OHIO-1 beta-lactamase mutants: the Arg244Ser mutant and resistance to beta-lactams and beta-lactamase inhibitors.

Mutations at residue 244 (Ambler numbering system) in the class A TEM beta-lactamase confer resistance to inactivation by beta-lactamase inhibitors and result in diminished turnover of beta-lactam substrates. The Arg244Ser mutant of the OHIO-1 beta-lactamase, an SHV family enzyme, demonstrates variable susceptibilities to beta-lactamase inhibitors and has significantly reduced catalytic efficiency. The minimum inhibitory concentrations (MICs) for Escherichia coli DH5alpha expressing the Arg244Ser beta-lactamase were reduced when compared to the strain bearing the OHIO-1 beta-lactamase: ampicillin, 512 vs. 8192 micrograms ml-1; cephaloridine, 4 vs. 32 micrograms ml-1, respectively. The MICs for the beta-lactam beta-lactamase inhibitor combinations demonstrated resistance only to ampicillin-clavulanate, 16/8 vs. 8/4 micrograms ml-1 respectively. In contrast, there was increased susceptibility to ampicillin-sulbactam, ampicillin-tazobactam, and piperacillin-tazobactam. When compared to the OHIO-1 beta-lactamase homogenous preparations of the Arg244Ser beta-lactamase enzyme demonstrated increased Km and decreased kcat values for benzylpenicillin (Km=17 vs. 50 microM, kcat=345 vs. 234 s-1) and cephaloridine (Km=97 vs. 202 microM, kcat=1023 vs. 202 s-1). Although the Ki and IC50 values were increased for each inhibitor when compared to OHIO-1 beta-lactamase, the turnover numbers (tn) required for inactivation were increased only for clavulanate. For the Arg244Ser mutant enzyme of OHIO-1, the increased Ki, decreased tn for the sulfones, and different partition ratio (kcat/kinact) support the notion that not all class A enzymes are inactivated in the same manner, and that certain class A beta-lactamase enzymes may react differently with identical substitutions in structurally conserved amino acids. The resistance phenotype of a specific mutations can vary depending on the enzyme.     

Broønsted analysis of aspartate aminotransferase via exogenous catalysis of reactions of an inactive mutant

Primary amines functionally replace lysine 258 by catalyzing both the 1,3-prototropic shift and external aldimine hydrolysis reactions with the inactive aspartate aminotransferase mutant K258A. This finding allows classical Brønsted analyses of proton transfer reactions to be applied to enzyme-catalyzed reactions. An earlier study of the reaction of K258A with cysteine sulfinate (Toney, M.D. & Kirsch, J.F., 1989, Science 243, 1485) provided a beta value of 0.4 for the 1,3-prototropic shift. The beta value reported here for the transamination of oxalacetate to aspartate is 0.6. The catalytic efficacy of primary amines is largely determined by basicity and molecular volume. The dependence of the rate constants for the reactions of K258A and K258M on amine molecular volume is nearly identical. This observation argues that the alkyl groups of the added amines do not occupy the position of the lysine 258 side chain in the wild type enzyme. Large primary C alpha and insignificant solvent deuterium kinetic isotope effects with amino acid substrates demonstrate that the amine nitrogen of the exogenous catalysts directly abstracts the labile proton in the rate-determining step.     

Selection for purine regulatory mutants in an E. coli hypoxanthine phosphoribosyl transferase-guanine phosphoribosyl transferase double mutant

Summary We have studied the relationship between purine salvage enzymes, 6-mercaptopurine resistance, and the purR phenotype in E. coli. Mutants resistant to 6-mercaptopurine were found to have defects in HPRT, the purR repressor, or in both. Analysis of these mutants led to the isolation of a hypoxanthine phosphoribosyl transferase-guanine phosphoribosyl transferase double mutant (hpt ^- gpt^-) that is extremely sensitive to adenine. Two classes of adenine resistant mutants were isolated from this strain. The first class was deficient in APRT (apt ^-) while the second class represented purine regulatory mutants (purR ^-). There is thus selection for the purR phenotype in a hpt ^- gpt^-background.Abbreviations FGAR formyl glycinamide ribotide - HPRT hypoxanthine phosphoribosyl transferase - GPRT guanine phosphoribosyl transferase - APRT adenine phosphoribosyl transferase - PRPP 5 phosphoribosyl-1 pyrophosphate - 6MP 6-mercaptopurine - FA 2-fluoroadenine     

Use of electrospray mass spectrometry to directly observe an acyl enzyme intermediate in beta-lactamase catalysis

Electrospray mass spectrometry was used to directly observe intact acyl enzyme complexes formed between a class C beta-lactamase (from Enterobacter cloacae P99) and four poor substrates/inhibitors. In each case the molecular weight difference between the unreacted and the reacted beta-lactamase was consistent with the formation of an acyl enzyme.     

