Bacterial degradation of 3-chloroacrylic acid and the characterization of cis- and trans-specific dehalogenases

A coryneform bacterium that is able to utilize cis- and trans-3-chloroacrylic acid as sole carbon source for growth was isolated from freshwater sediment. The organism was found to produce two inducible dehalogenases, one specific for the cis- and the other for trans-3-chloroacrylic acid. Both dehalogenases were purified to homogeneity from cells induced for dehalogenase synthesis with 3-chlorocrotonic acid. The enzymes produced muconic acid semialdehyde (3-oxopropionic acid) from their respective 3-chloroacrylic acid substrate. No other substrates were found. The cis-3-chloroacrylic acid dehalogenase consisted of two polypeptide chains of a molecular weight 16.2 kDa. Trans-3-chloroacrylic acid dehalogenase was a protein with subunits of 7.4 and 8.7 kDa. The subunit and amino acid compositions and the N-terminal amino acid sequences of the enzymes indicate that they are not closely related.     

Stereospecific alkylation of cis-3-chloroacrylic acid dehalogenase by (R)-oxirane-2-carboxylate: analysis and mechanistic implications

The enzymes trans-3-chloroacrylic acid dehalogenase (CaaD) and cis-3-chloroacrylic acid dehalogenase (cis-CaaD) represent the two major classes of bacterial, isomer-selective 3-chloroacrylic acid dehalogenases. They catalyze the hydrolytic dehalogenation of either trans- or cis-3-haloacrylates to yield malonate semialdehyde, presumably through unstable halohydrin intermediates. In view of a proposed general acid/base mechanism for these enzymes, (R)- and (S)-oxirane-2-carboxylate were investigated as potential irreversible inhibitors. Only cis-CaaD is irreversibly inhibited in a time- and concentration-dependent manner and only by the (R)-enantiomer of oxirane-2-carboxylate. The enzyme displays saturation kinetics and is protected from inactivation by the presence of substrate. These findings indicate that the inactivation process involves the initial formation of a reversibly bound enzyme-inhibitor complex at the active site followed by covalent modification. Mass spectral analysis of the inactivated cis-CaaD shows that Pro-1 is the site of modification. It has also been determined that Arg-70 and Arg-73 are required for covalent modification because incubation of either the R70A or R73A mutant with inhibitor does not result in enzyme alkylation. Studies of the pH dependence of the kinetic parameters of wild-type cis-CaaD reveal that a protonated group with a pK(a) of approximately 9.3 is essential for catalysis. The group is likely Pro-1, making it predominately a charged species under the conditions of the inactivation experiments. Two mechanisms could account for these observations. In one mechanism, the oxirane undergoes acid-catalyzed ring opening followed by alkylation of the conjugate base of Pro-1. Alternatively, the oxirane undergoes a nucleophilic substitution reaction where the conjugate base of Pro-1 functions as the nucleophile and an acid catalyst polarizes the carbon oxygen bond. The two arginine residues likely bind the carboxylate group and position the inhibitor in a favorable orientation for the alkylation reaction. These findings set the stage for a crystallographic analysis of the inactived enzyme to delineate further the roles of active site residues in both the inactivation process and the catalytic mechanism.     

Crystal structures of native and inactivated cis-3-chloroacrylic acid dehalogenase: Implications for the catalytic and inactivation mechanisms

The isomeric mixture of cis- and trans-1,3-dichloropropene constitutes the active component of a widely used nematocide known as Telone II®. The mixture is processed by various soil bacteria to acetaldehyde through the 1,3-dichloropropene catabolic pathway. The pathway relies on an isomer-specific hydrolytic dehalogenation reaction catalyzed by cis- or trans-3-chloroacrylic acid dehalogenase, known respectively as cis-CaaD and CaaD. Previous sequence analysis and crystallographic studies of the native and covalently modified enzymes identified Pro-1, His-28, Arg-70, Arg-73, Tyr-103, and Glu-114 as key binding and catalytic residues in cis-CaaD. Mutagenesis of these residues confirmed their importance to the dehalogenation reaction. Crystal structures of the native enzyme (2.01 Å resolution) and the enzyme covalently modified at the Pro-1 nitrogen by 2-hydroxypropanoate (1.65 Å resolution) are reported here. Both structures are at a resolution higher than previously reported (2.75 Å and 2.1 Å resolution, respectively). The conformation of the covalent adduct is strikingly different from that previously reported due to its interaction with a 7-residue loop (Thr-32 to Leu-38). The participation of another active site residue, Arg-117, in catalysis and inactivation was also examined. The implications of the combined findings for the mechanisms of catalysis and inactivation are discussed.     

