The crystal structure of bacillus cereus phosphonoacetaldehyde hydrolase: insight into catalysis of phosphorus bond cleavage and catalytic diversification within the HAD enzyme superfamily

Phosphonoacetaldehyde hydrolase (phosphonatase) catalyzes the hydrolysis of phosphonoacetaldehyde to acetaldehyde and phosphate using Mg(II) as cofactor. The reaction proceeds via a novel bicovalent catalytic mechanism in which an active-site nucleophile abstracts the phosphoryl group from the Schiff-base intermediate formed from Lys53 and phosphonoacetaldehyde. In this study, the X-ray crystal structure of the Bacillus cereus phosphonatase homodimer complexed with the phosphate (product) analogue tungstate (K(i) = 50 microM) and the Mg(II) cofactor was determined to 3.0 A resolution with an R(cryst) = 0.248 and R(free) = 0.284. Each monomer is made up of an alpha/beta core domain consisting of a centrally located six-stranded parallel beta-sheet surrounded by six alpha-helices. Two flexible, solvated linkers connect to a small cap domain (residues 21-99) that consists of an antiparallel, five-helix bundle. The subunit-subunit interface, formed by the symmetrical packing of the two alpha8 helices from the respective core domains, is stabilized through the hydrophobic effect derived from the desolvation of paired Met171, Trp164, Tyr162, Tyr167, and Tyr176 side chains. The active site is located at the domain-domain interface of each subunit. The Schiff base forming Lys53 is positioned on the cap domain while tungstate and Mg(II) are bound to the core domain. Mg(II) ligands include two oxygens of the tungstate ligand, one oxygen of the carboxylates of Asp12 and Asp186, the backbone carbonyl oxygen of Ala14, and a water that forms a hydrogen bond with the carboxylate of Asp190 and Thr187. The guanidinium group of Arg160 binds tungstate and the proposed nucleophile Asp12, which is suitably positioned for in-line attack at the tungsten atom. The side chains of the core domain residue Tyr128 and the cap domain residues Cys22 and Lys53 are located nearby. The identity of Asp12 as the active-site nucleophile was further evidenced by the observed removal of catalytic activity resulting from Asp12Ala substitution. The similarity of backbone folds observed in phosphonatase and the 2-haloacid dehalogenase of the HAD enzyme superfamily indicated common ancestry. Superposition of the two structures revealed a conserved active-site scaffold having distinct catalytic stations. Analysis of the usage of polar amino acid residues at these stations by the dehalogenases, phosphonatases, phosphatases, and phosphomutases of the HAD superfamily suggests possible ways in which the active site of an ancient enzyme ancestor might have been diversified for catalysis of C-X, P-C, and P-O bond cleavage reactions.     

The X-ray crystallographic structure and activity analysis of a Pseudomonas-specific subfamily of the HAD enzyme superfamily evidences a novel biochemical function

The haloacid dehalogenase (HAD) superfamily is a large family of proteins dominated by phosphotransferases. Thirty-three sequence families within the HAD superfamily (HADSF) have been identified to assist in function assignment. One such family includes the enzyme phosphoacetaldehyde hydrolase (phosphonatase). Phosphonatase possesses the conserved Rossmanniod core domain and a C1-type cap domain. Other members of this family do not possess a cap domain and because the cap domain of phosphonatase plays an important role in active site desolvation and catalysis, the function of the capless family members must be unique. A representative of the capless subfamily, PSPTO_2114, from the plant pathogen Pseudomonas syringae, was targeted for catalytic activity and structure analyses. The X-ray structure of PSPTO_2114 reveals a capless homodimer that conserves some but not all of the intersubunit contacts contributed by the core domains of the phosphonatase homodimer. The region of the PSPTO_2114 that corresponds to the catalytic scaffold of phosphonatase (and other HAD phosphotransfereases) positions amino acid residues that are ill suited for Mg+2 cofactor binding and mediation of phosphoryl group transfer between donor and acceptor substrates. The absence of phosphotransferase activity in PSPTO_2114 was confirmed by kinetic assays. To explore PSPTO_2114 function, the conservation of sequence motifs extending outside of the HADSF catalytic scaffold was examined. The stringently conserved residues among PSPTO_2114 homologs were mapped onto the PSPTO_2114 three-dimensional structure to identify a surface region unique to the family members that do not possess a cap domain. The hypothesis that this region is used in protein-protein recognition is explored to define, for the first time, HADSF proteins which have acquired a function other than that of a catalyst.     

