Evolution of enzymatic activities in the enolase superfamily: crystallographic and mutagenesis studies of the reaction catalyzed by D-glucarate dehydratase from Escherichia coli

D-Glucarate dehydratase (GlucD) from Escherichia coli catalyzes the dehydration of both D-glucarate and L-idarate as well as their interconversion via epimerization. GlucD is a member of the mandelate racemase (MR) subgroup of the enolase superfamily, the members of which catalyze reactions that are initiated by abstraction of the alpha-proton of a carboxylate anion substrate. Alignment of the sequence of GlucD with that of MR reveals a conserved Lys-X-Lys motif and a His-Asp dyad homologous to the S- and R-specific bases in the active site of MR. Crystals of GlucD have been obtained into which the substrate D-glucarate and two competitive inhibitors, 4-deoxy-D-glucarate and xylarohydroxamate, could be diffused; D-glucarate is converted to the dehydration product, 5-keto-4-deoxy-D-glucarate (KDG). The structures of these complexes have been determined and reveal the identities of the ligands for the required Mg(2+) (Asp(235), Glu(266), and Asn(289)) as well as confirm the expected presence of Lys(207) and His(339), the catalytic bases that are properly positioned to abstract the proton from C5 of L-idarate and D-glucarate, respectively. Surprisingly, the C6 carboxylate group of KDG is a bidentate ligand to the Mg(2+), with the resulting geometry of the bound KDG suggesting that stereochemical roles of Lys(207) and His(339) are reversed from the predictions made on the basis of the established structure-function relationships for the MR-catalyzed reaction. The catalytic roles of these residues have been examined by characterization of mutant enzymes, although we were unable to use these to demonstrate the catalytic independence of Lys(207) and His(339) as was possible for the homologous Lys(166) and His(297) in the MR-catalyzed reaction.     

Evolution of enzymatic activities in the enolase superfamily: L-rhamnonate dehydratase

The l-rhamnonate dehydratase (RhamD) function was assigned to a previously uncharacterized family in the mechanistically diverse enolase superfamily that is encoded by the genome of Escherichia coli K-12. We screened a library of acid sugars to discover that the enzyme displays a promiscuous substrate specificity: l-rhamnonate (6-deoxy- l-mannonate) has the "best" kinetic constants, with l-mannonate, l-lyxonate, and d-gulonate dehydrated less efficiently. Crystal structures of the RhamDs from both E. coli K-12 and Salmonella typhimurium LT2 (95% sequence identity) were obtained in the presence of Mg (2+); the structure of the RhamD from S. typhimurium was also obtained in the presence of 3-deoxy- l-rhamnonate (obtained by reduction of the product with NaBH 4). Like other members of the enolase superfamily, RhamD contains an N-terminal alpha + beta capping domain and a C-terminal (beta/alpha) 7beta-barrel (modified TIM-barrel) catalytic domain with the active site located at the interface between the two domains. In contrast to other members, the specificity-determining "20s loop" in the capping domain is extended in length and the "50s loop" is truncated. The ligands for the Mg (2+) are Asp 226, Glu 252 and Glu 280 located at the ends of the third, fourth and fifth beta-strands, respectively. The active site of RhamD contains a His 329-Asp 302 dyad at the ends of the seventh and sixth beta-strands, respectively, with His 329 positioned to function as the general base responsible for abstraction of the C2 proton of l-rhamnonate to form a Mg (2+)-stabilized enediolate intermediate. However, the active site does not contain other acid/base catalysts that have been implicated in the reactions catalyzed by other members of the MR subgroup of the enolase superfamily. Based on the structure of the liganded complex, His 329 also is expected to function as the general acid that both facilitates departure of the 3-OH group in a syn-dehydration reaction and delivers a proton to carbon-3 to replace the 3-OH group with retention of configuration.     