Characterization of a newly identified mycobacterial tautomerase with promiscuous dehalogenase and hydratase activities reveals a functional link to a recently diverged cis-3-chloroacrylic acid dehalogenase

The enzyme cis-3-chloroacrylic acid dehalogenase (cis-CaaD) is found in a bacterial pathway that degrades a synthetic nematocide, cis-1,3-dichloropropene, introduced in the 20th century. The previously determined crystal structure of cis-CaaD and its promiscuous phenylpyruvate tautomerase (PPT) activity link this dehalogenase to the tautomerase superfamily, a group of homologous proteins that are characterized by a catalytic amino-terminal proline and a β-α-β structural fold. The low-level PPT activity of cis-CaaD, which may be a vestige of the function of its progenitor, prompted us to search the databases for a homologue of cis-CaaD that was annotated as a putative tautomerase and test both its PPT and cis-CaaD activity. We identified a mycobacterial cis-CaaD homologue (designated MsCCH2) that shares key sequence and active site features with cis-CaaD. Kinetic and 1H NMR spectroscopic studies show that MsCCH2 functions as an efficient PPT and exhibits low-level promiscuous dehalogenase activity, processing both cis- and trans-3-chloroacrylic acid. To further probe the active site of MsCCH2, the enzyme was incubated with 2-oxo-3-pentynoate (2-OP). At pH 8.5, MsCCH2 is inactivated by 2-OP due to the covalent modification of Pro-1, suggesting that Pro-1 functions as a nucleophile at pH 8.5 and attacks 2-OP in a Michael-type reaction. At pH 6.5, however, MsCCH2 exhibits hydratase activity and converts 2-OP to acetopyruvate, which implies that Pro-1 is cationic at pH 6.5 and not functioning as a nucleophile. At pH 7.5, the hydratase and inactivation reactions occur simultaneously. From these results, it can be inferred that Pro-1 of MsCCH2 has a pKa value that lies in between that of a typical tautomerase (pKa of Pro-1～6) and that of cis-CaaD (pKa of Pro-1～9). The shared activities and structural features, coupled with the intermediate pKa of Pro-1, suggest that MsCCH2 could be characteristic of an evolutionary intermediate along the past route for the divergence of cis-CaaD from an unknown superfamily tautomerase. This makes MsCCH2 an ideal candidate for laboratory evolution of its promiscuous dehalogenase activity, which could identify additional features necessary for a fully active cis-CaaD. Such results will provide insight into pathways that could lead to the rapid divergent evolution of an efficient cis-CaaD enzyme.     

Characterization of Cg10062 from Corynebacterium glutamicum: implications for the evolution of cis-3-chloroacrylic acid dehalogenase activity in the tautomerase superfamily

A 149-amino acid protein designated Cg10062 is encoded by a gene from Corynebacterium glutamicum. The physiological function of Cg10062 is unknown, and the gene encoding this protein has no obvious genomic context. Sequence analysis links Cg10062 to the cis-3-chloroacrylic acid dehalogenase ( cis-CaaD) family, one of the five known families of the tautomerase superfamily. The characterized tautomerase superfamily members have two distinctive characteristics: a beta-alpha-beta structure motif and a catalytic amino-terminal proline. Pro-1 is present in the Cg10062 amino acid sequence along with His-28, Arg-70, Arg-73, Tyr-103, and Glu-114, all of which have been implicated as critical residues for cis-CaaD activity. The gene for Cg10062 has been cloned and the protein overproduced, purified, and subjected to kinetic and mechanistic characterization. Like cis-CaaD, Cg10062 functions as a hydratase: it converts 2-oxo-3-pentynoate to acetopyruvate and processes 3-bromopropiolate to a species that inactivates the enzyme by acylation of Pro-1. Kinetic and (1)H NMR spectroscopic studies also show that Cg10062 processes both isomers of 3-chloroacrylic acid at low levels with a clear preference for the cis isomer. Pro-1 is critical for the dehalogenase and hydratase activities because the P1A mutant no longer catalyzes either reaction. The presence of the six key catalytic residues and the hydratase activity coupled with the absence of an efficient cis-CaaD activity and the lack of isomer specificity implicate factors beyond this core set of residues in cis-CaaD catalysis and specificity. This work sets the stage for in-depth mechanistic and structural studies of Cg10062, which could identify the additional features necessary for a fully active and highly specific cis-CaaD. Such results will also shed light on how cis-CaaD emerged in the tautomerase superfamily because Cg10062 could be characteristic of an intermediate along the evolutionary pathway for this dehalogenase.     