Diversification of function in the haloacid dehalogenase enzyme superfamily: The role of the cap domain in hydrolytic phosphoruscarbon bond cleavage

Phosphonatase functions in the 2-aminoethylphosphonate (AEP) degradation pathway of bacteria, catalyzing the hydrolysis of the C-P bond in phosphonoacetaldehyde (Pald) via formation of a bi-covalent Lys53ethylenamine/Asp12 aspartylphosphate intermediate. Because phosphonatase is a member of the haloacid dehalogenase superfamily, a family predominantly comprised of phosphatases, the question arises as to how this new catalytic activity evolved. The source of general acid-base catalysis for Schiff-base formation and aspartylphosphate hydrolysis was probed using pH-rate profile analysis of active-site mutants and X-ray crystallographic analysis of modified forms of the enzyme. The 2.9 A X-ray crystal structure of the mutant Lys53Arg complexed with Mg2+ and phosphate shows that the equilibrium between the open and the closed conformation is disrupted, favoring the open conformation. Thus, proton dissociation from the cap domain Lys53 is required for cap domain-core domain closure. The likely recipient of the Lys53 proton is a water-His56 pair that serves to relay the proton to the carbonyl oxygen of the phosphonoacetaldehyde (Pald) substrate upon addition of the Lys53. The pH-rate profile analysis of active-site mutants was carried out to test this proposal. The proximal core domain residues Cys22 and Tyr128 were ruled out, and the role of cap domain His56 was supported by the results. The X-ray crystallographic structure of wild-type phosphonatase reduced with NaBH4 in the presence of Pald was determined at 2.4A resolution to reveal N epsilon-ethyl-Lys53 juxtaposed with a sulfate ligand bound in the phosphate site. The position of the C2 of the N-ethyl group in this structure is consistent with the hypothesis that the cap domain N epsilon-ethylenamine-Lys53 functions as a general base in the hydrolysis of the aspartylphosphate bi-covalent enzyme intermediate. Because the enzyme residues proposed to play a key role in P-C bond cleavage are localized on the cap domain, this domain appears to have evolved to support the diversification of the HAD phosphatase core domain for catalysis of hydrolytic P-C bond cleavage.     

Analysis of the substrate specificity loop of the HAD superfamily cap domain

The haloacid dehalogenase (HAD) superfamily includes a variety of enzymes that catalyze the cleavage of substrate C-Cl, P-C, and P-OP bonds via nucleophilic substitution pathways. All members possess the alpha/beta core domain, and many also possess a small cap domain. The active site of the core domain is formed by four loops (corresponding to sequence motifs 1-4), which position substrate and cofactor-binding residues as well as the catalytic groups that mediate the "core" chemistry. The cap domain is responsible for the diversification of chemistry within the family. A tight beta-turn in the helix-loop-helix motif of the cap domain contains a stringently conserved Gly (within sequence motif 5), flanked by residues whose side chains contribute to the catalytic site formed at the domain-domain interface. To define the role of the conserved Gly in the structure and function of the cap domain loop of the HAD superfamily members phosphonoacetaldehyde hydrolase and beta-phosphoglucomutase, the Gly was mutated to Pro, Val, or Ala. The catalytic activity was severely reduced in each mutant. To examine the impact of Gly substitution on loop 5 conformation, the X-ray crystal structure of the Gly50Pro phosphonoacetaldehyde hydrolase mutant was determined. The altered backbone conformation at position 50 had a dramatic effect on the spatial disposition of the side chains of neighboring residues. Lys53, the Schiff Base forming lysine, had rotated out of the catalytic site and the side chain of Leu52 had moved to fill its place. On the basis of these studies, it was concluded that the flexibility afforded by the conserved Gly is critical to the function of loop 5 and that it is a marker by which the cap domain substrate specificity loop can be identified within the amino acid sequence of HAD family members.     

Caught in the act: the structure of phosphorylated beta-phosphoglucomutase from Lactococcus lactis

