Molecular dynamics simulations of class C beta-lactamase from Citrobacter freundii: insights into the base catalyst for acylation

Herein, we present results from molecular dynamics (MD) simulations of the class C beta-lactamase from Citrobacter freundii and its Michaelis complex with aztreonam. Four different configurations of the active site were modeled in aqueous solution, and their relative stability was estimated by means of quantum mechanical energy calculations. For the free enzyme, the energetically most stable configurations present a neutral Lys67 residue or an anionic Tyr150 side chain. Our calculations predict that these two configurations are quite close in terms of free energy, the anionic Tyr150 state being favored by approximately 1 kcal/mol. In contrast, for the noncovalent complex formed between the C. freundii enzyme and aztreonam, the energetic analyses predict that the configuration with the neutral Lys67 residue is much more stable than the anionic Tyr150 one (approximately 20 kcal/mol). Moreover, the MD simulations reveal that the neutral Lys67 state results in a proper enzyme-aztreonam orientation for nucleophilic attack and in a very stable contact between the nucleophilic hydroxyl group of Ser64 and the neutral amino side chain of Lys67. Thus, both the computed free energies and the structural analyses support the assignation of Lys67 as the base catalyst for the acylation step in the native form of the C. freundii enzyme.     

Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase: theoretical and experimental study of the effect of glutamic acid 284 on the protonation state of lysine 213

The crystal structure of Escherichia coli phosphoenolpyruvate (PEP) carboxykinase shows Lys213 is one of the ligands of enzyme-bound Mn2+ [Nat. Struct. Biol. 4 (1997) 990]. The direct coordination of Mn2+ by N(epsilon) of Lys213 is only consistent with a neutral (uncharged) Lys213, suggesting a low pKa for this residue. This work shows, through theoretical calculations and experimental analyses on homologous Saccharomyces cerevisiae PEP carboxykinase, how the microenvironment affects Mn2+ binding and the protonation state of Lys213. We show that Glu284, a residue close to Lys212, is required for correct protonation states of Lys212 and Lys213, and for Mn2+ binding. deltaG and deltaH values for the proton reorganization processes were calculated to analyze the energetic stability of the two different protonation states of Lys212 and Lys213 in wild-type and Glu284Gln S. cerevisiae PEP carboxykinase. Calculations were done using two modeling approaches, ab-initio density functional calculations and free energy perturbation (FEP) calculations. Both methods suggest that Lys212 must be protonated and Lys213 neutral in the wild-type enzyme. On the other hand, the calculations on the Glu284Gln mutant suggest a more stable neutral Lys212 and protonated Lys213. Experimental measurements showed 3 orders of magnitude lower activity and a threefold increase in Km for Mn2+ for Glu284Gln S. cerevisiae PEP carboxykinase when compared to wild type. The data here presented suggest that Glu284 is required for Mn2+ binding by S. cerevisiae PEP carboxykinase. We propose that Glu284 modulates the pKa value of Lys213 through electrostatic effects mediated by     

Catalytic mechanism of the Streptomyces K15 DD-transpeptidase/penicillin-binding protein probed by site-directed mutagenesis and structural analysis

The Streptomyces K15 penicillin-binding DD-transpeptidase is presumed to be involved in peptide cross-linking during bacterial cell wall peptidoglycan assembly. To gain insight into the catalytic mechanism, the roles of residues Lys38, Ser96, and Cys98, belonging to the structural elements defining the active site cleft, have been investigated by site-directed mutagenesis, biochemical studies, and X-ray diffraction analysis. The Lys38His and Ser96Ala mutations almost completely abolished the penicillin binding and severely impaired the transpeptidase activities while the geometry of the active site was essentially the same as in the wild-type enzyme. It is proposed that Lys38 acts as the catalytic base that abstracts a proton from the active serine Ser35 during nucleophilic attack and that Ser96 is a key intermediate in the proton transfer from the Ogamma of Ser35 to the substrate leaving group nitrogen. The role of these two residues should be conserved among penicillin-binding proteins containing the Ser-Xaa-Asn/Cys sequence in motif 2. Conversion of Cys98 into Asn decreased the transpeptidase activity and increased hydrolysis of the thiolester substrate and the acylation rate with most beta-lactam antibiotics. Cys98 is proposed to play the same role as Asn in motif 2 of other penicilloyl serine transferases in properly positioning the substrate for the catalytic process.     