Reactions of trans-3-chloroacrylic acid dehalogenase with acetylene substrates: consequences of and evidence for a hydration reaction

Various soil bacteria use 1,3-dichloropropene, a component of the commercially available fumigants Shell D-D and Telone II, as a sole source of carbon and energy. One enzyme involved in the catabolism of 1,3-dichloropropene is trans-3-chloroacrylic acid dehalogenase (CaaD), which converts the trans-isomers of 3-bromo- and 3-chloroacrylate to malonate semialdehyde. Sequence analysis suggested a relationship between the heterohexameric CaaD and the bacterial isomerase, 4-oxalocrotonate tautomerase (4-OT), thereby distinguishing CaaD from a number of dehalogenases whose mechanisms proceed through an alkyl- or aryl-enzyme intermediate. In this study, the genes for the alpha- and beta-subunits of CaaD have been synthesized using a polymerase chain reaction-based strategy, cloned into separate plasmids, and the proteins expressed and purified as the functional heterohexamer. Subsequently, the product of the reaction was confirmed to be malonate semialdehyde by (1)H and (13)C NMR spectroscopy, and kinetic constants were determined using a UV spectrophotometric assay. In view of the proposed hydrolytic nature of the CaaD-catalyzed reaction, three acetylene compounds were investigated as substrates for the enzyme. One compound, 2-oxo-3-pentynoate, a potent active site-directed irreversible inhibitor of 4-OT, is a substrate for CaaD, and was processed to acetopyruvate with kinetic constants similar to those determined for the trans-isomers of 3-bromo- and 3-chloroacrylate. The remaining two compounds, 3-bromo- and 3-chloropropiolic acid, were transformed into potent irreversible inhibitors of CaaD. The inactivation observed for 3-bromopropiolic acid is due to the covalent modification of Pro-1 of the beta-subunit. The results provide evidence for a hydratase activity and set the stage to use the 3-halopropiolic acids as ligands in inactivated CaaD complexes that can be studied by X-ray crystallography.     

The roles of active-site residues in the catalytic mechanism of trans-3-chloroacrylic acid dehalogenase: a kinetic, NMR, and mutational analysis