Phosphoglucomutases catalyze the interconversion of D-glucose 1-phosphate and D-glucose 6-phosphate, a reaction central to energy metabolism in all cells and to the synthesis of cell wall polysaccharides in bacterial cells. Two classes of phosphoglucomutases (alpha-PGM and beta-PGM) are distinguished on the basis of their specificity for alpha- and beta-glucose-1-phosphate. beta-PGM is a member of the haloacid dehalogenase (HAD) superfamily, which includes the sarcoplasmic Ca(2+)-ATPase, phosphomannomutase, and phosphoserine phosphatase. beta-PGM is unusual among family members in that the common phosphoenzyme intermediate exists as a stable ground-state complex in this enzyme. Herein we report, for the first time, the three-dimensional structure of a beta-PGM and the first view of the true phosphoenzyme intermediate in the HAD superfamily. The crystal structure of the Mg(II) complex of phosphorylated beta-phosphoglucomutase (beta-PGM) from Lactococcus lactis has been determined to 2.3 A resolution by multiwavelength anomalous diffraction (MAD) phasing on selenomethionine, and refined to an R(cryst) = 0.24 and R(free) = 0.28. The active site of beta-PGM is located between the core and the cap domain and is freely solvent accessible. The residues within a 6 A radius of the phosphorylated Asp8 include Asp10, Thr16, Ser114, Lys145, Glu169, and Asp170. The cofactor Mg(2+) is liganded with octahedral coordination geometry by the carboxylate side chains of Asp8, Glu169, Asp170, and the backbone carbonyl oxygen of Asp10 along with one oxygen from the Asp8-phosphoryl group and one water ligand. The phosphate group of the phosphoaspartyl residue, Asp8, interacts with the side chains of Ser114 and Lys145. The absence of a base residue near the aspartyl phosphate group accounts for the persistence of the phosphorylated enzyme under physiological conditions. Substrate docking shows that glucose-6-P can bind to the active site of phosphorylated beta-PGM in such a way as to position the C(1)OH near the phosphoryl group of the phosphorylated Asp8 and the C(6) phosphoryl group near the carboxylate group of Asp10. This result suggests a novel two-base mechanism for phosphoryl group transfer in a phosphorylated sugar.     

Investigation of the Bacillus cereus phosphonoacetaldehyde hydrolase. Evidence for a Schiff base mechanism and sequence analysis of an active-site peptide containing the catalytic lysine residue

Reaction of Bacillus cereus phosphonoacetaldehyde hydrolase (phosphonatase) with phosphonoacetaldehyde or acetaldehyde in the presence of NaBH4 resulted in complete loss of enzymatic activity. Treatment of phosphonatase with NaBH4 in the absence of substrate or product had no effect on catalysis. Inactivation of phosphonatase with [3H]NaBH4 and phosphonoacetaldehyde, NaBH4 and [14C]acetaldehyde, or NaBH4 and [2-3H]phosphonoacetaldehyde produced in each instance radiolabeled enzyme. The nature of the covalent modification was investigated by digesting the radiolabeled enzyme preparations with trypsin and by separating the tryptic peptides with HPLC. Analysis of the peptide fractions revealed that incorporation of the 3H- or 14C-radiolabel into the protein was reasonably selective for an amino acid residue found in a peptide fragment observed in each of the three trypsin digests. Sequence analysis of the 3H-labeled peptide fragment isolated from the digest of the [2-3H]phosphonoacetaldehyde/NaBH4-treated enzyme identified N epsilon-ethyllysine as the radiolabeled amino acid. The ability of the phosphonatase competitive inhibitor (Ki = 230 +/- 20 microM) acetonylphosphonate to protect the enzyme from phosphonoacetaldehyde/NaBH4-induced inactivation suggested that the reactive lysine residue is located in the enzyme active site. Comparison of the relative effectiveness of phosphonoacetaldehyde and acetaldehyde as phosphonatase inactivators showed that the N-ethyllysine imine that is reduced by the NaBH4 is derived from the corresponding N-(phosphonoethyl) imine. On the basis of these findings, a catalytic mechanism for for phosphonatase is proposed in which phosphonoacetaldehyde is activated for P-C bond cleavage by formation of a Schiff base with an active-site lysine. Accordingly, an N-ethyllsysine enamine rather than the high-energy acetaldehyde enolate anion is displaced from the phosphorus.     

Protein engineering of xylose (glucose) isomerase from Actinoplanes missouriensis. 3. Changing metal specificity and the pH profile by site-directed mutagenesis.

Aldose-ketose isomerization by xylose isomerase requires bivalent cations such as Mg2+, Mn2+, or Co2+. The active site of the enzyme from Actinoplanes missouriensis contains two metal ions that are involved in substrate binding and in catalyzing a hydride shift between the C1 and C2 substrate atoms. Glu 186 is a conserved residue located near the active site but not in contact with the substrate and not with a metal ligand. The E186D and E186Q mutant enzymes were prepared. Both are active, and their metal specificity is different from that of the wild type. The E186Q enzyme is most active with Mn2+ and has a drastically shifted pH optimum. The X-ray analysis of E186Q was performed in the presence of xylose and either Mn2+ or Mg2+. The Mn2+ structure is essentially identical to that of the wild type. In the presence of Mg2+, the carboxylate group of residue Asp 255, which is part of metal site 2 and a metal ligand, turns toward Gln 186 and hydrogen bonds to its side-chain amide. Mg2+ is not bound at metal site 2, explaining the low activity of the mutant with this cation. Movements of Asp 255 also occur in the wild-type enzyme. We propose that they play a role in the O1 to O2 proton relay accompanying the hydride shift.     