Electrostatic interactions play a significant role in the affinity of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase for Mn

Phosphoenolpyruvate (PEP) carboxykinases catalyse the reversible formation of oxaloacetate (OAA) and ATP (or GTP) from PEP, ADP (or GDP) and CO₂. They are activated by Mn²⁺, a metal ion that coordinates to the protein through the ?-amino group of a lysine residue, the N_?-2-imidazole of a histidine residue, and the carboxylate from an aspartic acid residue. Neutrality in the ?-amino group of Lys213 of Saccharomyces cerevisiae PEP carboxykinase is expected to be favoured by the vicinity of ionised Lys212. Glu272 and Glu284, located close to Lys212, should, in turn, electrostatically stabilise its positive charge and hence assist in keeping the ?-amino group of Lys213 in a neutral state. The mutations Glu272Gln, Glu284Gln, and Lys212Met increased the activation constant for Mn²⁺ in the main reaction of the enzyme up to seven-fold. The control mutation Lys213Gln increased this constant by ten-fold, as opposed to control mutation Lys212Arg, which did not affect the Mn²⁺ affinity of the enzyme. These observations indicate a role for Glu272, Glu284, and Lys212 in assisting Lys213 to properly bind Mn²⁺. In an unexpected result, the mutations Glu284Gln, Lys212Met and Lys213Gln changed the nucleotide-independent OAA decarboxylase activity of S. cerevisiae PEP carboxykinase into an ADP-requiring activity, implying an effect on the OAA binding characteristics of PEP carboxykinase.     

Site-directed mutagenesis study of the microenvironment characteristics of Lys213 of Saccharomyces cerevisiae phosphoenolpyruvate carboxykinase

Saccharomyces cerevisiae phosphoenolpyruvate (PEP) carboxykinase catalyzes the reversible formation of oxaloacetate and adenosine triphosphate from PEP, adenosine diphosphate and carbon dioxide, and uses Mn(2+) as the activating metal ion. Comparison with the crystalline structure of homologous Escherichia coli PEP carboxykinase [Tari et al. Nature Struct. Biol. 4 (1997) 990-994] shows that Lys(213) is one of the ligands to Mn(2+) at the enzyme active site. Coordination of Mn(2+) to a lysyl residue is infrequent and suggests a low pK(a) value for the epsilon-NH(2) group of Lys(213). In this work, we evaluate the role of neighboring Phe(416) in contributing to provide a low polarity microenvironment suitable to keep the epsilon-NH(2) of Lys(213) in the unprotonated form. Mutation Phe416Tyr shows that the introduction of a hydroxyl group in the lateral chain of the residue produces a substantial loss in the enzyme affinity for Mn(2+), suggesting an increase of the pK(a) of Lys(213). A study of the effect of pH on K(m) for Mn(2+) indicate that the affinity of recombinant wild type enzyme for the metal ion is dependent on deprotonation of a group with pK(a) of 7.1+/-0.2, compatible with the low pK(a) expected for Lys(213). This pK(a) value increases at least 1.5 pH units upon Phe416Tyr mutation, in agreement with the expected effect of an increase in the polarity of Lys(213) microenvironment. Theoretical calculations of the pK(a) of Lys(213) indicate a value of 6.5+/-0.9, and it increases to 8.2+/-1.6 upon Phe416Tyr mutation. Additionally, mutation Phe416Tyr causes a loss of 1.3 kcal mol(-1) in the affinity of the enzyme for PEP, an effect perhaps related to the close proximity of Phe(416) to Arg(70), a residue previously shown to be important for PEP binding.     