trans-3-Chloroacrylic acid dehalogenase (CaaD) converts trans-3-chloroacrylic acid to malonate semialdehyde by the addition of H(2)O to the C-2, C-3 double bond, followed by the loss of HCl from the C-3 position. Sequence similarity between CaaD, an (alphabeta)(3) heterohexamer (molecular weight 47,547), and 4-oxalocrotonate tautomerase (4-OT), an (alpha)(6) homohexamer, distinguishes CaaD from those hydrolytic dehalogenases that form alkyl-enzyme intermediates. The recently solved X-ray structure of CaaD demonstrates that betaPro-1 (i.e., Pro-1 of the beta subunit), alphaArg-8, alphaArg-11, and alphaGlu-52 are at or near the active site, and the >or=10(3.4)-fold decreases in k(cat) on mutating these residues implicate them as mechanistically important. The effect of pH on k(cat)/K(m) indicates a catalytic base with a pK(a) of 7.6 and an acid with a pK(a) of 9.2. NMR titration of (15)N-labeled wild-type CaaD yielded pK(a) values of 9.3 and 11.1 for the N-terminal prolines, while the fully active but unstable alphaP1A mutant showed a pK(a) of 9.7 (for the betaPro-1), implicating betaPro-1 as the acid catalyst, which may protonate C-2 of the substrate. These results provide the first evidence for an amino-terminal proline, conserved in all known tautomerase superfamily members, functioning as a general acid, rather than as a general base as in 4-OT. Hence, a reasonable candidate for the general base in CaaD is the active site residue alphaGlu-52. CaaD has 10 arginine residues, six in the alpha-subunit (Arg-8, Arg-11, Arg-17, Arg-25, Arg-35, and Arg-43), and four in the beta-subunit (Arg-15, Arg-21, Arg-55, and Arg-65). (1)H-(15)N-heteronuclear single quantum coherence (HSQC) spectra of CaaD showed seven to nine Arg-NepsilonH resonances (denoted R(A) to R(I)) depending on the protein concentration and pH. One of these signals (R(D)) disappeared in the spectrum of the largely inactive alphaR11A mutant (deltaH = 7.11 ppm, deltaN = 89.5 ppm), and another one (R(G)) disappeared in the spectrum of the inactive alphaR8A mutant (deltaH = 7.48 ppm, deltaN = 89.6 ppm), thereby assigning these resonances to alphaArg-11NepsilonH, and alphaArg-8NepsilonH, respectively. (1)H-(15)N-HSQC titration of the enzyme with the substrate analogue 3-chloro-2-butenoic acid (3-CBA), a competitive inhibitor (K(I)(slope) = 0.35 +/- 0.06 mM), resulted in progressive downfield shifts of the alphaArg-8Nepsilon resonance yielding a K(D) = 0.77 +/- 0.44 mM, comparable to the (K(I)(slope), suggestive of active site binding. Increasing the pH of free CaaD to 8.9 at 5 degrees C resulted in the disappearance of all nine Arg-NepsilonH resonances due to base-catalyzed NepsilonH exchange. Saturating the enzyme with 3-CBA (16 mM) induced the reappearance of two NepsilonH signals, those of alphaArg-8 and alphaArg-11, indicating that the binding of the substrate analogue 3-CBA selectively slows the NepsilonH exchange rates of these two arginine residues. The kinetic and NMR data thus indicate that betaPro-1 is the acid catalyst, alphaGlu-52 is a reasonable candidate for the general base, and alphaArg-8 and alphaArg-11 participate in substrate binding and in stabilizing the aci-carboxylate intermediate in a Michael addition mechanism.     

Inactivation of Cg10062, a cis-3-chloroacrylic acid dehalogenase homologue in Corynebacterium glutamicum, by (R)- and (S)-oxirane-2-carboxylate: analysis and implications

( R)- and ( S)-oxirane-2-carboxylate were determined to be active site-directed irreversible inhibitors of the cis-3-chloroacrylic acid dehalogenase ( cis-CaaD) homologue Cg10062 found in Corynebacterium glutamicum. Kinetic analysis indicates that the ( R) enantiomer binds more tightly and is the more potent inhibitor, likely reflecting more favorable interactions with active site residues. Pro-1 is the sole site of covalent modification by the ( R) and ( S) enantiomers. Pro-1, Arg-70, Arg-73, and Glu-114, previously identified as catalytic residues in Cg10062, have also been implicated in the inactivation mechanism. Pro-1, Arg-70, and Arg-73 are essential residues for the process as indicated by the observation that the enzymes with the corresponding alanine mutations are not covalently modified by either enantiomer. The E114Q mutant slows covalent modification of Cg10062 but does not prevent it. The results are comparable to those found for the irreversible inactivation of cis-CaaD by ( R)-oxirane-2-carboxylate with two important distinctions: the alkylation of cis-CaaD is stereospecific, and Glu-114 does not take part in the cis-CaaD inactivation mechanism. Cg10062 exhibits low-level cis-CaaD and trans-3-chloroacrylic acid dehalogenase (CaaD) activities, with the cis-CaaD activity predominating. Hence, the preference of Cg10062 for the cis isomer correlates with the observation that the ( R) enantiomer is the more potent inactivator. Moreover, the factors responsible for the relaxed substrate specificity of Cg10062 may account for the stereoselective inactivation by the enantiomeric epoxides. Delineation of these factors would provide a more complete picture of the substrate specificity determinants for cis-CaaD. This study represents an important step toward this goal by setting the stage for a crystallographic analysis of inactivated Cg10062.     

