or enzyme activity of ODC. A multiple sequence alignment of ODC and its homologous protein antizyme inhibitor revealed that 5 of 9 residues (residues 165, 277, 331, 332 and 389) are divergent, whereas 4 (134, 169, 294 and 322) are conserved. Analytical ultracentrifugation analysis suggested that some dimer-interface amino acid residues contribute to formation of the dimer of ODC and that this dimerization results from the cooperativity of these interface residues. The quaternary structure of the sextuple mutant Y331S/Y389D/R277S/D332E/V322D/D134A was changed to a monomer rather than a dimer, and the K _d value of the mutant was 52.8 µM, which is over 500-fold greater than that of the wild-type ODC (ODC_WT). In addition, most interface mutants showed low but detectable or negligible enzyme activity. Therefore, the protein stability of these interface mutants was measured by differential scanning calorimetry. These results indicate that these dimer-interface residues are important for dimer formation and, as a consequence, are critical for enzyme catalysis.     

Manipulation of the active site loops of D-hydantoinase, a (beta/alpha)8-barrel protein, for modulation of the substrate specificity

We previously proposed that the stereochemistry gate loops (SGLs) constituting the substrate binding pocket of D-hydantoinase, a (beta/alpha)(8)-barrel enzyme, might be major structural determinants of the substrate specificity [Cheon, Y. H., et al. (2002) Biochemistry 41, 9410-9417]. To construct a mutant D-hydantoinase with favorable substrate specificity for the synthesis of commercially important non-natural amino acids, the SGL loops of the enzyme were rationally manipulated on the basis of the structural analysis and sequence alignment of three hydantoinases with distinct substrate specificities. In the SGLs of D-hydantoinase from Bacillus stearothermophilus SD1, mutations of hydrophobic and bulky residues Met 63, Leu 65, Phe 152, and Phe 159, which interact with the exocyclic substituent of the substrate, induced remarkable changes in the substrate specificities. In particular, the substrate specificity of mutant F159A toward aromatic substrate hydroxyphenylhydantoin (HPH) was enhanced by approximately 200-fold compared with that of the wild-type enzyme. Saturation mutagenesis at position 159 revealed that k(cat) for aromatic substrates increased gradually as the size of the amino acid side chain decreased, and this seems to be due to reduced steric hindrance between the bulky exocyclic group of the substrate and the amino acid side chains. When site-directed random mutagenesis of residues 63 and 65 was conducted with the wild type and mutant F159A, the selected enzymes (M63F/L65V and L65F/F159A) exhibited approximately 10-fold higher k(cat) values for HPH than the wild-type counterpart, which is likely to result from reorganization of the active site for efficient turnover. These results indicate that the amino acid residues of SGLs forming the substrate binding pocket are critical for the substrate specificity of D-hydantoinase, and the results also imply that substrate specificities of cyclic amidohydrolase family enzymes can be modulated by rational design of these SGLs.     

Structural and Mechanistic Studies on Klebsiella pneumoniae 2-Oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline Decarboxylase

The stereospecific oxidative degradation of uric acid to (S)-allantoin was recently shown to proceed via three enzymatic steps. The final conversion is a decarboxylation of the unstable intermediate 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) and is catalyzed by OHCU decarboxylase. Here we present the structures of Klebsiella pneumoniae OHCU decarboxylase in unliganded form and with bound allantoin. These structures provide evidence that ligand binding organizes the active site residues for catalysis. Modeling of the substrate and intermediates provides additional support for this hypothesis. In addition we characterize the steady state kinetics of this enzyme and report the first OHCU decarboxylase inhibitor, allopurinol, a structural isomer of hypoxanthine. This molecule is a competitive inhibitor of K. pneumoniae OHCU decarboxylase with a K_i of 30 ± 2 μm. Circular dichroism measurements confirm structural observations that this inhibitor disrupts the necessary organization of the active site. Our structural and biochemical studies also provide further insights into the mechanism of catalysis of OHCU decarboxylation.     

Crystal Structure and Substrate Specificity of Drosophila 3,4-Dihydroxyphenylalanine Decarboxylase

Background

3,4-Dihydroxyphenylalanine decarboxylase (DDC), also known as aromatic L-amino acid decarboxylase, catalyzes the decarboxylation of a number of aromatic L-amino acids. Physiologically, DDC is responsible for the production of dopamine and serotonin through the decarboxylation of 3,4-dihydroxyphenylalanine and 5-hydroxytryptophan, respectively. In insects, both dopamine and serotonin serve as classical neurotransmitters, neuromodulators, or neurohormones, and dopamine is also involved in insect cuticle formation, eggshell hardening, and immune responses.

Principal Findings

In this study, we expressed a typical DDC enzyme from Drosophila melanogaster, critically analyzed its substrate specificity and biochemical properties, determined its crystal structure at 1.75 Angstrom resolution, and evaluated the roles residues T82 and H192 play in substrate binding and enzyme catalysis through site-directed mutagenesis of the enzyme. Our results establish that this DDC functions exclusively on the production of dopamine and serotonin, with no activity to tyrosine or tryptophan and catalyzes the formation of serotonin more efficiently than dopamine.

Conclusions

The crystal structure of Drosophila DDC and the site-directed mutagenesis study of the enzyme demonstrate that T82 is involved in substrate binding and that H192 is used not only for substrate interaction, but for cofactor binding of drDDC as well. Through comparative analysis, the results also provide insight into the structure-function relationship of other insect DDC-like proteins.     

Crystal structures of isoorotate decarboxylases reveal a novel catalytic mechanism of 5-carboxyl-uracil decarboxylation and shed light on the search for DNA decarboxylase

DNA methylation and demethylation regulate many crucial biological processes in mammals and are linked to many diseases. Active DNA demethylation is believed to be catalyzed by TET proteins and a putative DNA decarboxylase that may share some similarities in sequence, structure and catalytic mechanism with isoorotate decarboxylase (IDCase) that catalyzes decarboxylation of 5caU to U in fungi. We report here the structures of wild-type and mutant IDCases from Cordyceps militaris and Metarhizium anisopliae in apo form or in complexes with 5caU, U, and an inhibitor 5-nitro-uracil. IDCases adopt a typical (β/α)₈ barrel fold of the amidohydrolase superfamily and function as dimers. A Zn²⁺ is bound at the active site and coordinated by four strictly conserved residues, one Asp and three His. The substrate is recognized by several strictly conserved residues. The functional roles of the key residues at the active site are validated by mutagenesis and biochemical studies. Based on the structural and biochemical data, we present for the first time a novel catalytic mechanism of decarboxylation for IDCases, which might also apply to other members of the amidohydrolase superfamily. In addition, our biochemical data show that IDCases can catalyze decarboxylation of 5caC to C albeit with weak activity, which is the first in vitro evidence for direct decarboxylation of 5caC to C by an enzyme. These findings are valuable in the identification of potential DNA decarboxylase in mammals.