Analysis of the NH2-Terminal 83rd Amino Acid of Escherichia coli GyrA in Quinolone-Resistance

Artificial mutations of Gyrase A protein (GyrA) in Escherichia coli by site-directed mutagenesis were generated to analyze quinolone-resistant mechanisms. By genetic analysis of gyrA genes in a gyrA temperature sensitive (Ts) background, exchange of Ser at the NH₂-terminal 83rd position of GyrA to Trp, Leu, Phe, Tyr, Ala, Val, and Ile caused bacterial resistance to the quinolones, while exchange to Gly, Asn, Lys, Arg and Asp did not confer resistance. These results indicate that it is the most important for the 83rd amino acid residue to be hydrophobic in expressing the phenotype of resistance to the quinolones. These findings also suggest that the hydroxyl group of Ser would not play a major role in the quinolone-gyrase interaction and Ser83 would not interact directly with other amino acid residues.     

Purification and characterization of a novel biosurfactant produced by <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> MS3

The physical properties and chemical structure of a new biosurfactant (licheniformin) produced by Bacillus licheniformis MS3 were investigated. The purified biosurfactant was identified as a lipopeptide with amino acid sequence of Gly, Ala, Val, Asp, Ser, Gly, Tyr and a lactone linkage between the carboxyl group of Aspargine and hydroxyl group of Tyrosine residue. The fatty acid moiety was attached to N-terminal amino acid residue through an amide bond. The purified licheniformin could lower the surface tension of water from 72 to 38 mN/m at concentrations higher than 15 μg/mL and its relative emulsion volume (EV%) was equal to 36%. It also showed stable surface activity over a wide range of temperature (45–85°C) and pH (3–11).     

Identification of conserved prolyl residue important for transport activity and the substrate specificity range of yeast plasma membrane Na+/H+ antiporters

Yeast plasma membrane Na+/H+ antiporters are divided according to their substrate specificity in two distinct subfamilies. To identify amino acid residues responsible for substrate specificity determination (recognition of K+), the Zygosaccharomyces rouxii Sod2-22 antiporter (non-transporting K+) was mutagenized and a collection of ZrSod2-22 mutants that improved the KCl tolerance of a salt-sensitive Saccharomyces cerevisiae strain was isolated. Several independent ZrSod2-22 mutated alleles contained the replacement of a highly conserved proline 145 with a residue containing a hydroxyl group (Ser, Thr). Site-directed mutagenesis of Pro145 proved that an amino acid with a hydroxyl group at this position is enough to enable ZrSod2-22p to transport K+. Simultaneously, the P145(S/T) mutation decreased the antiporter transport activity for both Na+ and Li+. Replacement of Pro145 with glycine resulted in a ZrSod2-22p with extremely low activity only for Na+, and the exchange of a charged residue (Asp, Lys) for Pro145 completely stopped the activity. Mutagenesis of the corresponding proline in the S. cerevisiae Nha1 antiporter (Pro146) confirmed that this proline of the fifth transmembrane domain is a critical residue for antiporter function. This is the first evidence that a non-polar amino acid residue is important for the substrate specificity and activity of yeast Nha antiporters.     

Roles of active site residues in Pseudomonas aeruginosa phosphomannomutase/phosphoglucomutase