Metal ion requirements for structure and catalysis of an RNA ligase ribozyme

The class I ligase, a ribozyme previously isolated from random sequence, catalyzes a reaction similar to RNA polymerization, positioning its 5'-nucleotide via a Watson-Crick base pair, forming a 3',5'-phosphodiester bond between its 5'-nucleotide and the substrate, and releasing pyrophosphate. Like most ribozymes, it requires metal ions for structure and catalysis. Here, we report the ionic requirements of this self-ligating ribozyme. The ligase requires at least five Mg(2+) for activity and has a [Mg(2+)](1/2) of 70-100 mM. It has an unusual specificity for Mg(2+); there is only marginal activity in Mn(2+) and no detectable activity in Ca(2+), Sr(2+), Ba(2+), Zn(2+), Co(2+), Cd(2+), Pb(2+), Co(NH(3))(6)(3+), or spermine. All tested cations other than Mg(2+), including Mn(2+), inhibit the ribozyme. Hill analysis in the presence of inhibitory cations suggested that Ca(2+) and Co(NH(3))(6)(3+) inhibit by binding at least two sites, but they appear to productively fill a subset of the required sites. Inhibition is not the result of a significant structural change, since the ribozyme assumes a nativelike structure when folded in the presence of Ca(2+) or Co(NH(3))(6)(3+), as observed by hydroxyl-radical mapping. As further support for a nativelike fold in Ca(2+), ribozyme that has been prefolded in Ca(2+) can carry out the self-ligation very quickly upon the addition of Mg(2+). Ligation rates of the prefolded ribozyme were directly measured and proceed at 800 min(-1) at pH 9.0.     

Cofactor requirements of BamHI mutant endonuclease E77K and its suppressor mutants

A mutant BamHI endonuclease, E77K, belongs to a class of catalytic mutants that bind DNA efficiently but cleave DNA at a rate more than 10(3)-fold lower than that of the wild-type enzyme (S. Y. Xu and I. Schildkraut, J. Biol. Chem. 266:4425-4429, 1991). The preferred cofactor for the wild-type BamHI is Mg2+. BamHI is 10-fold less active with Mn2+ as the cofactor. In contrast, the E77K variant displays an increased activity when Mn2+ is substituted for Mg2+ in the reaction buffer. Mutations that partially suppress the E77K mutation were isolated by using an Escherichia coli indicator strain containing the dinD::lacZ fusion. These pseudorevertant endonucleases induce E. coli SOS response (as evidenced by blue colony formation) and thus presumably nick or cleave chromosomal DNA in vivo. Consistent with the in vivo result, the pseudorevertant endonucleases in the crude cell extract display site-specific partial DNA cleavage activity. DNA sequencing revealed two unique suppressing mutations that were located within two amino acid residues of the original mutation. Both pseudorevertant proteins were purified and shown to increase specific activity at least 50-fold. Like the wild-type enzyme, both pseudorevertant endonucleases prefer Mg2+ as the cofactor. Thus, the second-site mutation not only restores partial cleavage activity but also suppresses the metal preference as well. These results suggest that the Glu-77 residue may play a role in metal ion binding or in enzyme activation (allosteric transition) following sequence-specific recognition.     

Primary structure and properties of an inactive mutant aspartate transcarbamoylase.