Structures of bacterial polynucleotide kinase in a Michaelis complex with GTP?Mg2+ and 5′-OH oligonucleotide and a product complex with GDP?Mg2+ and 5′-PO4 oligonucleotide reveal a mechanism of general acid-base catalysis and the determinants of phosphoacceptor recognition

Clostridium thermocellum polynucleotide kinase (CthPnk), the 5′ end-healing module of a bacterial RNA repair system, catalyzes reversible phosphoryl transfer from an NTP donor to a 5′-OH polynucleotide acceptor. Here we report the crystal structures of CthPnk-D38N in a Michaelis complex with GTP•Mg²⁺ and a 5′-OH oligonucleotide and a product complex with GDP•Mg²⁺ and a 5′-PO₄ oligonucleotide. The O5′ nucleophile is situated 3.0 Å from the GTP γ phosphorus in the Michaelis complex, where it is coordinated by Asn38 and is apical to the bridging β phosphate oxygen of the GDP leaving group. In the product complex, the transferred phosphate has undergone stereochemical inversion and Asn38 coordinates the 5′-bridging phosphate oxygen of the oligonucleotide. The D38N enzyme is poised for catalysis, but cannot execute because it lacks Asp38—hereby implicated as the essential general base catalyst that abstracts a proton from the 5′-OH during the kinase reaction. Asp38 serves as a general acid catalyst during the ‘reverse kinase’ reaction by donating a proton to the O5′ leaving group of the 5′-PO₄ strand. The acceptor strand binding mode of CthPnk is distinct from that of bacteriophage T4 Pnk.     

Crystal structure of pyridoxine 4-oxidase from Mesorhizobium loti

Pyridoxine 4-oxidase (PNOX) from Mesorhizobium loti is a monomeric glucose–methanol–choline (GMC) oxidoreductase family enzyme, catalyzes FAD-dependent oxidation of pyridoxine (PN) into pyridoxal, and is the first enzyme in pathway I for the degradation of PN. The tertiary structures of PNOX with a C-terminal His₆-tag and PNOX–pyridoxamine (PM) complex were determined at 2.2 Å and at 2.1 Å resolutions, respectively. The overall structure consisted of FAD-binding and substrate-binding domains. In the active site, His460, His462, and Pro504 were located on the re-face of the isoalloxazine ring of FAD. PM binds to the active site through several hydrogen bonds. The side chains of His462 and His460 are located at 2.7 and 3.1 Å from the N4′ atom of PM. The activities of His460Ala and His462Ala mutant PNOXs were very low, and 460Ala/His462Ala double mutant PNOX exhibited no activity. His462 may act as a general base for the abstraction of a proton from the 4′-hydroxyl of PN. His460 may play a role in the binding and positioning of PN. The C4′ atom in PM is located at 3.2 Å, and the hydride ion from the C4′ atom may be transferred to the N5 atom of the isoalloxazine ring. The comparison of active site residues in GMC oxidoreductase shows that Pro504 in PNOX corresponds to Asn or His of the conserved His–Asn or His–His pair in other GMC oxidoreductases. The function of the novel proline residue was discussed.     

Chemical mechanism of DNA cleavage by the homing endonuclease I-PpoI

Homing endonucleases are distinguished by their ability to catalyze the cleavage of double-stranded DNA with extremely high specificity. I-PpoI endonuclease, a homing endonuclease from the slime mold Physarum polycephalum, is a small enzyme (2 x 20 kDa) of known three-dimensional structure that catalyzes the cleavage of a long target DNA sequence (15 base pairs). Here, a detailed chemical mechanism for catalysis of DNA cleavage by I-PpoI endonuclease is proposed and tested by creating six variants in which active-site residues are replaced with alanine. The side chains of three residues (Arg61, His98, and Asn119) are found to be important for efficient catalysis of DNA cleavage. This finding is consistent with the proposed mechanism in which His98 abstracts a proton from an attacking water molecule bound by an adjacent phosphoryl oxygen, Arg61 and Asn119 stabilize the pentavalent transition state, and Asn119 also binds to the essential divalent metal cation (e.g., Mg(2+) ion), which interacts with the 3'-oxygen leaving group. Because Mg(2+) is required for cleavage of a substrate with a good leaving group (p-nitrophenolate), Mg(2+) likely stabilizes the pentavalent transition state. The pH-dependence of k(cat) for catalysis by I-PpoI reveals a macroscopic pK(a) of 8.4 for titratable groups that modulate product release. I-PpoI appears to be unique among known restriction endonucleases and homing endonucleases in its use of a histidine residue to activate the attacking water molecule for in-line displacement of the 3'-leaving group.     

