Dimeric structure of nucleoside diphosphate kinase from moderately halophilic bacterium: contrast to the tetrameric Pseudomonas counterpart

Light scattering and chemical cross-linking analyses of nucleoside diphosphate kinase (NDK) from moderate halophile, Halomonas sp. 593 (HaNDK), unambiguously demonstrated that this enzyme formed a dimeric structure, in contrast to the Pseudomonas NDK (PaNDK), a nonhalophilic counterpart, and other NDKs from Gram-negative bacteria, which all formed a tetrameric structure. Comparison of HaNDK and PaNDK showed that the HaNDK was less thermally stable than the PaNDK: the optimum temperature of PaNDK enzyme activity was 20 degrees C higher than that of HaNDK. However, the HaNDK readily refolded and reassembled back to the active dimeric structure, upon heat denaturation at 0.2 M NaCl, as soon as the temperature was lowered. On the contrary, the thermally more stable PaNDK was irreversibly denatured at its melting temperature.     

A structural mechanism for dimeric to tetrameric oligomer conversion in Halomonas sp. nucleoside diphosphate kinase

Nucleoside diphosphate kinase (NDK) is known to form homotetramers or homohexamers. To clarify the oligomer state of NDK from moderately halophilic Halomonas sp. 593 (HaNDK), the oligomeric state of HaNDK was characterized by light scattering followed by X‐ray crystallography. The molecular weight of HaNDK is 33,660, and the X‐ray crystal structure determination to 2.3 and 2.7 Å resolution showed a dimer form which was confirmed in the different space groups of R3 and C2 with an independent packing arrangement. This is the first structural evidence that HaNDK forms a dimeric assembly. Moreover, the inferred molecular mass of a mutant HaNDK (E134A) indicated 62.1–65.3 kDa, and the oligomerization state was investigated by X‐ray crystallography to 2.3 and 2.5 Å resolution with space groups of P2₁ and C2. The assembly form of the E134A mutant HaNDK was identified as a Type I tetramer as found in Myxococcus NDK. The structural comparison between the wild‐type and E134A mutant HaNDKs suggests that the change from dimer to tetramer is due to the removal of negative charge repulsion caused by the E134 in the wild‐type HaNDK. The higher ordered association of proteins usually contributes to an increase in thermal stability and substrate affinity. The change in the assembly form by a minimum mutation may be an effective way for NDK to acquire molecular characteristics suited to various circumstances.     

Engineering of halophilic enzymes: two acidic amino acid residues at the carboxy-terminal region confer halophilic characteristics to Halomonas and Pseudomonas nucleoside diphosphate kinases

Nucleoside diphosphate kinase from Halomonas sp. 593 (HaNDK) exhibits halophilic characteristics. Residues 134 and 135 in the carboxy-terminal region of HaNDK are Glu-Glu, while those of its homologous counterpart of non-halophilic Pseudomonas NDK (PaNDK) are Ala-Ala. The double mutation, E134A-E135A, in HaNDK results in the loss of the halophilic characteristics, and, conversely, the double mutation of A134E-A135E in PaNDK confers halophilic characters to this enzyme, indicating that the charged state of these two residues that are located in the C-terminal region plays a critical role in determining halophilic characteristics. The importance of these two residues versus the net negative charges will be discussed in relation to the halophilicity of NDK.     

A Carboxyl Terminal Leucine Zipper Is Required for Tyrosine Hydroxylase Tetramer Formation

Abstract: Tyrosine hydroxylase catalyzes the rate-limiting reaction in the biosynthesis of the catecholamine neurotransmitters and hormones (dopamine, norepinephrine, and epinephrine). Rat tyrosine hydroxylase exists, in its native form, as a tetramer composed of identical 498 amino acid subunits. There is currently no information describing the molecular interactions by which the four monomeric tyrosine hydroxylase subunits assemble into an active tetramer. Mutational analysis was performed on bacterially expressed enzyme to assess the role of a putative C-terminal leucine zipper in the assembly of subunits into the tetrameric holoenzyme. Deletion of the C-terminal 19 amino acids, or mutation of a leucine residue (to an alanine), converts the enzyme from a tetrameric to a dimeric form that exhibits greater structural heterogeneity. This change in macromolecular form is accompanied by a 75% (deletion mutation) to 20% (Leu → Ala mutation) reduction in specific activity of the enzyme. This represents the first report of the functional involvement of a region containing a leucine zipper motif in the assembly and activity of a neuronal enzyme.     