In Pseudomonas aeruginosa, the dual-specificity enzyme phosphomannomutase/phosphoglucomutase catalyzes the transfer of a phosphoryl group from serine 108 to the hydroxyl group at the 1-position of the substrate, either mannose 6-P or glucose 6-P. The enzyme must then catalyze transfer of the phosphoryl group on the 6-position of the substrate back to the enzyme. Each phosphoryl transfer is expected to require general acid-base catalysis, provided by amino acid residues at the enzyme active site. An extensive survey of the active site residues by site-directed mutagenesis failed to identify a single key residue that mediates the proton transfers. Mutagenesis of active site residues Arg20, Lys118, Arg247, His308, and His329 to residues that do not contain ionizable groups produced proteins for which V(max) was reduced to 4-12% of that of the wild type. The fact that no single residue decreased catalytic activity more significantly, and that several residues had similar effects on V(max), suggested that the ensemble of active site amino acids act by creating positive electrostatic potential, which serves to depress the pK of the substrate hydroxyl group so that it binds in ionized form at the active site. In this way, the necessity of positioning the reactive hydroxyl group near a specific amino acid residue is avoided, which may explain how the enzyme is able to promote catalysis of both phosphoryl transfers, even though the 1- and 6-positions do not occupy precisely the same position when the substrate binds in the two different orientations in the active site. When Ser108 is mutated, the enzyme retains a surprising amount of activity, which has led to the suggestion that an alternative residue becomes phosphorylated in the absence of Ser108. (31)P NMR spectra of the S108A protein confirm that it is phosphorylated. Although the S108A/H329N protein had no detectable catalytic activity, the (31)P NMR spectra were not consistent with a phosphohistidine residue.     

Catalytic sites of medicinal leech enzyme destabilase-lysozyme (Mldl). Structure-functional correlation

Based on three-dimensional model of the bifunctional enzyme Destabilase-Lysozyme (mlDL-Ds2) in complex with trimer of N-acetylglucosoamine (NAG)3 the functional role of the stereochemically based group of amino acids (Glu14, Asp26, Ser 29, Ser31, Lys38, His92), in manifestation of glycosidase and isopeptidase activities has been elucidated. By method of site-directed mutagenesis it has been shown that mlDL glycosidase active site includes catalytic Glu14 and Asp26, and isopeptidase site functions as Ser/Lys dyad presented by catalytic residues Lys38 and Ser29. Thus, among the invertebrate lysozymes mlDL presents first example of the bifunctional enzyme with identified position of the isopeptidase active site and localization of the corresponding catalytic residues.     

Characterization of the S1 binding site of the glutamic acid-specific protease from Streptomyces griseus.

The glutamic acid-specific protease from Streptomyces griseus (SGPE) is an 18.4-kDa serine protease with a distinct preference for Glu in the P1 position. Other enzymes characterized by a strong preference for negatively charged residues in the P1 position, e.g., interleukin-1 beta converting enzyme (ICE), use Arg or Lys residues as counterions within the S1 binding site. However, in SGPE, this function is contributed by a His residue (His 213) and two Ser residues (Ser 192 and S216). It is demonstrated that proSGPE is activated autocatalytically and dependent on the presence of a Glu residue in the -1 position. Based on this observation, the importance of the individual S1 residues is evaluated considering that enzymes unable to recognize a Glu in the P1 position will not be activated. Among the residues constituting the S1 binding site, it is demonstrated that His 213 and Ser 192 are essential for recognition of Glu in the P1 position, whereas Ser 216 is less important for catalysis out has an influence on stabilization of the ground state. From the three-dimensional structure, it appears that His 213 is linked to two other His residues (His 199 and His 228), forming a His triad extending from the S1 binding site to the back of the enzyme. This hypothesis has been tested by substitution of His 199 and His 228 with other amino acid residues. The catalytic parameters obtained with the mutant enzymes, as well as the pH dependence, do not support this theory; rather, it appears that His 199 is responsible for orienting His 213 and that His 228 has no function associated with the recognition of Glu in P1.     

Novel cyclization chemistry especially suited for biologically derived, unprotected peptides.