Mechanistic diversity in the RuBisCO superfamily: the "enolase" in the methionine salvage pathway in Geobacillus kaustophilus

D-Ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO), the most abundant enzyme, is the paradigm member of the recently recognized mechanistically diverse RuBisCO superfamily. The RuBisCO reaction is initiated by abstraction of the proton from C3 of the d-ribulose 1,5-bisphosphate substrate by a carbamate oxygen of carboxylated Lys 201 (spinach enzyme). Heterofunctional homologues of RuBisCO found in species of Bacilli catalyze the tautomerization ("enolization") of 2,3-diketo-5-methylthiopentane 1-phosphate (DK-MTP 1-P) in the methionine salvage pathway in which 5-methylthio-d-ribose (MTR) derived from 5'-methylthioadenosine is converted to methionine [Ashida, H., Saito, Y., Kojima, C., Kobayashi, K., Ogasawara, N., and Yokota, A. (2003) A functional link between RuBisCO-like protein of Bacillus and photosynthetic RuBisCO, Science 302, 286-290]. The reaction catalyzed by this "enolase" is accomplished by abstraction of a proton from C1 of the DK-MTP 1-P substrate to form the tautomerized product, a conjugated enol. Because the RuBisCO- and "enolase"-catalyzed reactions differ in the regiochemistry of proton abstraction but are expected to share stabilization of an enolate anion intermediate by coordination to an active site Mg2+, we sought to establish structure-function relationships for the "enolase" reaction so that the structural basis for the functional diversity could be established. We determined the stereochemical course of the reaction catalyzed by the "enolases" from Bacillus subtilis and Geobacillus kaustophilus. Using stereospecifically deuterated samples of an alternate substrate derived from d-ribose (5-OH group instead of the 5-methylthio group in MTR) as well as of the natural DK-MTP 1-P substrate, we determined that the "enolase"-catalyzed reaction involves abstraction of the 1-proS proton. We also determined the structure of the activated "enolase" from G. kaustophilus (carboxylated on Lys 173) liganded with Mg2+ and 2,3-diketohexane 1-phosphate, a stable alternate substrate. The stereospecificity of proton abstraction restricts the location of the general base to the N-terminal alpha+beta domain instead of the C-terminal (beta/alpha)8-barrel domain that contains the carboxylated Lys 173. Lys 98 in the N-terminal domain, conserved in all "enolases", is positioned to abstract the 1-proS proton. Consistent with this proposed function, the K98A mutant of the G. kaustophilus "enolase" is unable to catalyze the "enolase" reaction. Thus, we conclude that this functionally divergent member of the RuBisCO superfamily uses the same structural strategy as RuBisCO for stabilizing the enolate anion intermediate, i.e., coordination to an essential Mg2+, but the proton abstraction is catalyzed by a different general base.     

Evolution of enzymatic activities in the enolase superfamily: D-tartrate dehydratase from Bradyrhizobium japonicum

We focus on the assignment of function to and elucidation of structure-function relationships for a member of the mechanistically diverse enolase superfamily encoded by the Bradyrhizobium japonicum genome (bll6730; GI:27381841). As suggested by sequence alignments, the active site contains the same functional groups found in the active site of mandelate racemase (MR) that catalyzes a 1,1-proton transfer reaction: two acid/base catalysts, Lys 184 at the end of the second beta-strand, and a His 322-Asp 292 dyad at the ends of the seventh and sixth beta-strands, respectively, as well as ligands for an essential Mg2+, Asp 213, Glu 239, and Glu 265 at the ends of the third, fourth, and fifth beta-strands, respectively. We screened a library of 46 acid sugars and discovered that only d-tartrate is dehydrated, yielding oxaloacetate as product. The kinetic constants (kcat = 7.3 s(-1); kcat/KM = 8.5 x 10(4) M(-1) s(-1)) are consistent with assignment of the d-tartrate dehydratase (TarD) function. The kinetic phenotypes of mutants as well as the structures of liganded complexes are consistent with a mechanism in which Lys 184 initiates the reaction by abstraction of the alpha-proton to generate a Mg2+-stabilized enediolate intermediate, and the vinylogous beta-elimination of the 3-OH group is general acid-catalyzed by the His 322, accomplishing the anti-elimination of water. The replacement of the leaving group by solvent-derived hydrogen is stereorandom, suggesting that the enol tautomer of oxaloacetate is the product; this expectation was confirmed by its observation by 1H NMR spectroscopy. Thus, the TarD-catalyzed reaction is a "simple" extension of the two-step reaction catalyzed by MR: base-catalyzed proton abstraction to generate a Mg2+-stabilized enediolate intermediate followed by acid-catalyzed decomposition of that intermediate to yield the product.     