The critical role of the 185-189-loop in the factor Xa interaction with Na+ and factor Va in the prothrombinase complex

The S1 site (Asp(189)) of factor Xa (fXa) is located on a loop (residues 185-189) that contains three solvent-exposed charged residues (Asp(185), Lys(186), and Glu(188)) below the active-site pocket of the protease. To investigate the role of these residues in the catalytic function of fXa, we expressed three mutants of the protease in which the charges of these residues were neutralized by their substitutions with Ala (D185A, K186A, and E188A). Kinetic studies revealed that E188A has a normal catalytic activity toward small synthetic and natural substrates and inhibitors of fXa; however, the same activities were slightly ( approximately 2-fold) and dramatically ( approximately 20-50-fold) impaired for the D185A and K186A mutants, respectively. Further studies revealed that the affinity of D185A and K186A for interaction with Na(+) has also been altered, with a modest impairment ( approximately 2-fold) for the former and a dramatic impairment for the latter mutant. Both prothrombinase and direct binding studies indicated that K186A also has an approximately 6-fold impaired affinity for factor Va. Interestingly, a saturating concentration of factor Va restored the catalytic defect of K186A in reactions with prothrombin and the recombinant tick anticoagulant peptide that is known to interact with the Na(+) loop of fXa, but not with other substrates. These results suggest that factor Va interacts with 185-189-loop for fXa, which is energetically linked to the Na(+)-binding site of the protease.     

Identification of Asp218 and Asp326 as the principal Mg2+ binding ligands of the homing endonuclease PI-SceI

The monomeric homing endonuclease PI-SceI harbors two catalytic centers which cooperate in the cleavage of the two strands of its extended recognition sequence. Structural and biochemical data suggest that catalytic center I contains Asp218, Asp229, and Lys403, while catalytic center II contains Asp326, Thr341, and Lys301. The analogy with I-CreI, for which the cocrystal structure with the DNA substrate has been determined, suggests that Asp218 and Asp229 in catalytic center I and Asp326 and Thr341 in catalytic center II serve as ligands for Mg(2+), the essential divalent metal ion cofactor which can be replaced by Mn(2+) in vitro. We have carried out a mutational analysis of these presumptive Mg(2+) ligands. The variants carrying an alanine or asparagine substitution bind DNA, but (with the exception of the D229N variant) are inactive in DNA cleavage in the presence of Mg(2+), demonstrating that these residues are important for cleavage. Our finding that the PI-SceI variants carrying single cysteine substitutions at these positions are inactive in the presence of the oxophilic Mg(2+) but active in the presence of the thiophilic Mn(2+) suggests that the amino acid residues at these positions are involved in cofactor binding. From the fact that in the presence of Mn(2+) the D218C and D326C variants are even more active than the wild-type enzyme, it is concluded that Asp218 and Asp326 are the principal Mg(2+) ligands of PI-SceI. On the basis of these findings and the available structural information, a model for the composition of the two Mg(2+) binding sites of PI-SceI is proposed.     

Catalytic cycling in beta-phosphoglucomutase: a kinetic and structural analysis

Lactococcus lactis beta-phosphoglucomutase (beta-PGM) catalyzes the interconversion of beta-d-glucose 1-phosphate (beta-G1P) and beta-d-glucose 6-phosphate (G6P), forming beta-d-glucose 1,6-(bis)phosphate (beta-G16P) as an intermediate. Beta-PGM conserves the core domain catalytic scaffold of the phosphatase branch of the HAD (haloalkanoic acid dehalogenase) enzyme superfamily, yet it has evolved to function as a mutase rather than as a phosphatase. This work was carried out to identify the structural basis underlying this diversification of function. In this paper, we examine beta-PGM activation by the Mg(2+) cofactor, beta-PGM activation by Asp8 phosphorylation, and the role of cap domain closure in substrate discrimination. First, the 1.90 A resolution X-ray crystal structure of the Mg(2+)-beta-PGM complex is examined in the context of previously reported structures of the Mg(2+)-alpha-d-galactose-1-phosphate-beta-PGM, Mg(2+)-phospho-beta-PGM, and Mg(2+)-beta-glucose-6-phosphate-1-phosphorane-beta-PGM complexes to identify conformational changes that occur during catalytic turnover. The essential role of Asp8 in nucleophilic catalysis was confirmed by demonstrating that the D8A and D8E mutants are devoid of catalytic activity. Comparison of the ligands to Mg(2+) in the different complexes shows that a single Mg(2+) coordination site must alternatively accommodate water, phosphate, and the phosphorane intermediate during catalytic turnover. Limited involvement of the HAD family metal-binding loop in Mg(2+) anchoring in beta-PGM is consistent with the relatively loose binding indicated by the large K(m) for Mg(2+) activation (270 +/- 20 microM) and with the retention of activity found in the E169A/D170A double loop mutant. Comparison of the relative positions of cap and core domains in the different complexes indicated that interaction of cap domain Arg49 with the "nontransferring" phosphoryl group of the substrate ligand might stabilize the cap-closed conformation, as required for active site desolvation and alignment of Asp10 for acid-base catalysis. Kinetic analyses of the specificity of beta-PGM toward phosphoryl group donors and the specificity of phospho-beta-PGM toward phosphoryl group acceptors were carried out. The results support a substrate induced-fit mechanism of beta-PGM catalysis, which allows phosphomutase activity to dominate over the intrinsic phosphatase activity. Last, we present evidence that the autophosphorylation of beta-PGM by the substrate beta-G1P accounts for the origin of phospho-beta-PGM in the cell.     