A mutant form of Escherichia coli aspartate transcarbamoylase (ATCase) which lacks catalytic activity has been purified and characterized (Wall, K.A., Flatgaard, J.E., Gibbons, I., and Schachman, H.K. (1979) J. Biol. Chem 254, 11910-11916). Peptide mapping of the mutant and wild type catalytic chains followed by the determination of the amino acid sequence of the one altered peptide in the mutant indicated that a glycyl residue was replaced by aspartic acid. This substitution is located at position 125 in the tentative sequence kindly provided by W. Konigsberg (personal communication). The mutant protein has an overall secondary structure similar to that of the wild type as indicated by circular dichroism spectroscopy. However, marked changes in the reactivity of several amino acid residues were demonstrated. Lysyl residue 84 which in the wild type subunits reacts specifically with pyridoxal 5'-phosphate is only slightly reactive in the mutant even though the peptide containing that residue was not altered in amino acid composition. Another residue, cysteinyl 46, which is thought to be in the active site, is much more reactive toward p-hydroxymercuribenzoate in the mutant subunit than in the wild type protein. Finally, tyrosyl residue 213, which according to recent crystallographic studies is not near the active site and which exhibits an unusually low pK (9.1) in the wild type catalytic subunits, appears to have its pK shifted to 10.5 or higher as a result of the mutation. The evidence indicates that the substitution of an aspartyl for a glycyl residue at a region of the amino acid sequence remote from those residues in the active site causes sufficient modification of the tertiary structure to cause the loss of enzyme activity and to affect the reactivity of other residues in the protein. Moreover, the quaternary structure of the intact enzyme is altered as well since the subunit interactions are greatly weakened.     

The use of simple model systems to study spliceosomal catalysis

Since direct analysis of many aspects of spliceosomal function is greatly hindered by the daunting complexity of the spliceosome, the development of functionally validated simple model systems can be of great value. The critical role played by a base-paired complex of U6 and U2 snRNAs in splicing in vivo suggests that this complex could be a suitable starting point for the development of such a simple model system. However, several criteria must be satisfied before such a snRNA-based in vitro system can be considered a valid model for the spliceosomal catalytic core, including similarities at the level of reaction chemistry and cationic and sequence requirements. Previous functional analyses of in vitro assembled base-paired complexes of human U2 and U6 snRNAs have been promising, providing insight into catalysis. Furthermore, they strongly suggest that with further optimization, these RNAs might indeed be able to recapitulate the function of the spliceosomal catalytic core, thus opening the door to several lines of study not previously possible.     

Identification of an arsenic tolerant double mutant with a thiol-mediated component and increased arsenic tolerance in phyA mutants

A genetic screen was performed to isolate mutants showing increased arsenic tolerance using an Arabidopsis thaliana population of activation tagged lines. The most arsenic-resistant mutant shows increased arsenate and arsenite tolerance. Genetic analyses of the mutant indicate that the mutant contains two loci that contribute to arsenic tolerance, designated ars4 and ars5. The ars4ars5 double mutant contains a single T-DNA insertion, ars4, which co-segregates with arsenic tolerance and is inserted in the Phytochrome A (PHYA) gene, strongly reducing the expression of PHYA. When grown under far-red light conditions ars4ars5 shows the same elongated hypocotyl phenotype as the previously described strong phyA-211 allele. Three independent phyA alleles, ars4, phyA-211 and a new T-DNA insertion allele (phyA-t) show increased tolerance to arsenate, although to a lesser degree than the ars4ars5 double mutant. Analyses of the ars5 single mutant show that ars5 exhibits stronger arsenic tolerance than ars4, and that ars5 is not linked to ars4. Arsenic tolerance assays with phyB-9 and phot1/phot2 mutants show that these photoreceptor mutants do not exhibit phyA-like arsenic tolerance. Fluorescence HPLC analyses show that elevated levels of phytochelatins were not detected in ars4, ars5 or ars4ars5, however increases in the thiols cysteine, gamma-glutamylcysteine and glutathione were observed. Compared with wild type, the total thiol levels in ars4, ars5 and ars4ars5 mutants were increased up to 80% with combined buthionine sulfoximine and arsenic treatments, suggesting the enhancement of mechanisms that mediate thiol synthesis in the mutants. The presented findings show that PHYA negatively regulates a pathway conferring arsenic tolerance, and that an enhanced thiol synthesis mechanism contributes to the arsenic tolerance of ars4ars5.     

Recovery of catalytic activity from an inactive aggregated mutant of l-aspartase.