Insight into the phosphodiesterase mechanism from combined QM/MM free energy simulations

Molecular dynamics simulations employing a combined quantum mechanical and molecular mechanical potential have been carried out to elucidate the reaction mechanism of the hydrolysis of a cyclic nucleotide cAMP substrate by phosphodiesterase 4B (PDE4B). PDE4B is a member of the PDE superfamily of enzymes that play crucial roles in cellular signal transduction. We have determined a two-dimensional potential of mean force (PMF) for the coupled phosphoryl bond cleavage and proton transfer through a general acid catalysis mechanism in PDE4B. The results indicate that the ring-opening process takes place through an S(N)2 reaction mechanism, followed by a proton transfer to stabilize the leaving group. The computed free energy of activation for the PDE4B-catalyzed cAMP hydrolysis is about 13 kcal·mol(-1) and an overall reaction free energy is about -17 kcal·mol(-1), both in accord with experimental results. In comparison with the uncatalyzed reaction in water, the enzyme PDE4B provides a strong stabilization of the transition state, lowering the free energy barrier by 14 kcal·mol(-1). We found that the proton transfer from the general acid residue His234 to the O3' oxyanion of the ribosyl leaving group lags behind the nucleophilic attack, resulting in a shallow minimum on the free energy surface. A key contributing factor to transition state stabilization is the elongation of the distance between the divalent metal ions Zn(2+) and Mg(2+) in the active site as the reaction proceeds from the Michaelis complex to the transition state.     

Evolution of enzymatic activities in the enolase superfamily: L-talarate/galactarate dehydratase from Salmonella typhimurium LT2

We assigned l-talarate dehydratase (TalrD) and galactarate dehydratase (GalrD) functions to a group of orthologous proteins in the mechanistically diverse enolase superfamily, focusing our characterization on the protein encoded by the Salmonella typhimurium LT2 genome (GI:16766982; STM3697). Like the homologous mandelate racemase, l-fuconate dehydratase, and d-tartrate dehydratase, the active site of TalrD/GalrD contains a general acid/base Lys 197 at the end of the second beta-strand in the (beta/alpha)7beta-barrel domain, Asp 226, Glu 252, and Glu 278 as ligands for the essential Mg2+ at the ends of the third, fourth, and fifth beta-strands, a general acid/base His 328-Asp 301 dyad at the ends of the seventh and sixth beta-strands, and an electrophilic Glu 348 at the end of the eighth beta-strand. We discovered the function of STM3697 by screening a library of acid sugars; it catalyzes the efficient dehydration of both l-talarate (kcat = 2.1 s-1, kcat/Km = 9.1 x 10(3) M-1 s-1) and galactarate (kcat = 3.5 s-1, kcat/Km = 1.1 x 10(4) M-1 s-1). Because l-talarate is a previously unknown metabolite, we demonstrated that S. typhimurium LT2 can utilize l-talarate as carbon source. Insertional disruption of the gene encoding STM3697 abolishes this phenotype; this disruption also diminishes, but does not eliminate, the ability of the organism to utilize galactarate as carbon source. The dehydration of l-talarate is accompanied by competing epimerization to galactarate; little epimerization to l-talarate is observed in the dehydration of galactarate. On the basis of (1) structures of the wild type enzyme complexed with l-lyxarohydroxamate, an analogue of the enolate intermediate, and of the K197A mutant complexed with l-glucarate, a substrate for exchange of the alpha-proton, and (2) incorporation of solvent deuterium into galactarate in competition with dehydration, we conclude that Lys 197 functions as the galactarate-specific base and His 328 functions as the l-talarate-specific base. The epimerization of l-talarate to galactarate that competes with dehydration can be rationalized by partitioning of the enolate intermediate between dehydration (departure of the 3-OH group catalyzed by the conjugate acid of His 328) and epimerization (protonation on C2 by the conjugate acid of Lys 197). The promiscuous catalytic activities discovered for STM3697 highlight the evolutionary potential of a "conserved" active site architecture.     