The X-ray structure of trans-3-chloroacrylic acid dehalogenase reveals a novel hydration mechanism in the tautomerase superfamily

Isomer-specific 3-chloroacrylic acid dehalogenases function in the bacterial degradation of 1,3-dichloropropene, a compound used in agriculture to kill plant-parasitic nematodes. The crystal structure of the heterohexameric trans-3-chloroacrylic acid dehalogenase (CaaD) from Pseudomonas pavonaceae 170 inactivated by 3-bromopropiolate shows that Glu-52 in the alpha-subunit is positioned to function as the water-activating base for the addition of a hydroxyl group to C-3 of 3-chloroacrylate and 3-bromopropiolate, whereas the nearby Pro-1 in the beta-subunit is positioned to provide a proton to C-2. Two arginine residues, alphaArg-8 and alphaArg-11, interact with the C-1 carboxylate groups, thereby polarizing the alpha,beta-unsaturated acids. The reaction with 3-chloroacrylate results in the production of an unstable halohydrin, 3-chloro-3-hydroxypropanoate, which decomposes into the products malonate semialdehyde and HCl. In the inactivation mechanism, however, malonyl bromide is produced, which irreversibly alkylates the betaPro-1. CaaD is related to 4-oxalocrotonate tautomerase, with which it shares an N-terminal proline. However, in 4-oxalocrotonate tautomerase, Pro-1 functions as a base participating in proton transfer within a hydrophobic active site, whereas in CaaD, the acidic proline is stabilized in a hydrophilic active site. The altered active site environment of CaaD thus facilitates a previously unknown reaction in the tautomerase superfamily, the hydration of the alpha,beta-unsaturated bonds of trans-3-chloroacrylate and 3-bromopropiolate. The mechanism for these hydration reactions represents a novel catalytic strategy that results in carbon-halogen bond cleavage.     

Crystal structures of native and inactivated cis-3-chloroacrylic acid dehalogenase. Structural basis for substrate specificity and inactivation by (R)-oxirane-2-carboxylate

The bacterial degradation pathways for the nematocide 1,3-dichloropropene rely on hydrolytic dehalogenation reactions catalyzed by cis- and trans-3-chloroacrylic acid dehalogenases (cis-CaaD and CaaD, respectively). X-ray crystal structures of native cis-CaaD and cis-CaaD inactivated by (R)-oxirane-2-carboxylate were elucidated. They locate four known catalytic residues (Pro-1, Arg-70, Arg-73, and Glu-114) and two previously unknown, potential catalytic residues (His-28 and Tyr-103'). The Y103F and H28A mutants of these latter two residues displayed reductions in cis-CaaD activity confirming their importance in catalysis. The structure of the inactivated enzyme shows covalent modification of the Pro-1 nitrogen atom by (R)-2-hydroxypropanoate at the C3 position. The interactions in the complex implicate Arg-70 or a water molecule bound to Arg-70 as the proton donor for the epoxide ring-opening reaction and Arg-73 and His-28 as primary binding contacts for the carboxylate group. This proposed binding mode places the (R)-enantiomer, but not the (S)-enantiomer, in position to covalently modify Pro-1. The absence of His-28 (or an equivalent) in CaaD could account for the fact that CaaD is not inactivated by either enantiomer. The cis-CaaD structures support a mechanism in which Glu-114 and Tyr-103' activate a water molecule for addition to C3 of the substrate and His-28, Arg-70, and Arg-73 interact with the C1 carboxylate group to assist in substrate binding and polarization. Pro-1 provides a proton at C2. The involvement of His-28 and Tyr-103' distinguishes the cis-CaaD mechanism from the otherwise parallel CaaD mechanism. The two mechanisms probably evolved independently as the result of an early gene duplication of a common ancestor.     

3D cryo-EM imaging of bacterial flagella: Novel structural and mechanistic insights into cell motility