Two highly conserved lysyl residues have been replaced with an arginine to examine their role in the mechanism of l-aspartase from Escherichia coli. Replacement of an active-site lysine results in a significant loss of catalytic efficiency [A. S. Saribas, J. F. Schindler, and R. E. Viola (1994) J. Biol. Chem. 269, 6313-6319], while replacement of the second lysine leads to a completely inactive and insoluble protein. Fluorescence spectral evidence has suggested that the loss of activity is due to the misfolding of this aspartase mutant. Some catalytic activity is recovered when the mutant is treated with varying levels of denaturants, and extended treatment with high levels of guanidine.HCl results in the recovery of a substantial fraction of the wild-type activity from this inactive mutant. However, upon removal of the denaturant this mutant enzyme slowly reverts to its inactive and insoluble form. Treatment with an artificial chaperone system in which solubilization by detergent is followed by its removal with beta-cyclodextrin leads to a stable enzyme under nondenaturing conditions with about half the catalytic activity of the wild-type enzyme. These results confirm a structural role for lysine-55 in l-aspartase and demonstrate that additional characterization is required before conclusions can be drawn from the production of an inactive mutant.     

Site-directed mutagenesis of beta-lactamase I. Single and double mutants of Glu-166 and Lys-73.

Two single mutants and the corresponding double mutant of beta-lactamase I from Bacillus cereus 569/H were constructed and their kinetics investigated. The mutants have Lys-73 replaced by arginine (K73R), or Glu-166 replaced by aspartic acid (E166D), or both (K73R + E166D). All four rate constants in the acyl-enzyme mechanism were determined for the E166D mutant by the methods described by Christensen, Martin & Waley [(1990) Biochem. J. 266, 853-861]. Both the rate constants for acylation and deacylation for the hydrolysis of benzylpenicillin were decreased about 2000-fold in this mutant. In the K73R mutant, and in the double mutant, the rate constants for acylation were decreased about 100-fold and 10,000-fold respectively. All three mutants also had lowered values for the rate constants for the formation and dissociation of the non-covalent enzyme-substrate complex. The specificities of the mutants did not differ greatly from those of wild-type beta-lactamase, but the hydrolysis of cephalosporin C by the K73R mutant gave 'burst' kinetics.     

Selection of allosteric beta-lactamase mutants featuring an activity regulation by transition metal ions

Libraries of phage-displayed beta-lactamase mutants in which up to three loops have been engineered by genetic introduction of random peptide sequences or by randomization of the wild-type sequence have been submitted to selection protocols designed to find mutants in which binding of transition metal ions to the engineered secondary binding site leads to significant effects on the enzymatic activity. A double-selection protocol was applied: The phage-displayed libraries were first selected for transition metal ions affinity by panning on IMAC support, then a second selection step was applied to isolate mutants that have retained significant catalytic activity. The analysis of the kinetic properties of mutants in the presence of nickel, copper, or zinc ions allowed isolation of a few mutants whose activity was either enhanced or inhibited by factors up to three and >10, respectively, in a metal-specific manner. A remarkable mutant exhibiting differential allosteric regulation depending on the metal was found. Its activity was activated by nickel ion binding, inhibited by cupric ion binding, and nearly unaffected by zinc ions. These observations point to an interesting potential for up- or down-regulation of activity within a monomeric enzyme by binding to an "allosteric site" relatively remote from the active site.     

Crystal structure of an inactive duck delta II crystallin mutant with bound argininosuccinate

Delta-crystallin, the major soluble protein component of avian and reptilian eye lenses, is highly homologous to the urea cycle enzyme, argininosuccinate lyase (ASL). In duck lenses, there are two highly homologous delta crystallins, delta I and delta II, that are 94% identical in amino acid sequence. While delta II crystallin has been shown to exhibit ASL activity in vitro, delta I is enzymatically inactive. The X-ray structure of a His to Asn mutant of duck delta II crystallin (H162N) with bound argininosuccinate has been determined to 2.3 A resolution using the molecular replacement technique. The overall fold of the protein is similar to other members of the superfamily to which this protein belongs, with the active site located in a cleft formed by three different monomers in the tetramer. The active site of the H162N mutant structure reveals that the side chain of Glu 296 has a different orientation relative to the homologous residue in the H91N mutant structure [Abu-Abed et al. (1997) Biochemistry 36, 14012-14022]. This shift results in the loss of the hydrogen bond between His 162 and Glu 296 seen in the H91N and turkey delta I crystallin structures; this H-bond is believed to be crucial for the catalytic mechanism of ASL/delta II crystallin. Argininosuccinate was found to be bound to residues in each of the three monomers that form the active site. The fumarate moiety is oriented toward active site residues His 162 and Glu 296 and other residues that are part of two of the three highly conserved regions of amino acid sequence in the superfamily, while the arginine moiety of the substrate is oriented toward residues which belong to either domain 1 or domain 2. The analysis of the structure reveals that significant conformational changes occur on substrate binding. The comparison of this structure with the inactive turkey delta I crystallin reveals that the conformation of domain 1 is crucial for substrate affinity and that the delta I protein is almost certainly inactive because it can no longer bind the substrate.     