A novel method is described for the cyclization of peptides--or segments of polypeptides--which requires a free N-terminal alpha-amino group and a distal amino acid residue containing a nucleophilic side chain. The reaction is conducted in two steps, both in the aqueous phase. The first step involves acylation of the N-terminal alpha-amino group with iodoacetic anhydride at pH 6. This acylation reaction has greater than 90% specificity for peptide alpha-amino groups and gives no alkylation of Arg, His, Lys or Met by the iodoacetate side product (R. Wetzel et al., Bioconjugate Chem., 1, 114-122, 1990). In the second step, the acylation reaction mixture or the isolated iodoacetyl-peptide is incubated at room temperature to give the cyclic peptide formed by reaction of the nucleophilic side chain with the iodoacetyl moiety. The pH dependence of the cyclization reaction by Met, Lys, Arg or His is consistent with the pKa of the nucleophilic side chain. Thus, peptides containing Met plus other nucleophilic amino acids should preferentially cyclize via Met at low pH. In this paper, preparation of cyclic peptides containing 3-6 amino acids is described; the full range of ring sizes and sequences which can undergo this cyclization has not been further explored. Preliminary results suggest that this method is also fairly general with respect to the amino acid sequence being cyclized. The reaction appears to be particularly suited for cyclization via Lys and Met side chains. All of the cyclized products are sufficiently stable for many biological applications.(ABSTRACT TRUNCATED AT 250 WORDS)     

Acidic residues emulate a phosphorylation switch to enhance the activity of rat hepatic neutral cytosolic cholesterol esterase

Site-directed mutagenesis of rat hepatic neutral cytosolic cholesteryl ester hydrolase (rhncCEH) was used to substitute acidic, basic or neutral amino acid residues for Ser506, required for activation by protein kinase A. The substitution of acidic Asp506 resulted in esterase activities with cholesteryl oleate, p-nitrophenylcaprylate (PNPC) and p-nitrophenylacetate (PNPA) equivalent to those of native rhncCEH with Ser506. The substitution of 2 acidic residues (Asp505/506), emulating the 2 negative charges of phosphoserine, resulted in a 10-fold greater cholesterol esterase activity than that of native rhncCEH, similar to the activity of rhncCEH treated with protein kinase A. In contrast to mutants with Ser506, protein kinase A did not increase the specific activities of mutants with Asp505/506. The substitution of basic (Lys506) or neutral (Asn506) residues abolished activity with cholesteryl oleate but not PNPC or PNPA. The substitution of neutral Gln for basic residues Lys496/Arg503 also abolished cholesterol esterase activity but not PNPC- and PNPA-esterase activities. These structure-activity relationships are modeled by homology with a recently reported crystal structure for the homologous human triacylglycerol hydrolase. The results suggest that the cholesterol esterase activity of carboxylesterases is enhanced by interactions between one or more basic residues on helix alpha16 (residues 485-503) and acidic groups at residues 505-506 in the adjacent surface loop.     

Structures of the acyl-enzyme complexes of the Staphylococcus aureus beta-lactamase mutant Glu166Asp:Asn170Gln with benzylpenicillin and cephaloridine

The serine-beta-lactamases hydrolyze beta-lactam antibiotics in a reaction that proceeds via an acyl-enzyme intermediate. The double mutation, E166D:N170Q, of the class A enzyme from Staphylococcus aureus results in a protein incapable of deacylation. The crystal structure of this beta-lactamase, determined at 2.3 A resolution, shows that except for the mutation sites, the structure is very similar to that of the native protein. The crystal structures of two acyl-enzyme adducts, one with benzylpenicillin and the other with cephaloridine, have been determined at 1.76 and 1.86 A resolution, respectively. Both acyl-enzymes show similar key features, with the carbonyl carbon atom of the cleaved beta-lactam bond covalently bound to the side chain of the active site Ser70, and the carbonyl oxygen atom in an oxyanion hole. The thiadolizine ring of the cleaved penicillin is located in a slightly different position than the dihydrothiazine ring of cephaloridine. Consequently, the carboxylate moieties attached to the rings form different sets of interactions. The carboxylate group of benzylpenicillin interacts with the side chain of Gln237. The carboxylate group of cephaloridine is located between Arg244 and Lys234 side chains and also interacts with Ser235 hydroxyl group. The interactions of the cephaloridine resemble those seen in the structure of the acyl-enzyme of beta-lactamase from Escherichia coli with benzylpenicillin. The side chains attached to the cleaved beta-lactam rings of benzylpenicillin and cephaloridine are located in a similar position, which is different than the position observed in the E. coli benzylpenicillin acyl-enzyme complex. The three modes of binding do not show a trend that explains the preference for benzylpenicillin over cephaloridine in the class A beta-lactamases. Rather, the conformational variation arises because cleavage of the beta-lactam bond provides additional flexibility not available when the fused rings are intact. The structural information suggests that specificity is determined prior to the cleavage of the beta-lactam ring, when the rigid fused rings of benzylpenicillin and cephaloridine each form different interactions with the active site.     