The His23 and Lys79 pair determines the high catalytic efficiency of the inorganic pyrophosphatase of the haloacid dehalogenase superfamily

Haloacid dehalogenase (HAD) superfamily members are mainly phosphomonoesterases, while BT2127 from Bacteroides thetaiotaomicron of the HAD superfamily is identified as an inorganic pyrophosphatase. In this study, to explore the roles of the Lys79 and His23 pair in the hydrolysis reaction of inorganic pyrophosphate (PP_i) catalyzed by BT2127, a series of models were designed. Calculations were performed by using the density functional theory (DFT) method with the dispersion energy D3-B3LYP. The His23 and Lys79 pair plays a key role in the high catalytic efficiency of BT2127 with PPi. First, the His23 and Lys79 pair prompts Asp13 to easily provide a proton to the leaving group, which remarkably reduces the energy barrier of the phospho-transfer step; then, Lys79 provides a proton to the first leaving phosphate group via His23, produces a more electrically stabilized phosphate (H₃PO₄), makes this step exothermal, and further promotes the subsequent phospho-enzyme intermediate hydrolysis. The results suggest that the Lys79-His23 pair helps BT2127 reach high catalytic efficiency by strengthening the acid catalysis. Our study provides detailed chemical insights into the evolution of the inorganic pyrophosphatase function of BT2127 from the phosphomonoesterase of the HAD superfamily and the biomimetic enzyme design.     

The structural determination of phosphosulfolactate synthase from Methanococcus jannaschii at 1.7-A resolution: an enolase that is not an enolase

Members of the enolase mechanistically diverse superfamily catalyze a wide variety of chemical reactions that are related by a common mechanistic feature, the abstraction of a proton adjacent to a carboxylate group. Recent investigations into the function and mechanism of the phosphosulfolactate synthase encoded by the ComA gene in Methanococcus jannaschii have suggested that ComA, which catalyzes the stereospecific Michael addition of sulfite to phosphoenolpyruvate to form phosphosulfolactate, may be a member of the enolase superfamily. The ComA-catalyzed reaction, the first step in the coenzyme M biosynthetic pathway, likely proceeds via a Mg2+ ion-stabilized enolate intermediate in a manner similar to that observed for members of the enolase superfamily. ComA, however, has no significant sequence similarity to any known enolase. Here we report the x-ray crystal structure of ComA to 1.7-A resolution. The overall fold for ComA is an (alpha/beta)8 barrel that assembles with two other ComA molecules to form a trimer in which three active sites are created at the subunit interfaces. From the positions of two ordered sulfate ions in the active site, a model for the binding of phosphoenolpyruvate and sulfite is proposed. Despite its mechanistic similarity to the enolase superfamily, the overall structure and active site architecture of ComA are unlike any member of the enolase superfamily, which suggests that ComA is not a member of the enolase superfamily but instead acquired an enolase-type mechanism through convergent evolution.     

The structure of 3-methylaspartase from Clostridium tetanomorphum functions via the common enolase chemical step.

Methylaspartate ammonia-lyase (3-methylaspartase, MAL; EC ) catalyzes the reversible anti elimination of ammonia from L-threo-(2S,3S)-3-methylaspartic acid to give mesaconic acid. This reaction lies on the main catabolic pathway for glutamate in Clostridium tetanomorphum. MAL requires monovalent and divalent cation cofactors for full catalytic activity. The enzyme has attracted interest because of its potential use as a biocatalyst. The structure of C. tetanomorphum MAL has been solved to 1.9-A resolution by the single-wavelength anomalous diffraction method. A divalent metal ion complex of the protein has also been determined. MAL is a homodimer with each monomer consisting of two domains. One is an alpha/beta-barrel, and the other smaller domain is mainly beta-strands. The smaller domain partially occludes the C terminus of the barrel and forms a large cleft. The structure identifies MAL as belonging to the enolase superfamily of enzymes. The metal ion site is located in a large cleft between the domains. Potential active site residues have been identified based on a combination of their proximity to a metal ion site, molecular modeling, and sequence homology. In common with all members of the enolase superfamily, the carboxylic acid of the substrate is co-ordinated by the metal ions, and a proton adjacent to a carboxylic acid group of the substrate is abstracted by a base. In MAL, it appears that Lys(331) removes the alpha-proton of methylaspartic acid. This motif is the defining mechanistic characteristic of the enolase superfamily of which all have a common fold. The degree of structural conservation is remarkable given only four residues are absolutely conserved.     