Structural requirements for the stability of novel cephalosporins to AmpC beta-lactamase based on 3D-structure

AmpC beta-lactamase is one of the leading causes of Pseudomonas aeruginosa (P. aeruginosa) resistance to cephalosporins. FR259647 is a cephalosporin having a novel pyrazolium substituent at the 3-position and exhibits excellent activity (MIC=1 microg/mL) against the AmpC beta-lactamase overproducing P. aeruginosa FP1380 strain in comparison with the third-generation cephalosporins FK518 [Abstracts of Papers, 30th Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta, GA, October 21-24, 1990, Abs. 454; Abstracts of Papers, 30th Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta, GA, October 21-24, 1990, Abs. 455; Abstracts of Papers, 30th Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta, GA, October 21-24, 1990, Abs. 456; Abstracts of Papers, 30th Interscience Conference on Antimicrobial Agents and Chemotherapy, Atlanta, GA, October 21-24, 1990, Abs. 457] (MIC=16 microg/mL) and ceftazidime (CAZ) (MIC=128 microg/mL). The stability of FR259647 and FK518 to AmpC beta-lactamase was evaluated using MIC assays against both the P. aeruginosa PAO1 strain and a PAO1 mutant strain overproducing AmpC beta-lactamase as a differential assay, which indicates that the main difference derives from their stability to AmpC beta-lactamase. A structural analysis using computer simulations indicated that the difference in stability may be due to steric hindrance of the 3-position substituents causing differential affinity. This steric hindrance may disturb entry of the cephalosporins into the binding pocket. We predicted the possibility of inhibition of entry as a potential means of enhancing stability by conformational analysis. In order to validate this speculation, novel FR259647 derivatives 4-9 were designed, calculated, synthesized, and evaluated. As a result, we demonstrated that their probability of entry correlated with the MIC ratio of the mutant strain to the parent strain and supports the validity of our model.     

Conversion of the proximal histidine ligand to glutamine restores activity to an inactive mutant of cytochrome c peroxidase.

Using site-directed mutagenesis, a double mutant in yeast cytochrome c peroxidase (CCP) has been constructed where the proximal ligand, His175, has been converted to glutamine and the neighboring Trp191 has been converted to phenylalanine. The refined 2.4-A crystal structure of the double mutant shows that the Gln175 side chain is within coordination distance of the heme iron atom and that Phe191 occupies the same position as Trp191 in the native enzyme with very little rearrangement outside the immediate vicinity of the mutations. Consistent with earlier work, we find that the single mutant, His175-->Gln, is fully active under steady state assay conditions and that as reported earlier (Mauro et al., 1988), the Trp191-->Phe mutant exhibits only < 0.05% activity. However, the double mutant, His175-->Gln/Phe191-->Phe, exhibits 20% wild type activity. Since it is known that the Trp191-->Phe mutant is inactive because it can no longer transfer electrons from ferrocytochrome c, changing the nature of the proximal ligand is able to restore this activity. These results raise interesting questions regarding the mechanism of interprotein electron transfer reactions.     

The use of gas-phase substrates to study enzyme catalysis at low hydration

Although there are varying estimates as to the degree of enzyme hydration required for activity, a threshold value of ca. 0.2 g of water per gram of protein has been widely accepted. The evidence upon which this is based is reviewed here. In particular, results from the use of gas-phase substrates are discussed. Results using solid-phase enzyme-substrate mixtures are not altogether in accord with those obtained using gas-phase substrates. The use of gaseous substrates and products provides an experimental system in which the hydration of the enzyme can be easily controlled, but which is not limited by diffusion. All the results show that increasing hydration enhances activity. The results using gas-phase substrates do not support the existence of a critical hydration value below which enzymatic activity is absent, and suggest that enzyme activity is possible at much lower hydrations than previously thought; they do not support the notion that significant hydration of the surface polar groups is required for activity. However, the marked improvement of activity as hydration is increased suggests that water does play a role, perhaps in optimizing the structure or facilitating the flexibility required for maximal activity.