Bacterial flagella are nanomachines that enable cells to move at high speeds. Comprising 25 and more different types of proteins, the flagellum is a large supramolecular assembly organized into three widely conserved substructures: a basal body including the rotary motor, a connecting hook, and a long filament. The whole flagellum from Escherichia coli weighs ∼20 MDa, without considering its filament portion, which is by itself a ∼1.6 GDa structure arranged as a multimer of ∼30,000 flagellin protomers. Breakthroughs regarding flagellar structure and function have been achieved in the last few years, mainly because of the revolutionary improvements in 3D cryo-EM methods. This review discusses novel structures and mechanistic insights derived from such high-resolution studies, advancing our understanding of each one of the three major flagellar segments. The rotation mechanism of the motor has been unveiled with unprecedented detail, showing a two-cogwheel machine propelled by a Brownian ratchet device. In addition, by imaging the flagellin-like protomers that make up the hook in its native bent configuration, their unexpected conformational plasticity challenges the paradigm of a two-state conformational rearrangement mechanism for flagellin-fold proteins. Finally, imaging of the filaments of periplasmic flagella, which endow Spirochete bacteria with their singular motility style, uncovered a strikingly asymmetric protein sheath that coats the flagellin core, challenging the view of filaments as simple homopolymeric structures that work as freely whirling whips. Further research will shed more light on the functional details of this amazing nanomachine, but our current understanding has definitely come a long way.     

Serine hydroxymethyltransferase: a model enzyme for mechanistic, structural, and evolutionary studies

Serine hydroxymethyltransferase is a ubiquitous representative of the family of fold type I, pyridoxal 5'-phosphate-dependent enzymes. The reaction catalyzed by this enzyme, the reversible transfer of the Cβ of serine to tetrahydropteroylglutamate, represents a link between amino acid and folates metabolism and operates as a major source of one-carbon units for several essential biosynthetic processes. Serine hydroxymethyltransferase has been intensively investigated because of the interest aroused by the complex mechanism of the hydroxymethyltransferase reaction and its broad substrate and reaction specificity. Although the increasing availability of crystallographic data and the characterization of several site-specific mutants helped in understanding previous functional and structural studies, they also represent the starting point of novel investigations. This review will focus on recently highlighted catalytic, structural, and evolutionary aspects of serine hydroxymethyltransferase. This article is part of a Special Issue entitled: Pyridoxal phosphate Enzymology.     

Cloning and expression of a lombricine kinase from an echiuroid worm: insights into structural correlates of substrate specificity

Phosphagen kinases constitute a large family of enzymes catalyzing the reversible phosphorylation of guanidino acceptor compounds. These guanidino substrates differ substantially in size and chemical properties. In spite of the appearance of X-ray crystal structures for two members of this family, creatine kinase (CK) and arginine kinase (AK), the structural correlates of substrate specificity remain to be fully elucidated. We have determined the cDNA and deduced amino acid sequences for lombricine (guanidinethylphosphoserine) kinase (LK) from the echiuroid worm Urechis caupo and expressed the cDNA in Escherichia coli. The recombinant protein was purified by affinity chromatography and showed high capacity for phosphorylation of lombricine. Phosphagen kinases consist of a small, N-terminal domain and a much larger domain connected by a linker sequence. A key event in catalysis in CK and AK, and certainly all other phosphagen kinases, is a large conformational change involving involving a rotation of the two domains and the movement of two highly conserved flexible loops (one located in the small domain; the other located in the large domain of these enzymes) which clamp down on the substrates. Multiple sequence alignments of Urechis LK with the only other LK sequence available and CK, AK and glycocyamine kinase sequences, confirm the importance of the small flexible loop located in the N-terminal domain of phosphagen kinases as one component of the structural determinants of guanidine specificity. The role of the other flexible loop in the large domain in terms of substrate specificity remains questionable.     

The Aspergillus fumigatus sialidase is a 3-deoxy-D-glycero-D-galacto-2-nonulosonic acid hydrolase (KDNase): structural and mechanistic insights

Aspergillus fumigatus is a filamentous fungus that can cause severe respiratory disease in immunocompromised individuals. A putative sialidase from A. fumigatus was recently cloned and shown to be relatively poor in cleaving N-acetylneuraminic acid (Neu5Ac) in comparison with bacterial sialidases. Here we present the first crystal structure of a fungal sialidase. When the apo structure was compared with bacterial sialidase structures, the active site of the Aspergillus enzyme suggested that Neu5Ac would be a poor substrate because of a smaller pocket that normally accommodates the acetamido group of Neu5Ac in sialidases. A sialic acid with a hydroxyl in place of an acetamido group is 2-keto-3-deoxynononic acid (KDN). We show that KDN is the preferred substrate for the A. fumigatus sialidase and that A. fumigatus can utilize KDN as a sole carbon source. A 1.45-Å resolution crystal structure of the enzyme in complex with KDN reveals KDN in the active site in a boat conformation and nearby a second binding site occupied by KDN in a chair conformation, suggesting that polyKDN may be a natural substrate. The enzyme is not inhibited by the sialidase transition state analog 2-deoxy-2,3-dehydro-N-acetylneuraminic acid (Neu5Ac2en) but is inhibited by the related 2,3-didehydro-2,3-dideoxy-d-glycero-d-galacto-nonulosonic acid that we show bound to the enzyme in a 1.84-Å resolution crystal structure. Using a fluorinated KDN substrate, we present a 1.5-Å resolution structure of a covalently bound catalytic intermediate. The A. fumigatus sialidase is therefore a KDNase with a similar catalytic mechanism to Neu5Ac exosialidases, and this study represents the first structure of a KDNase.     