The structural basis for substrate promiscuity in 2-keto-3-deoxygluconate aldolase from the Entner-Doudoroff pathway in Sulfolobus solfataricus

The hyperthermophilic Archaea Sulfolobus solfataricus grows optimally above 80 degrees C and metabolizes glucose by a non-phosphorylative variant of the Entner-Doudoroff pathway. In this pathway glucose dehydrogenase and gluconate dehydratase catalyze the oxidation of glucose to gluconate and the subsequent dehydration of gluconate to D-2-keto-3-deoxygluconate (KDG). KDG aldolase (KDGA) then catalyzes the cleavage of KDG to D-glyceraldehyde and pyruvate. It has recently been shown that all the enzymes of this pathway exhibit a catalytic promiscuity that also enables them to be used for the metabolism of galactose. This phenomenon, known as metabolic pathway promiscuity, depends crucially on the ability of KDGA to cleave KDG and D-2-keto-3-deoxygalactonate (KDGal), in both cases producing pyruvate and D-glyceraldehyde. In turn, the aldolase exhibits a remarkable lack of stereoselectivity in the condensation reaction of pyruvate and D-glyceraldehyde, forming a mixture of KDG and KDGal. We now report the structure of KDGA, determined by multiwavelength anomalous diffraction phasing, and confirm that it is a member of the tetrameric N-acetylneuraminate lyase superfamily of Schiff base-forming aldolases. Furthermore, by soaking crystals of the aldolase at more than 80 degrees C below its temperature activity optimum, we have been able to trap Schiff base complexes of the natural substrates pyruvate, KDG, KDGal, and pyruvate plus D-glyceraldehyde, which have allowed rationalization of the structural basis of promiscuous substrate recognition and catalysis. It is proposed that the active site of the enzyme is rigid to keep its thermostability but incorporates extra functionality to be promiscuous.     

Role of His159 in yeast enolase catalysis.

There are presently several proposed catalytic mechanisms of yeast enolase, all of which have emerged from separate structural investigations of enolase from yeast and lobster muscle. However, the identities of the residues functioning as the general acid/base pair are not yet established unambiguously. In the Mn(2+)-phosphoglycolate complex of lobster muscle enolase, the imidazole group of His157 (His159 in the yeast enolase numbering system) is in van der Waals contact (4.5 A) with the C(2) of the inhibitor [Duquerroy et al. (1995) Biochemistry 34, 12513-12523]. To gain further information about the role played by His159 in the catalytic mechanism of yeast enolase this residue has been mutated to Ala. The gene encoding for the H159A mutation has been constructed and the mutant protein has been expressed in Escherichia coli. The purified mutant protein is folded properly as indicated by near- and far-UV circular dichroism and fluorescence data, and the mutation has no significant effect on the formation of ternary and quaternary enzyme-ligand complexes. In a typical assay, H159A showed 0.01% of wild-type specific activity, which corresponds to a reduction in k(cat) of 4 orders of magnitude. The H159A fails to ionize the C-2 proton of either 2-PGA or phosphoglycolate. These findings are consistent with His159 serving as a potential catalytic base in the enolase reaction. We have suggested that His159 could also serve as a metal ligand at the third, inhibitory, metal binding site. This proposal is consistent with the catalytic mechanism of yeast enolase. Binding of metal ion at site III interferes with His159 reacting as the catalytic base, i.e., abstracting the C(2) proton from 2-PGA. Metal binding studies support the above proposal. Mn(2+) binding at sites I and II for the His159Ala mutant is identical to that of wild type. The binding of Mn(2+) at the third, inhibitory site of H159A is a factor of 3 weaker compared to wild-type enolase. The factor of 3 in binding is reasonable for the contribution to binding strength of a single nondominant ligand in a chelate [Klemba, M., and Regan, L. (1995) Biochemistry 34, 10094-10100. Regan, L. (1993) Annu. Rev. Biophys. Biomol. Struct. 22, 257-281. Cha et al. (1994) J. Biol. Chem. 269, 2687-2694].     

Multiple active site histidine protonation states in Acetobacter aceti N5-carboxyaminoimidazole ribonucleotide mutase detected by REDOR NMR

Class I PurE (N5-carboxyaminoimidazole mutase) catalyzes a chemically unique mutase reaction. A working mechanistic hypothesis involves a histidine (His45 in Escherichia coli PurE) functioning as a general acid, but no evidence for multiple protonation states has been obtained. Solution NMR is a peerless tool for this task but has had limited application to enzymes, most of which are larger than its effective molecular size limit. Solid-state NMR is not subject to this limit. REDOR NMR studies of a 151 kDa complex of uniformly 15N-labeled Acetobacter aceti PurE (AaPurE) and the active site ligand [6-13C]citrate probed a single ionization equilibrium associated with the key histidine (AaPurE His59). In the AaPurE complex, the citrate central carboxylate C6 13C peak moves upfield, indicating diminution of negative charge, and broadens, indicating heterogeneity. Histidine 15N chemical shifts indicate His59 exists in approximately equimolar amounts of an Ndelta-unprotonated (pyridine-like) form and an Ndelta-protonated (pyrrole-like) form, each of which is approximately 4 A from citrate C6. The spectroscopic data are consistent with proton transfers involving His59 Ndelta that are invoked in the class I PurE mechanism.     