Kinetic and mutational analyses of the major cytosolic exopolyphosphatase from Saccharomyces cerevisiae

Yeast exopolyphosphatase (scPPX) processively splits off the terminal phosphate group from linear polyphosphates longer than pyrophosphate. scPPX belongs to the DHH phosphoesterase superfamily and is evolutionarily close to the well characterized family II pyrophosphatase (PPase). Here, we used steady-state kinetic and binding measurements to elucidate the metal cofactor requirement for scPPX catalysis over the pH range 4.2-9.5. A single tight binding site for Mg(2+) (K(d) of 24 microm) was detected by equilibrium dialysis. Steady-state kinetic analysis of tripolyphosphate hydrolysis revealed a second site that binds Mg(2+) in the millimolar range and modulates substrate binding. This step requires two protonated and two deprotonated enzyme groups with pK(a) values of 5.0-5.3 and 7.6-8.2, respectively. The catalytic step requiring two deprotonated groups (pK(a) of 4.6 and 5.6) is modulated by ionization of a third group (pK(a) of 8.7). Conservative mutations of Asp(127), His(148), His(149) (conserved in scPPX and PPase), and Asn(35) (His in PPase) reduced activity by a factor of 600-5000. N35H and D127E substitutions reduced the Mg(2+) affinity of the tight binding site by 25-60-fold. Contrary to expectations, the N35H variant was unable to hydrolyze pyrophosphate, but markedly altered metal cofactor specificity, displaying higher catalytic activity with Co(2+) bound to the weak binding site versus the Mg(2+)- or Mn(2+)-bound enzyme. These results provide an initial step toward understanding the dynamics of scPPX catalysis and reveal significant functional differences between structurally similar scPPX and family II PPase.     

Insight into the mechanism of phosphoenolpyruvate mutase catalysis derived from site-directed mutagenesis studies of active site residues

PEP mutase catalyzes the conversion of phosphoenolpyruvate (PEP) to phosphonopyruvate in biosynthetic pathways leading to phosphonate secondary metabolites. A recent X-ray structure [Huang, K., Li, Z., Jia, Y., Dunaway-Mariano, D., and Herzberg, O. (1999) Structure (in press)] of the Mytilus edulis enzyme complexed with the Mg(II) cofactor and oxalate inhibitor reveals an alpha/beta-barrel backbone-fold housing an active site in which Mg(II) is bound by the two carboxylate groups of the oxalate ligand and the side chain of D85 and, via bridging water molecules, by the side chains of D58, D85, D87, and E114. The oxalate ligand, in turn, interacts with the side chains of R159, W44, and S46 and the backbone amide NHs of G47 and L48. Modeling studies identified two feasible PEP binding modes: model A in which PEP replaces oxalate with its carboxylate group interacting with R159 and its phosphoryl group positioned close to D58 and Mg(II) shifting slightly from its original position in the crystal structure, and model B in which PEP replaces oxalate with its phosphoryl group interacting with R159 and Mg(II) retaining its original position. Site-directed mutagenesis studies of the key mutase active site residues (R159, D58, D85, D87, and E114) were carried out in order to evaluate the catalytic roles predicted by the two models. The observed retention of low catalytic activity in the mutants R159A, D85A, D87A, and E114A, coupled with the absence of detectable catalytic activity in D58A, was interpreted as evidence for model A in which D58 functions in nucleophilic catalysis (phosphoryl transfer), R159 functions in PEP carboxylate group binding, and the carboxylates of D85, D87 and E114 function in Mg(II) binding. These results also provide evidence against model B in which R159 serves to mediate the phosphoryl transfer. A catalytic motif, which could serve both the phosphoryl transfer and the C-C cleavage enzymes of the PEP mutase superfamily, is proposed.     