The E46K mutation in alpha-synuclein increases amyloid fibril formation

The identification of a novel mutation (E46K) in one of the KTKEGV-type repeats in the amino-terminal region of alpha-synuclein suggests that this region and, more specifically, Glu residues in the repeats may be important in regulating the ability of alpha-synuclein to polymerize into amyloid fibrils. It was demonstrated that the E46K mutation increased the propensity of alpha-synuclein to fibrillize, but this effect was less than that of the A53T mutation. The substitution of Glu(46) for an Ala also increased the assembly of alpha-synuclein, but the polymers formed can have different ultrastructures, further indicating that this amino acid position has a significant effect on the assembly process. The effect of residue Glu(83) in the sixth repeat of alpha-synuclein, which lies closest to the amino acid stretch critical for filament assembly, was also studied. Mutation of Glu(83) to a Lys or Ala increased polymerization but perturbed some of the properties of mature amyloid. These results demonstrated that some of the Glu residues within the repeats can have significant effects on modulating the assembly of alpha-synuclein to form amyloid fibrils. The greater effect of the A53T mutation, even when compared with what may be predicted to be a more dramatic mutation such as E46K, underscores the importance of protein microenvironment in affecting protein structure. Moreover, the relative effects of the A53T and E46K mutations are consistent with the age of onset of disease. These findings support the notion that aberrant alpha-synuclein polymerization resulting in the formation of pathological inclusions can lead to disease.     

Role of Cys-295 on subunit interactions and allosteric regulation of phosphofructokinase-2 from Escherichia coli

In a previous work, chemical modification of Cys-238 of Escherichia coli Pfk-2 raised concerns on the importance of the dimeric state of Pfk-2 for enzyme activity, whereas modification of Cys-295 impaired the enzymatic activity and the MgATP-induced tetramerization of the enzyme. The results presented here demonstrate that the dimeric state of Pfk-2 is critical for the stability and the activity of the enzyme. The replacement of Cys-238 by either Ala or Phe shows no effect on the kinetic parameters, allosteric inhibition, dimer stability and oligomeric structure of Pfk-2. However, the mutation of Cys-295 by either Ala or Phe provokes a decrease in the k(cat) value and an increment in the K(m) values for both substrates. We suggest that the Cys-295 residue participates in intersubunit interactions in the tetramer since the Cys-295-Phe mutant exhibits higher tetramer stability, which in turn results in an increase in the fructose-6-P concentration required for the reversal of the MgATP inhibition relative to the wild type enzyme.     

Stability of a 24-meric homopolymer: comparative studies of assembly-defective mutants of Rhodobacter capsulatus bacterioferritin and the native protein