Analysis of the active-site mechanism of tyrosyl-DNA phosphodiesterase I: a member of the phospholipase D superfamily

Tyrosyl-DNA phosphodiesterase I (Tdp1) is a member of the phospholipase D superfamily that hydrolyzes 3'-phospho-DNA adducts via two conserved catalytic histidines-one acting as the lead nucleophile and the second acting as a general acid/base. Substitution of the second histidine specifically to arginine contributes to the neurodegenerative disease spinocerebellar ataxia with axonal neuropathy (SCAN1). We investigated the catalytic role of this histidine in the yeast protein (His432) using a combination of X-ray crystallography, biochemistry, yeast genetics, and theoretical chemistry. The structures of wild-type Tdp1 and His432Arg both show a phosphorylated form of the nucleophilic histidine that is not observed in the structure of His432Asn. The phosphohistidine is stabilized in the His432Arg structure by the guanidinium group that also restricts the access of nucleophilic water molecule to the Tdp1-DNA intermediate. Biochemical analyses confirm that His432Arg forms an observable and unique Tdp1-DNA adduct during catalysis. Substitution of His432 by Lys does not affect catalytic activity or yeast phenotype, but substitutions with Asn, Gln, Leu, Ala, Ser, and Thr all result in severely compromised enzymes and DNA topoisomerase I-camptothecin dependent lethality. Surprisingly, His432Asn did not show a stable covalent Tdp1-DNA intermediate that suggests another catalytic defect. Theoretical calculations revealed that the defect resides in the nucleophilic histidine and that the pK(a) of this histidine is crucially dependent on the second histidine and on the incoming phosphate of the substrate. This represents a unique example of substrate-activated catalysis that applies to the entire phospholipase D superfamily.     

Analysis of the roles of amino acid residues in the flavoprotein tryptophan 2-monooxygenase modified by 2-oxo-3-pentynoate: characterization of His338, Cys339, and Cys511 mutant enzymes

The flavoprotein tryptophan 2-monooxygenase catalyzes the oxidative decarboxylation of tryptophan to indoleacetamide. His338, Cys339, and Cys511 of the Pseudomonas savastanoi enzyme were previously identified as possible active-site residues by modification with 2-oxo-3-pentynoate ([G. Gadda, L.J. Dangott, W.H. Johnson Jr., C.P. Whitman, P.F. Fitzpatrick, Biochemistry 38 (1999) 5822-5828]). The H338N, C339A, and C511S enzymes have been characterized to determine the roles of these residues in catalysis. The steady-state kinetic parameters with both tryptophan and methionine decrease only slightly in the case of the H338N and C339A enzymes; the decrease in activity is greater for the C511S enzyme. Only in the case of the C511S enzyme do deuterium kinetic isotope effects on kinetic parameters indicate a significant change in catalytic rates. The structural bases for the effects of the mutations can be interpreted by identification of L-amino acid oxidase and tryptophan monooxygenase as homologous proteins.     

Structural and kinetic evidence for an extended hydrogen-bonding network in catalysis of methyl group transfer. Role of an active site asparagine residue in activation of methyl transfer by methyltransferases

The methyltetrahydrofolate (CH(3)-H(4)folate) corrinoid-iron-sulfur protein (CFeSP) methyltransferase (MeTr) catalyzes transfer of the methyl group of CH(3)-H(4)folate to cob(I)amide. This key step in anaerobic CO and CO(2) fixation is similar to the first half-reaction in the mechanisms of other cobalamin-dependent methyltransferases. Methyl transfer requires electrophilic activation of the methyl group of CH(3)-H(4)folate, which includes proton transfer to the N5 group of the pterin ring and poises the methyl group for reaction with the Co(I) nucleophile. The structure of the binary CH(3)-H(4)folate/MeTr complex (revealed here) lacks any obvious proton donor near the N5 group. Instead, an Asn residue and water molecules are found within H-bonding distance of N5. Structural and kinetic experiments described here are consistent with the involvement of an extended H-bonding network in proton transfer to N5 of the folate that includes an Asn (Asn-199 in MeTr), a conserved Asp (Asp-160), and a water molecule. This situation is reminiscent of purine nucleoside phosphorylase, which involves protonation of the purine N7 in the transition state and is accomplished by an extended H-bond network that includes water molecules, a Glu residue, and an Asn residue (Kicska, G. A., Tyler, P. C., Evans, G. B., Furneaux, R. H., Shi, W., Fedorov, A., Lewandowicz, A., Cahill, S. M., Almo, S. C., and Schramm, V. L. (2002) Biochemistry 41, 14489-14498). In MeTr, the Asn residue swings from a distant position to within H-bonding distance of the N5 atom upon CH(3)-H(4)folate binding. An N199A variant exhibits only approximately 20-fold weakened affinity for CH(3)-H(4)folate but a much more marked 20,000-40,000-fold effect on catalysis, suggesting that Asn-199 plays an important role in stabilizing a transition state or high energy intermediate for methyl transfer.     