Structural and functional analysis of the phosphoryl transfer reaction mediated by the human small C‐terminal domain phosphatase,Scp1

Human small C‐terminal domain phosphatase 1 (Scp1) modulates the phosphorylation state of the C‐terminal domain (CTD) of eukaryotic RNA polymerase II (RNAP II), with preference for phosphorylated Ser5 in the tandem heptad repeats of the CTD. Additionally, Scp1 was identified as a conserved regulator of neuronal stem cell development. Scp1 is a member of haloacid dehalogenase (HAD) superfamily, whose catalysis depends on a Mg²⁺ ion and a DXDX(T/V) motif. The first Asp of the motif is identified as the nucleophile that is subject to phosphorylation leading to a phosphoryl‐aspartate intermediate. This high‐energy mixed anhydride intermediate is subsequently hydrolyzed to regenerate the enzyme. In the present study, we successfully captured the phosphoryl‐aspartate intermediate in the crystal structure of a Scp1D206A mutant soaked with para‐nitrophenyl phosphate (pNPP), providing strong evidence for the proposed mechanism. Furthermore, steady‐state kinetic analysis of a variety of Scp1 mutants revealed the importance of Asp206 in Mg²⁺ coordination mediated by a water molecule. Overall, we captured the snapshots of the phosphoryl transfer reaction at each stage of Scp1‐mediated catalysis. Through structural‐based sequence alignment, we show that the spatial position of the D206 side chain is strictly conserved throughout HAD family. Our results strongly suggest that Asp206 and its equivalent residues in other HAD family members play important structural and possible mechanistic roles.     

Divalent metal cofactor binding in the kinetic folding trajectory of Escherichia coli ribonuclease HI

Proteins often require cofactors to perform their biological functions and must fold in the presence of their cognate ligands. Using circular dichroism spectroscopy. we investigated the effects of divalent metal binding upon the folding pathway of Escherichia coli RNase HI. This enzyme binds divalent metal in its active site, which is proximal to the folding core of RNase HI as defined by hydrogen/deuterium exchange studies. Metal binding increases the apparent stability of native RNase HI chiefly by reducing the unfolding rate. As with the apo-form of the protein, refolding from high denaturant concentrations in the presence of Mg2+ follows three-state kinetics: formation of a rapid burst phase followed by measurable single exponential kinetics. Therefore, the overall folding pathway of RNase HI is minimally perturbed by the presence of metal ions. Our results indicate that the metal cofactor enters the active site pocket only after the enzyme reaches its native fold, and therefore, divalent metal binding stabilizes the protein by decreasing its unfolding rate. Furthermore, the binding of the cofactor is dependent upon a carboxylate critical for activity (Asp10). A mutation in this residue (D10A) alters the folding kinetics in the absence of metal ions such that they are similar to those observed for the unaltered enzyme in the presence of metal.     

X-ray crystallographic and site-directed mutagenesis analysis of the mechanism of Schiff-base formation in phosphonoacetaldehyde hydrolase catalysis

Phosphonoacetaldehyde hydrolase (phosphonatase) catalyzes the hydrolytic P-C bond cleavage of phosphonoacetaldehyde (Pald) to form orthophosphate and acetaldehyde. The reaction proceeds via a Schiff-base intermediate formed between Lys-53 and the Pald carbonyl. The x-ray crystal structures of the wild-type phosphonatase complexed with Mg(II) alone or with Mg(II) plus vinylsulfonate (a phosphonoethylenamine analog) were determined to 2.8 and 2.4 A, respectively. These structures were used to determine the identity and positions of active site residues surrounding the Lys-53 ammonium group and the Pald carbonyl. These include Cys-22, His-56, Tyr-128, and Met-49. Site-directed mutagenesis was then employed to determine whether or not these groups participate in catalysis. Based on rate contributions, Tyr-128 and Cys-22 were eliminated as potential catalytic groups. The Lys-53 epsilon-amino group, positioned for reaction with the Pald carbonyl, forms a hydrogen bond with water 120. Water 120 is also within hydrogen bond distance of an imidazole nitrogen of His-56 and the sulfur atom of Met-49. Kinetic constants for mutants indicated that His-56 (1000-fold reduction in k(cat)/K(m) upon Ala substitution) and Met-49 (17,000-fold reduction in k(cat)/K(m) upon Leu substitution) function in catalysis of Schiff-base formation. Based on these results, it is proposed that a network of hydrogen bonds among Lys-53, water 120, His-56, and Met-49 facilitate proton transfer from Lys-53 to the carbinolamine intermediate. Comparison of the vinylsulfonate complex versus unliganded structures indicated that association of the cap and core domains is essential for the positioning of the Lys-53 for attack at the Pald carbonyl and that substrate binding at the core domain stabilizes cap domain binding.     