The stability of Rhodobacter capsulatus bacterioferritin, a 24-meric homopolymer, toward denaturation on variation in pH and temperature, and increasing concentrations of urea and guanidine.HCl was investigated with native PAGE, and CD and fluorescence spectroscopies. With temperature and urea, the wild-type protein denatured without discernible intermediates in the equilibrium experiments, but with guanidine.HCl (Gnd.HCl) one or more intermediate species were apparent at relatively low Gnd.HCl concentrations. Dissociated subunit monomers, or aggregates smaller than 24-mers containing the high alpha-helical content characteristic of the native protein were not obtained at any pH without a high proportion of the 24-mer being present, and taken together with the other denaturation experiments and the construction of stable subunit dimers by site-directed mutagenesis, this observation indicates that folding of the bacterioferritin monomer could be coupled to its association into a dimer. Glu 128 and Glu 135 were replaced by alanine and arginine in a series of mutants to determine their role in stabilizing the 24-meric oligomer. The Glu128Ala, Glu135Ala and Glu135Arg variants retained a 24-meric structure, but the Glu128Ala/Glu135Ala and Glu128Arg/Glu135Arg variants were stable subunit dimers. CD spectra of the Glu135Arg, Glu128Ala/Glu135Ala, and Glu128Arg/Glu135Arg variants showed that they retained the high alpha-helical content of the wild-type protein. The 24-meric Glu135Arg variant was less stable than the wild-type protein (T(m), [Urea](50%) and [Gnd.HCl](50%) of 59 degrees C, 4.9 M and 3.2 M compared with 73 degrees C, approximately 8 M and 4.3 M, respectively), and the dimeric Glu128Arg/Glu135Arg variant was less stable still (T(m), [Urea](50%) and [Gnd.HCl](50%) of 43 degrees C, approximately 3.2 M and 1.8 M, respectively). The differences in stability are roughly additive, indicating that the salt-bridges formed by Glu 128 and Glu 135 in the native oligomer, with Arg 61 and the amino-terminal amine of neighboring subunits, respectively, contribute equally to the stability of the subunit assembly. The additivity and assembly states of the variant proteins suggest that the interactions involving Glu 128 and Glu 135 contribute significantly to stabilizing the 24-mer relative to the subunit dimer.     

Rational proteomics V: structure-based mutagenesis has revealed key residues responsible for substrate recognition and catalysis by the dehydrogenase and isomerase activities in human 3beta-hydroxysteroid dehydrogenase/isomerase type 1

Mammalian 3beta-hydroxysteroid dehydrogenase/isomerase (3beta-HSD) is a member of the short chain dehydrogenase/reductase. It is a key steroidogenic enzyme that catalyzes the first step of the multienzyme pathway conversion of circulating dehydroepiandrosterone and pregnenolone to active steroid hormones. A three dimensional model of a ternary complex of human 3beta-HSD type 1 (3beta-HSD_1) with an NAD cofactor and androstenedione product has been developed based upon X-ray structures of the ternary complex of E. coli UDP-galactose 4-epimerase (UDPGE) with an NAD cofactor and substrate (PDB_AC: 1NAH) and the ternary complex of human type 1 17beta-hydroxysteroid dehydrogenase (17beta-HSD_1) with an NADP cofactor and androstenedione (PDB_AC: 1QYX). The dimeric structure of the enzyme was built from two monomer models of 3beta-HSD_1 by respective 3D superposition with A and B subunits of the dimeric structure of Streptococcus suis DTDP-D-glucose 4,6-dehydratase (PDB_AC: 1KEP). The 3D model structure of 3beta-HSD_1 has been successfully used for the rational design of mutagenic experiments to further elucidate the key substrate binding residues in the active site as well as the basis for dual function of the 3beta-HSD_1 enzyme. The structure based mutant enzymes, Asn100Ser, Asn100Ala, Glu126Leu, His232Ala, Ser322Ala and Asn323Leu, have been constructed and functionally characterized. The mutagenic experiments have confirmed the predicted roles of the His232 and Asn323 residues in recognition of the 17-keto group of the substrate and identified Asn100 and Glu126 residues as key residues that participate for the dehydrogenase and isomerization reactions, respectively.     

Electrostatic repulsion, compensatory mutations, and long-range non-additive effects at the dimerization interface of the HIV capsid protein

In previous studies, thermodynamic dissection of the dimerization interface in CA-C, the C-terminal domain of the capsid protein of human immunodeficiency virus type 1, revealed that individual mutation to alanine of Ser178, Glu180, Glu187 or Gln192 led to significant increases in dimerization affinity. Four related aspects derived from this observation have been now addressed, and the results can be summarized as follows: (i) thermodynamic analyses indicate the presence of an intersubunit electrostatic repulsion between both Glu180 residues. (ii) The mutation Glu180 to Ala was detected in nearly all type 2 human immunodeficiency virus variants, and in several simian immunodeficiency viruses analyzed. However, this mutation was strictly co-variant with mutations Ser178Asp in a neighboring residue, and Glu187Gln. Thermodynamic analysis of multiple mutants showed that Ser178Asp compensated, alone or together with Glu187Gln, the increase in affinity caused by the mutation Glu180Ala, and restored a lower dimerization affinity. (iii) The increase in the affinity constant caused by the multiple mutation to Ala of Ser178, Glu180, Glu187 and Gln192 was more than one order of magnitude lower than predicted if additivity were present, despite the fact that the 178/180 pair and the two other residues were located more than 10A apart. (iv) Mutations in CA-C that caused non-additive increases in dimerization affinity also caused a non-additive increase in the capacity of the isolated CA-C domain to inhibit the assembly of capsid-like HIV-1 particles in kinetic assays. In summary, the study of a protein-protein interface involved in the building of a viral capsid has revealed unusual features, including intersubunit electrostatic repulsions, co-variant, compensatory mutations that may evolutionarily preserve a low association constant, and long-range, large magnitude non-additive effects on association.     