Cloning, expression, and characterization of sialic acid synthases

The most commonly occurring sialic acid, N-acetylneuraminic acid, is the repeating unit in polysialic acid chain of human neuronal cell adhesion molecule as well as in capsular polysialic acid of neuroinvasive bacteria, Escherichia coli K1 and Neisseria meningitidis. Sialic acid synthesis and polymerization occur in slightly different pathways in animals and bacteria. N-Acetylneuraminic acid (NeuNAc) is synthesized by the condensation of phosphoenolpyruvate and N-acetylmannosamine by NeuNAc synthase in bacteria. The mammalian homologue N-acetylneuraminic acid-9-phosphate (NeuNAc-9-P) synthase uses N-acetylmannosamine-6-phosphate in the condensation reaction to produce NeuNAc-9-P. Both subfamilies of sialic acid synthases possess N-terminal triosephosphate isomerase barrel domain and C-terminal antifreeze protein domain. We report cloning of the genes, expression, purification, and characterization of human NeuNAc-9-P synthase and N. meningitidis NeuNAc synthase. Stability of the purified enzymes and effects of pH and temperature on their activities were evaluated. Enzyme kinetics and preliminary mutagenesis experiments reveal the importance of C-terminal antifreeze protein domain and a conserved cysteine residue for the enzyme activities.     

The precursor of a psychrophilic alpha-amylase: structural characterization and insights into cold adaptation

The alpha-amylase precursor from the bacterium Pseudoalteromonas haloplanktis possesses a propeptide at the C-terminus possibly responsible for outer membrane translocation. Unlike the predicted beta-barrel of autotransporters, this C-terminal propeptide displays a noticeable alpha-helix content. It is connected to the enzyme by a disordered linker and has no significant interaction with the catalytic domain. The microcalorimetric pattern of the precursor also demonstrates that the stability of protein domains may evolve differently.     

Cloning, expression, and preliminary structural characterization of RTN-1C

Reticulons (RTNs) are endoplasmic reticulum-associated proteins widely distributed in plants, yeast, and animals. They are characterized by unique N-terminal parts and a common 200 amino acid C-terminal domain containing two long hydrophobic sequences. Despite their implication in many cellular processes, their molecular structure and function are still largely unknown. In this study, the reticulon family member RTN-1C has been expressed and purified in Escherichia coli and its molecular structure has been analysed by fluorescence and CD spectroscopy in different detergents in order to obtain a good solubility and a relative stability. The isotopically enriched protein has been also produced to perform structural studies by NMR spectroscopy. The preliminary results obtained showed that RTN-1C protein possesses helical transmembrane segments when a membrane-like environment is produced by detergents. Moreover, fluorescence experiments indicated the exposure of tryptophan side chains as predicted by structure prediction programs. We also produced the isotopically labelled protein and the procedure adopted allowed us to plan future NMR studies to investigate the biochemical behaviour of reticulon-1C and of its peptides spanning out from the membrane.     

Picking a winner: new mechanistic insights into the design of effective siRNAs

Recent work has shown that the efficacy of a small interfering RNA (siRNA) for silencing gene expression is a function of how easy it is to unwind the siRNA from the 5'-antisense end. Based on these insights, one group has designed an algorithm that substantially improves the odds of picking an effective siRNA, and two groups have shown that 'forked' or 'frayed' siRNAs, which should be easier to unwind from the 5'-antisense end, are more effective than conventional siRNAs. These strategies represent important steps towards the rational design of effective siRNAs.