Distinct roles of the active-site Mg2+ ligands, Asp882 and Asp705, of DNA polymerase I (Klenow fragment) during the prechemistry conformational transitions

DNA polymerases catalyze the incorporation of deoxynucleoside triphosphates into a growing DNA chain using a pair of Mg(2+) ions, coordinated at the active site by two invariant aspartates, whose removal by mutation typically reduces the polymerase activity to barely detectable levels. Using two stopped-flow fluorescence assays that we developed previously, we have investigated the role of the carboxylate ligands, Asp(705) and Asp(882), of DNA polymerase I (Klenow fragment) in the early prechemistry steps that prepare the active site for catalysis. We find that neither carboxylate is required for an early conformational transition, reported by a 2-aminopurine probe, that takes place in the open ternary complex after binding of the complementary dNTP. However, the subsequent fingers-closing step requires Asp(882); this step converts the open ternary complex into the closed conformation, creating the active-site geometry required for catalysis. Crystal structures indicate that the Asp(882) position changes very little during fingers-closing; this side chain may therefore serve as an anchor point to receive the dNTP-associated metal ion as the nucleotide is delivered into the active site. The Asp(705) carboxylate is not required until after the fingers-closing step, and we suggest that its role is to facilitate the entry of the second Mg(2+) into the active site. The two early prechemistry steps that we have studied take place normally at very low Mg(2+) concentrations, although higher concentrations are needed for covalent nucleotide addition, consistent with the second metal ion entering the ternary complex after fingers-closing.     

Human flap endonuclease-1: conformational change upon binding to the flap DNA substrate and location of the Mg2+ binding site

Human flap endonuclease-1 (FEN-1) is a member of the structure-specific endonuclease family and is a key enzyme in DNA replication and repair. FEN-1 recognizes the 5'-flap DNA structure and cleaves it, a specialized endonuclease function essential for the processing of Okazaki fragments during DNA replication and for the repair of 5'-end single-stranded tails from nicked double-stranded DNA substrates. Magnesium is a cofactor required for nuclease activity. We have used Fourier transform infrared (FTIR) spectroscopy to better understand how Mg2+ and flap DNA interact with human FEN-1. FTIR spectroscopy provides three fundamentally new insights into the structural changes induced by the interaction of FEN-1 with substrate DNA and Mg2+. First, FTIR difference spectra in the amide I vibrational band (1600-1700 cm(-1)) reveal a change in the secondary structure of FEN-1 induced by substrate DNA binding. Quantitative analysis of the FTIR spectra indicates a 4% increase in helicity upon DNA binding or about 14 residues converted from disordered to helical conformations. The observation that the residues are disordered without DNA strongly implicates the flexible loop region. The conversion to helix also suggests a mechanism for locking the flexible loop region around the bound DNA. This is the first direct experimental evidence for a binding mechanism that involves a secondary structural change of the protein. Second, in contrast with DNA binding, no change is observed in the secondary structure of FEN-1 upon Mg2+ binding to the wild type or to the noncleaving D181A mutant. Third, the FTIR results provide direct evidence (via the carboxylate ligand band at 1535 cm(-1)) that not only is D181 a ligand to Mg2+ in the human enzyme but Mg2+ binding does not occur in the D181A mutant which lacks this ligand.     