Contribution of halophilic nucleoside diphosphate kinase sequence to the heat stability of chimeric molecule

A halophilic nucleoside diphosphate kinase from a moderate halophile, Halomonas sp. 593 (593NDK), was found to be resistant to heat treatment, as indicated by the high level of activity recovery after heating at high temperatures. This is due to reversibility of thermal unfolding, not the high melting temperature, of the protein. The highly homologous NDK from non-halophilic organism, Pseudomonas aeruginosa, showed instability against heat treatment. Chimeric molecules consisting of each half of these two NDKs were constructed and characterized for their heat stability. The results showed that the N-terminal half of 593NDK contributes to the heat stability of the proteins. We discuss the possible reason for the observed difference in resistance to heat treatment between the 593NDK and PaNDK and between two chimeric proteins.     

A single amino acid substitution deregulates a bacterial lactate dehydrogenase and stabilizes its tetrameric structure

We have engineered a variant of the lactate dehydrogenase enzyme from Bacillus stearothermophilus in which arginine-173 at the proposed regulatory site has been replaced by glutamine. Like the wild-type enzyme, this mutant undergoes a reversible, protein-concentration-dependent subunit assembly, from dimer to tetramer. However, the mutant tetramer is much more stable (by a factor of 400) than the wild type and is destabilized rather than stabilized by binding the allosteric regulator, fructose 1,6-biphosphate (Fru-1,6-P2). The mutation has not significantly changed the catalytic properties of the dimer (Kd NADH, Km pyruvate, Ki oxamate and kcat), but has weakened the binding of Fru-1,6-P2 to both the dimeric and tetrameric forms of the enzyme and has almost abolished any stimulatory effect. We conclude that the Arg-173 residue in the wild-type enzyme is directly involved in the binding of Fru-1,6-P2, is important for allosteric communication with the active site, and, in part, regulates the state of quaternary structure through a charge-repulsion mechanism.     

Role of a glutamate bridge spanning the dimeric interface of human manganese superoxide dismutase

The function in the structure, stability, and catalysis of the interfaces between subunits in manganese superoxide dismutase (MnSOD) is currently under scrutiny. Glu162 in homotetrameric human MnSOD spans a dimeric interface and forms a hydrogen bond with His163 of an adjacent subunit which is a direct ligand of the manganese. We have examined the properties of two site-specific mutants of human MnSOD in which Glu162 is replaced with Asp (E162D) and Ala (E162A). The X-ray crystal structures of E162D and E162A MnSOD reveal no significant structural changes compared with the wild type other than the removal of the hydrogen bond interaction with His163 in E162A MnSOD. In the case of E162D MnSOD, an intervening solvent molecule fills the void created by the mutation to conserve the hydrogen bond interaction between His163 and residue 162. These mutants retain their tetrameric structure and their specificity for manganese over iron. Each has catalytic activity in the disproportionation of superoxide that is typically 5-25% of that of the wild-type enzyme and a level of product inhibition greater by approximately 2-fold. Differential scanning calorimetry indicates that the hydrogen bond between Glu162 and His163 contributes to the stability of MnSOD, with the major unfolding transition occurring at 81 degrees C for E162A compared to 90 degrees C for wild-type MnSOD. These results suggest that Glu162 at the tetrameric interface in human MnSOD supports stability and efficient catalysis and has a significant role in regulating product inhibition.     