Improvement in the alkaline stability of subtilisin using an efficient random mutagenesis and screening procedure

An efficient random mutagenesis procedure coupled to a replica plate screen facilitated the isolation of mutant subtilisins from Bacillus amyloliquefaciens that had altered autolytic stability under alkaline conditions. Out of about 4000 clones screened, approximately 70 produced subtilisins with reduced stability (negatives). Two clones produced a more stable subtilisin (positives) and were identified as having a single mutation, either Ile107Val or Lys213Arg (the wild-type amino acid is followed by the codon position and the mutant amino acid). One of the negative mutants, Met50Val, was at a site where other homologous subtilisins contained a Phe. When the Met50Phe mutation was introduced into the B. amyloliquefaciens gene, the mutant subtilisin was more alkaline stable. The double mutant (Ile107Val/Lys213Arg) was more stable than the isolated single mutant parents. The triple mutant (Met50Phe/Ile107Val/Lys213Arg) was even more stable than Ile107Val/Lys213Arg (up to two times the autolytic half-time of wild-type at pH 12). These studies demonstrate the feasibility for improving the alkaline stability of proteins by random mutagenesis and identifying potential sites where substitutions from homologous proteins can improve alkaline stability.     

Three-dimensional structure of 4-amino-4-deoxychorismate lyase from Escherichia coli

4-Amino-4-deoxychorismate lyase (ADCL) is a member of the fold-type IV of PLP dependent enzymes that converts 4-amino-4-deoxychorismate (ADC) to p-aminobenzoate and pyruvate. The crystal structure of ADCL from Escherichia coli has been solved using MIR phases in combination with density modification. The structure has been refined to an R-factor of 20.6% at 2.2 A resolution. The enzyme is a homo dimer with a crystallographic twofold axis, and the polypeptide chain is folded into small and large domains with an interdomain loop. The coenzyme, pyridoxal 5'-phosphate, resides at the domain interface, its re-face facing toward the protein. Although the main chain folding of the active site is homologous to those of D-amino acid and L-branched-chain amino acid aminotransferases, no residues in the active site are conserved among them except for Arg59, Lys159, and Glu193, which directly interact with the coenzyme and play critical roles in the catalytic functions. ADC was modeled into the active site of the unliganded enzyme on the basis of the X-ray structures of the unliganded and liganded forms in the D-amino acid and L-branched-chain amino acid aminotransferases. According to this model, the carboxylates of ADC are recognized by Asn256, Arg107, and Lys97, and the cyclohexadiene moiety makes van der Waals contact with the side chain of Leu258. ADC forms a Schiff base with PLP to release the catalytic residue Lys159, which forms a hydrogen bond with Thr38. The neutral amino group of Lys159 eliminates the a-proton of ADC to give a quinonoid intermediate to release a pyruvate in accord with the proton transfer from Thr38 to the olefin moiety of ADC.     