Binding and activation of thiamin diphosphate in acetohydroxyacid synthase

Acetohydroxyacid synthases (AHASs) are biosynthetic thiamin diphosphate- (ThDP) and FAD-dependent enzymes. They are homologous to pyruvate oxidase and other members of a family of ThDP-dependent enzymes which catalyze reactions in which the first step is decarboxylation of a 2-ketoacid. AHAS catalyzes the condensation of the 2-carbon moiety, derived from the decarboxylation of pyruvate, with a second 2-ketoacid, to form acetolactate or acetohydroxybutyrate. A structural model for AHAS isozyme II (AHAS II) from Escherichia coli has been constructed on the basis of its homology with pyruvate oxidase from Lactobacillus plantarum (LpPOX). We describe here experiments which further test the model, and test whether the binding and activation of ThDP in AHAS involve the same structural elements and mechanism identified for homologous enzymes. Interaction of a conserved glutamate with the N1' of the ThDP aminopyrimidine moiety is involved in activation of the cofactor for proton exchange in several ThDP-dependent enzymes. In accord with this, the analogue N3'-pyridyl thiamin diphosphate does not support AHAS activity. Mutagenesis of Glu47, the putative conserved glutamate, decreases the rate of proton exchange at C-2 of bound ThDP by nearly 2 orders of magnitude and decreases the turnover rate for the mutants by about 10-fold. Mutant E47A also has altered substrate specificity, pH dependence, and other changes in properties. Mutagenesis of Asp428, presumed on the basis of the model to be the crucial carboxylate ligand to Mg(2+) in the "ThDP motif", leads to a decrease in the affinity of AHAS II for Mg(2+). While mutant D428N shows ThDP affinity close to that of the wild-type on saturation with Mg(2+), D428E has a decreased affinity for ThDP. These mutations also lead to dependence of the enzyme on K(+). These experiments demonstrate that AHAS binds and activates ThDP in the same way as do pyruvate decarboxylase, transketolase, and other ThDP-dependent enzymes. The biosynthetic activity of AHAS also involves many other factors beyond the binding and deprotonation of ThDP; changes in the ligands to ThDP can have interesting and unexpected effects on the reaction.     

Effects of active site mutations on the metal binding affinity, catalytic competence, and stability of the family II pyrophosphatase from Bacillus subtilis

Family II inorganic pyrophosphatases (PPases) have been recently found in a variety of bacteria. Their primary and tertiary structures differ from those of the well-known family I PPases, although both have a binuclear metal center directly involved in catalysis. Here, we examined the effects of mutating one Glu, four His, and five Asp residues forming or close to the metal center on Mn(2+) binding affinity, catalysis, oligomeric structure, and thermostability of the family II PPase from Bacillus subtilis (bsPPase). Mutations H9Q, D13E, D15E, and D75E in two metal-binding subsites caused profound (10(4)- to 10(6)-fold) reductions in the binding affinity for Mn(2+). Most of the mutations decreased k(cat) for MgPP(i) by 2-3 orders of magnitude when measured with Mn(2+) or Mg(2+) bound to the high-affinity subsite and Mg(2+) bound to both the low-affinity subsite and pyrophosphate. In the E78D variant, the k(cat) for the Mn-bound enzyme was decreased 120-fold, converting bsPPase from an Mn-specific to an Mg-specific enzyme. K(m) values were less affected by the mutations, and, interestingly, were decreased in most cases. Mutations of His(97) and His(98) residues, which lie near the subunit interface, greatly destabilized the bsPPase dimer, whereas most other mutations stabilized it. Mn(2+), in sharp contrast to Mg(2+), conferred high thermostability to wild-type bsPPase, although this effect was reduced by all of the mutations except D203E. These results indicate that family II PPases have a more integrated active site structure than family I PPases and are consequently more sensitive to conservative mutations.     

Studies on the metal binding sites in the catalytic domain of beta1,4-galactosyltransferase

The catalytic domain of bovine beta1,4-galactosyltransferase (beta4Gal-T1) has been shown to have two metal binding sites, each with a distinct binding affinity. Site I binds Mn(2+) with high affinity and does not bind Ca(2+), whereas site II binds a variety of metal ions, including Ca(2+). The catalytic region of beta4Gal-T1 has DXD motifs, associated with metal binding in glycosyltransferases, in two separate sequences: D(242)YDYNCFVFSDVD(254) (region I) and W(312)GWGGEDDD(320) (region II). Recently, the crystal structure of beta4Gal-T1 bound with UDP, Mn(2+), and alpha-lactalbumin was determined in our laboratory. It shows that in the primary metal binding site of beta4Gal-T1, the Mn(2+) ion, is coordinated to five ligands, two supplied by the phosphates of the sugar nucleotide and the other three by Asp254, His347, and Met344. The residue Asp254 in the D(252)VD(254) sequence in region I is the only residue that is coordinated to the Mn(2+) ion. Region II forms a loop structure and contains the E(317)DDD(320) sequence in which residues Asp318 and Asp319 are directly involved in GlcNAc binding. This study, using site-directed mutagenesis, kinetic, and binding affinity analysis, shows that Asp254 and His347 are strong metal ligands, whereas Met344, which coordinates less strongly, can be substituted by alanine or glutamine. Specifically, substitution of Met344 to Gln has a less severe effect on the catalysis driven by Co(2+). Glu317 and Asp320 mutants, when partially activated by Mn(2+) binding to the primary site, can be further activated by Co(2+) or inhibited by Ca(2+), an effect that is the opposite of what is observed with the wild-type enzyme.