The salt bridge of calcineurin is important for transferring the effect of CNB binding to CNA

Calcineurin (CN) is a heterodimer consisting of a catalytic subunit (CNA) and a regulatory subunit (CNB). The crystal structure shows that three residues or regions of CNA are mainly responsible for the interaction with CNB: the CNB binding helix (BBH), the N-terminus, and Glu53 that forms a salt bridge with Lys134 of CNB. In this report, we try to find the role that the salt bridge plays in the interaction between CNA and CNB. We found that mutation of Glu53 greatly reduced its responsiveness to CNB in the phosphatase assay and also that mutation of Lys134 of CNB affected its ability to activate the phosphatase activity of CNA. Structural analysis showed that disruption of the salt bridge affected the compact association of CNA and CNB. Thus, the salt bridge appears to help to stabilize CN and transfer the effects of CNB binding to CNA to activate its phosphatase activity.     

Resonance Raman microprobe spectroscopy of rhodopsin mutants: effect of substitutions in the third transmembrane helix.

A microprobe system has been developed that can record Raman spectra from as little as 2 microL of solution containing only micrograms of biological pigments. The apparatus consists of a liquid nitrogen (l-N2)-cooled cold stage, an epi-illumination microscope, and a substractive-dispersion, double spectrograph coupled to a l-N2-cooled CCD detector. Experiments were performed on native bovine rhodopsin, rhodopsin expressed in COS cells, and four rhodopsin mutants: Glu134 replaced by Gln (E134Q), Glu122 replaced by Gln (E122Q), and Glu113 replaced by Gln (E113Q) or Ala (E113A). Resonance Raman spectra of photostationary steady-state mixtures of 11-cis-rhodopsin, 9-cis-isorhodopsin, and all-trans-bathorhodopsin at 77 K were recorded. The Raman spectra of E134Q and the wild-type are the same, indicating that Glu134 is not located near the chromophore. Substitution at Glu122 also does not affect the C = NH stretching vibration of the chromophore. The fingerprint and Schiff base regions of the Raman spectra of the 380-nm, pH 7 forms of E113Q and E113A are characteristic of unprotonated retinal Schiff bases. The C = NH modes of the approximately 500-nm, pH 5 forms of E113Q and E113A in H2O (D2O) are found at 1648 (1629) and 1645 (1630) cm-1, respectively. These frequencies indicate that the protonated Schiff base interacts more weakly with its protein counterion in the Glu113 mutants than it does in the native pigment. Furthermore, perturbations of the unique bathorhodopsin hydrogen out-of-plane (HOOP) vibrations in E113Q and E113A indicate that the strength of the protein perturbation near C12 is weakened compared to that in native bathorhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural determinant for cold inactivation of rodent L-xylulose reductase

L-Xylulose reductase (XR) is a homotetramer belonging to the short-chain dehydrogenase/reductase family. Human XR is stable at low temperature, whereas the enzymes of mouse, rat, guinea pig, and hamster are rapidly dissociated into their inactive dimeric forms. In order to identify amino acid residues that cause cold inactivation of the rodent XRs, we have here selected Asp238, Leu242, and Thr244 in the C-terminal regions of rodent XRs and performed site-directed mutagenesis of the residues of mouse XR to the corresponding residues (Glu, Trp, and Cys) of the human enzyme. Cold inactivation was prevented partially by the single mutation of L242W and the double mutation of L242W/T244C, and completely by the double mutation of D238E/L242W. The L242W and L242W/T244C mutants existed in both tetrameric and dimeric forms at low temperature and the D238E/L242W mutant retained its tetrameric structure. No preventive effect was exerted by the mutations of D238E and T244C, which were dissociated into their dimeric forms upon cooling. Crystallographic analysis of human XR revealed that Glu238 and Trp242 contribute to proper orientation of the guanidino group of Arg203 of the same subunit to the C-terminal carboxylate group of Cys244 of another subunit through the neighboring residues, Gln137 and Phe241. Thus, the determinants for cold inactivation of rodent XRs are Asp238 and Leu242 with small side chains, which weaken the salt bridges between Arg203 and the C-terminal carboxylate group, and lead to cold inactivation.     