Dissection of malonyl-coenzyme A decarboxylation from polyketide formation in the reaction mechanism of a plant polyketide synthase

Chalcone synthase (CHS) catalyzes formation of the phenylpropanoid chalcone from one p-coumaroyl-CoA and three malonyl-coenzyme A (CoA) thioesters. The three-dimensional structure of CHS [Ferrer, J.-L., Jez, J. M., Bowman, M. E., Dixon, R. A., and Noel, J. P. (1999) Nat. Struct. Biol. 6, 775-784] suggests that four residues (Cys164, Phe215, His303, and Asn336) participate in the multiple decarboxylation and condensation reactions catalyzed by this enzyme. Here, we functionally characterize 16 point mutants of these residues for chalcone production, malonyl-CoA decarboxylation, and the ability to bind CoA and acetyl-CoA. Our results confirm Cys164's role as the active-site nucleophile in polyketide formation and elucidate the importance of His303 and Asn336 in the malonyl-CoA decarboxylation reaction. We suggest that Phe215 may help orient substrates at the active site during elongation of the polyketide intermediate. To better understand the structure-function relationships in some of these mutants, we also determined the crystal structures of the CHS C164A, H303Q, and N336A mutants refined to 1.69, 2.0, and 2.15 A resolution, respectively. The structure of the C164A mutant reveals that the proposed oxyanion hole formed by His303 and Asn336 remains undisturbed, allowing this mutant to catalyze malonyl-CoA decarboxylation without chalcone formation. The structures of the H303Q and N336A mutants support the importance of His303 and Asn336 in polarizing the thioester carbonyl of malonyl-CoA during the decarboxylation reaction. In addition, both of these residues may also participate in stabilizing the tetrahedral transition state during polyketide elongation. Conservation of the catalytic functions of the active-site residues may occur across a wide variety of condensing enzymes, including other polyketide and fatty acid synthases.     

Identification of functional amino acids in the Nramp family by a combination of evolutionary analysis and biophysical studies of metal and proton cotransport in vivo

The natural resistance-associated macrophage protein (Nramp) family is functionally conserved in bacteria and eukarya; Nramp homologues function as proton-dependent membrane transporters of divalent metals. Sequence analyses indicate that five phylogenetic groups comprise the Nramp family, three bacterial and two eukaryotic, which are distinct from a more distantly related group of microbial sequences (Nramp outgroup). The Nramp family and outgroup share many conserved residues, suggesting they derived from a common ancestor and raising the possibility that the residues invariant in the Nramp family that correspond to residues which are different but also conserved in the outgroup represent candidate sites of functional divergence of the Nramp family. Four Nramp family-specific residues were identified within transmembrane domains 1, 6, and 11, and replaced by the corresponding invariant outgroup residues in the Escherichia coli Nramp ortholog (the proton-dependent manganese transporter, MntH of group A, EcoliA). The resulting mutants (Asp(34)Gly, Asn(37)Thr, His(211)Tyr, and Asn(401)Gly) were tested for both divalent metal uptake and proton transport; quasi-simultaneous analyses of uptake of metals and protons revealed for the first time protons and metals cotransport by a bacterial Nramp homologue. Additional mutations were studied for comparison (Asp(34)Asn, Asn(37)Asp and Asn(37)Val, Asn(401)Thr, His(211)Ala, His(216)Ala, and His(216)Arg). EcoliA activity was impaired after each of the Nramp/outgroup substitutions, as well as after more conservative replacements, showing that the tested sites are all important for metal uptake and metal-dependent H(+) transport. It is proposed that co-occurrence of these four Nramp-specific transmembrane residues may have contributed to the emergence of this family of metal and proton cotransporters.