Catalytic sites of medicinal leech enzyme destabilase-lysozyme (mlDL): Structure-function relationship

Based on the three-dimensional model of the bifunctional enzyme destabilase-lysozyme of the medicinal leech (mlDL) in complex with trimer of N-acetylglucosamine (NAG)₃ by site-directed mutagenesis method, the functional role of the group of amino acids (Glu14, Asp26, Ser29, Ser31, Lys38, His92) in manifestation of lysozyme (glycosidase, muramidase) and isopeptidase activities has been investigated by site-directed mutagenesis. The results obtained go well with hypothesis, that lysozyme active site of mlDL includes catalytic Glu14 and Asp26 residues, and isopeptidase site functions as Ser/Lys catalytic dyad presented by catalytic residues Ser29 and Lys38. Thus, among the invertebrate lysozymes, mlDL presents the first example of a bifunctional enzyme with identified position of the isopeptidase active site and localization of the corresponding catalytic residues.     

Crystal structure of the reduced Schiff-base intermediate complex of transaldolase B from Escherichia coli: mechanistic implications for class I aldolases.

Transaldolase catalyzes transfer of a dihydroxyacetone moiety from a ketose donor to an aldose acceptor. During catalysis, a Schiff-base intermediate between dihydroxyacetone and the epsilon-amino group of a lysine residue at the active site of the enzyme is formed. This Schiff-base intermediate has been trapped by reduction with potassium borohydride, and the crystal structure of this complex has been determined at 2.2 A resolution. The overall structures of the complex and the native enzyme are very similar; formation of the intermediate induces no large conformational changes. The dihydroxyacetone moiety is covalently linked to the side chain of Lys 132 at the active site of the enzyme. The Cl hydroxyl group of the dihydroxyacetone moiety forms hydrogen bonds to the side chains of residues Asn 154 and Ser 176. The C3 hydroxyl group interacts with the side chain of Asp 17 and Asn 35. Based on the crystal structure of this complex a reaction mechanism for transaldolase is proposed.     

pH-sensitive interactions between IgG and a mutated IgG-binding protein based upon two B domains of protein A from Staphylococcus aureus.

A fusion protein, consisting of the N-terminal 81 amino acids from an inactive bovine DNase I (Q38,E39-E38,Q39) and two sequential synthetic IgG-binding domains based upon domain B of Protein A from Staphylococcus aureus has been shown to bind to porcine IgG with a similar affinity and pH profile to Protein A. The same residue in each B domain (Tyr111 and Tyr169) has been mutated by cassette mutagenesis to Ser, Glu, His, Lys or Arg and the effect of the mutation on binding interactions with porcine IgG investigated. The evidence presented suggests that the interactions at the B domain are highly sensitive to the presence of a charged residue.     

Crystal structure of the human estrogen sulfotransferase-PAPS complex: evidence for catalytic role of Ser137 in the sulfuryl transfer reaction

Estrogen sulfotransferase (EST) transfers the sulfate group from 3'-phosphoadenosine 5'-phosphosulfate (PAPS) to estrogenic steroids. Here we report the crystal structure of human EST (hEST) in the context of the V269E mutant-PAPS complex, which is the first structure containing the active sulfate donor for any sulfotransferase. Superimposing this structure with the crystal structure of hEST in complex with the donor product 3'-phosphoadenosine 5'-phosphate (PAP) and the acceptor substrate 17beta-estradiol, the ternary structure with the PAPS and estradiol molecule, is modeled. These structures have now provided a more complete view of the S(N)2-like in-line displacement reaction catalyzed by sulfotransferases. In the PAPS-bound structure, the side chain nitrogen of the catalytic Lys(47) interacts with the side chain hydroxyl of Ser(137) and not with the bridging oxygen between the 5'-phosphate and sulfate groups of the PAPS molecule as is seen in the PAP-bound structures. This conformational change of the side chain nitrogen indicates that the interaction of Lys(47) with Ser(137) may regulate PAPS hydrolysis in the absences of an acceptor substrate. Supporting the structural data, the mutations of Ser(137) to cysteine and alanine decrease gradually k(cat) for PAPS hydrolysis and transfer activity. Thus, Ser(137) appears to play an important role in regulating the side chain interaction of Lys(47) with the bridging oxygen between the 5'-phosphate and the sulfate of PAPS.