Subunit interface mutants of rabbit muscle aldolase form active dimers.

We report the construction of subunit interface mutants of rabbit muscle aldolase A with altered quaternary structure. A mutation has been described that causes nonspherocytic hemolytic anemia and produces a thermolabile aldolase (Kishi H et al., 1987, Proc Natl Acad Sci USA 84:8623-8627). The disease arises from substitution of Gly for Asp-128, a residue at the subunit interface of human aldolase A. To elucidate the role of this residue in the highly homologous rabbit aldolase A, site-directed mutagenesis is used to replace Asp-128 with Gly, Ala, Asn, Gln, or Val. Rabbit aldolase D128G purified from Escherichia coli is found to be similar to human D128G by kinetic analysis, CD, and thermal inactivation assays. All of the mutant rabbit aldolases are similar to the wild-type rabbit enzyme in secondary structure and kinetic properties. In contrast, whereas the wild-type enzyme is a tetramer, chemical crosslinking and gel filtration indicate that a new dimeric species exists for the mutants. In sedimentation velocity experiments, the mutant enzymes as mixtures of dimer and tetramer at 4 degrees C. Sedimentation at 20 degrees C shows that the mutant enzymes are > 99.5% dimeric and, in the presence of substrate, that the dimeric species is active. Differential scanning calorimetry demonstrates that Tm values of the mutant enzymes are decreased by 12 degrees C compared to wild-type enzyme. The results indicate that Asp-128 is important for interface stability and suggest that 1 role of the quaternary structure of aldolase is to provide thermostability.     

Structural and dynamic requirements for optimal activity of the essential bacterial enzyme dihydrodipicolinate synthase

Dihydrodipicolinate synthase (DHDPS) is an essential enzyme involved in the lysine biosynthesis pathway. DHDPS from E. coli is a homotetramer consisting of a 'dimer of dimers', with the catalytic residues found at the tight-dimer interface. Crystallographic and biophysical evidence suggest that the dimers associate to stabilise the active site configuration, and mutation of a central dimer-dimer interface residue destabilises the tetramer, thus increasing the flexibility and reducing catalytic efficiency and substrate specificity. This has led to the hypothesis that the tetramer evolved to optimise the dynamics within the tight-dimer. In order to gain insights into DHDPS flexibility and its relationship to quaternary structure and function, we performed comparative Molecular Dynamics simulation studies of native tetrameric and dimeric forms of DHDPS from E. coli and also the native dimeric form from methicillin-resistant Staphylococcus aureus (MRSA). These reveal a striking contrast between the dynamics of tetrameric and dimeric forms. Whereas the E. coli DHDPS tetramer is relatively rigid, both the E. coli and MRSA DHDPS dimers display high flexibility, resulting in monomer reorientation within the dimer and increased flexibility at the tight-dimer interface. The mutant E. coli DHDPS dimer exhibits disorder within its active site with deformation of critical catalytic residues and removal of key hydrogen bonds that render it inactive, whereas the similarly flexible MRSA DHDPS dimer maintains its catalytic geometry and is thus fully functional. Our data support the hypothesis that in both bacterial species optimal activity is achieved by fine tuning protein dynamics in different ways: E. coli DHDPS buttresses together two dimers, whereas MRSA dampens the motion using an extended tight-dimer interface.     

Identification of single Mn(2+) binding sites required for activation of the mutant proteins of E.coli RNase HI at Glu48 and/or Asp134 by X-ray crystallography

Escherichia coli RNase HI has two Mn(2+)-binding sites. Site 1 is formed by Asp10, Glu48, and Asp70, and site 2 is formed by Asp10 and Asp134. Site 1 and site 2 have been proposed to be an activation site and an attenuation site, respectively. However, Glu48 and Asp134 are dispensable for Mn(2+)-dependent activity. In order to identify the Mn(2+)-binding sites of the mutant proteins at Glu48 and/or Asp134, the crystal structures of the mutant proteins E48A-RNase HI*, D134A-RNase HI*, and E48A/D134N-RNase HI* in complex with Mn(2+) were determined. In E48A-RNase HI*, Glu48 and Lys87 are replaced by Ala. In D134A-RNase HI*, Asp134 and Lys87 are replaced by Ala. In E48A/D134N-RNase HI*, Glu48 and Lys87 are replaced by Ala and Asp134 is replaced by Asn. All crystals had two or four protein molecules per asymmetric unit and at least two of which had detectable manganese ions. These structures indicated that only one manganese ion binds to the various positions around the center of the active-site pocket. These positions are different from one another, but none of them is similar to site 1. The temperature factors of these manganese ions were considerably larger than those of the surrounding residues. These results suggest that the first manganese ion required for activation of the wild-type protein fluctuates among various positions around the center of the active-site pockets. We propose that this fluctuation is responsible for efficient hydrolysis of the substrates by the protein (metal fluctuation model). The binding position of the first manganese ion is probably forced to shift to site 1 or site 2 upon binding of the second manganese ion.     

Characterization of a mutant Bacillus subtilis adenylosuccinate lyase equivalent to a mutant enzyme found in human adenylosuccinate lyase deficiency: asparagine 276 plays an important structural role

Adenylosuccinate lyase, an enzyme catalyzing two reactions in purine biosynthesis (the cleavage of either adenylosuccinate or succinylaminoimidazole carboxamide ribotide), has been implicated in a human disease arising from point mutations in the gene encoding the enzyme. Asn(276) of Bacillus subtilis adenylosuccinate lyase, a residue corresponding to the location of a human enzyme mutation, was replaced by Cys, Ser, Ala, Arg, and Glu. The mutant enzymes exhibit decreased V(max) values (2-400-fold lower) for both substrates compared to the wild-type enzyme and some changes in the pH dependence of V(max) but no loss in affinity for adenylosuccinate. Circular dichroism reveals no difference in secondary structure between the wild-type and mutant enzymes. We show here for the first time that wild-type adenylosuccinate lyase exhibits a protein concentration dependence of molecular weight, secondary structure, and specific activity. An equilibrium constant between the dimer and tetramer was measured by light scattering for the wild-type and mutant enzymes. The equilibrium is somewhat shifted toward the tetramer in the mutant enzymes. The major difference between the wild-type and mutant enzymes appears to be in quaternary structure, with many mutant enzymes exhibiting marked thermal instability relative to the wild-type enzyme. We propose that mutations at position 276 result in structurally impaired adenylosuccinate lyases which are assembled into defective tetramers.     

AdoMet-dependent methyl-transfer: Glu119 is essential for DNA C5-cytosine methyltransferase M.HhaI

The role of Glu119 in S-adenosyl-L-methionine-dependent DNA methyltransferase M.HhaI-catalyzed DNA methylation was studied. Glu119 belongs to the highly conserved Glu/Asn/Val motif found in all DNA C5-cytosine methyltransferases, and its importance for M.HhaI function remains untested. We show that formation of the covalent intermediate between Cys81 and the target cytosine requires Glu119, since conversion to Ala, Asp or Gln lowers the rate of methyl transfer 10(2)-10(6) fold. Further, unlike the wild-type M.HhaI, these mutants are not trapped by the substrate in which the target cytosine is replaced with the mechanism-based inhibitor 5-fluorocytosine. The DNA binding affinity for the Glu119Asp mutant is decreased 10(3)-fold. Thus, the ability of the enzyme to stabilize the extrahelical cytosine is coupled directly to tight DNA binding. The structures of the ternary protein/DNA/AdoHcy complexes for both the Glu119Ala and Glu119Gln mutants (2.70 A and 2.75 A, respectively) show that the flipped base is positioned nearly identically with that observed in the wild-type M.HhaI complex. A single water molecule in the Glu119Ala structure between Ala119 and the extrahelical cytosine N3 is lacking in the Glu119Gln and wild-type M.HhaI structures, and most likely accounts for this mutant's partial activity. Glu119 has essential roles in activating the target cytosine for nucleophilic attack and contributes to tight DNA binding.