©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
A New TEM -Lactamase Double Mutant with Broadened Specificity Reveals Substrate-dependent Functional Interactions (*)

(Received for publication, September 23, 1994)

Hector Viadiu (§) Joel Osuna Anthony L. Fink (1) Xavier Soberón (¶)

From the Instituto de Biotecnología, Universidad Nacional Autónoma de México, Apartado Postal 510-3, Cuernavaca, Morelos México Department of Chemistry and Biochemistry, The University of California, Santa Cruz, California 95064

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Using a random combinatorial mutagenesis of TEM beta-lactamase, directed against residues potentially involved in substrate discrimination, followed by selection on third generation cephalosporins, we obtained the double mutant E104M/G238S. Additionally, by using cloning strategies and site-directed mutagenesis we constructed the individual single mutants and also the single modification E104K and the double mutant E104K/G238S, which broaden the specificity of clinically isolated TEM beta-lactamase variants. The kinetic characterization of the purified double mutant E104M/G238S and its single counterparts E104M and G238S was carried out. The single mutant E104M exhibited increased k values against all substrates tested. K values remained similar to the values shown by the wild-type enzyme. The mutation at E104M was responsible for the increased hydrolysis rate against cefuroxime shown by the double mutant E104M/G238S. The effect of mutation G238S varied more pronouncedly, depending on the substrate. In general, a lower K was observed, but also a decreased k. The double mutant E104M/G238S exhibited a higher hydrolytic rate against cefotaxime compared with the corresponding single mutations. We observed nearly a 1000-fold greater k/K for the double mutant than for the wild type. This improvement in catalysis was the consequence of increased k and decreased K values. Computed contact interactions from modeling substrate complexes show reliable results only for benzylpenicillin. The modeling results with this substrate confirmed the observed enzyme activities for the different single and double mutants. Analysis of the apparent coupling energies, as calculated from the kinetic parameters of the single and double mutants, showed that the quantitative effect of a second mutation on a single mutant was either absent, additive, partially additive, or synergistic with respect to the first mutation, depending on the substrate analyzed.


INTRODUCTION

Class A beta-lactamases constitute a rich source of information about the relationship between the amino acid sequence and the function of the protein (Ambler et al., 1991). The class A, C, and D beta-lactamases represent three large families of active site serine enzymes (Coutere et al., 1992). Several crystallographic structures of class A enzymes are known (Herzberg, 1991; Knox and Moews, 1991; Jelsch et al., 1993). In addition, there is crystallographic work with some mutant variants (Herzberg et al., 1991; Knox et al., 1993), including one trapped in the acyl-enzyme intermediate (Strynadka et al., 1992). Also, there are reports about the structure of trapped intermediates of a class A enzyme in complex with an inhibitor molecule (Chen and Herzberg, 1992) or with a transition state analog (Chen et al., 1993).

Because of the extensive use of beta-lactam antibiotics, variant enzymes have emerged that are capable of hydrolyzing an extended spectrum of substrates, including third generation cephalosporins (Jacoby and Medeiros, 1991). The understanding of this extended spectrum mechanism is crucial for gaining insight in the design of new and better drugs. However, because only a few previous studies have included biochemical descriptions of the extended spectrum mutant enzymes (Sowek et al., 1991; Huletsky et al., 1993; Lee et al., 1991), more work is needed in order to dissect the individual contributions of the substituted residues present in the variants to the recognition of different substrates.

In this work we present data related to determining the contributions of individual residues to the substrate specificity of TEM beta-lactamase. We describe a new specificity mutant altered in positions 104 and 238, obtained through random combinatorial mutagenesis and selective approaches, and then we dissect the individual contributions of each residue in order to analyze the role of different parts of the active site in the substrate discrimination process.


EXPERIMENTAL PROCEDURES

Materials

Benzylpenicillin, ampicillin, 6-aminopenicillanic acid, cephaloridine, cefuroxime, ceftazidime, and cefotaxime (Fig. 1) were purchased from Sigma. Nitrocefin was a gift from Glaxo. Taq polymerase was purchased from Promega. Polymerase chain reactions were performed in a Biosycler Oven. Restriction enzymes, Klenow fragment and ligase were obtained from Boehringer Mannheim.


Figure 1: Structure of beta-lactam antibiotics.



Combinatorial Mutagenesis

Mutant beta-lactamases were made using the procedure described in Merino et al.(1992). Mutations were introduced via polymerase chain reaction and sequenced with Sequenase (U. S. Biochemical Corp.). The primers used for mutagenesis were synthesized using a strategy similar to that described by Hooft van Huijsduijnen et al.(1992). This consisted in the introduction, via removal and remixing of the solid support, of a 50% wild-type codon and a 50% of a NNG/C mixture representing all 20 amino acids (32 codons). The target codons included positions 104 and 105, and 237, 238, and 240, distributed in two different mutagenic oligonucleotides. N means an equimolar mixture of all four nucleotides and G/C means a mixture with 50% each G and C.

Determination of the Minimum Inhibitory Concentration

The minimum inhibitory concentrations were determined by a dilution method on Luria broth agar plates at 37 °C, with inocula of 30-50 colony-forming units dispensed in 5-µl drops. The plates contained the penicillin or cephalosporin antibiotic at several different concentrations, plus kanamycin at 25 µg/ml. The minimum concentration inhibiting the growth of cells was taken as the minimum inhibitory concentration.

Purification Procedure

The wild-type and mutant enzymes were purified as follows: beta-lactamase was expressed and purified from Escherichia coli. Fresh colonies were inoculated into 6 liters of Luria-Bertani media and grown on a 37 °C shaker for 12-14 h. After a cold shock treatment, cell debris was removed by centrifugation (6000 rpm for 10 min) and the cell-free supernatant mixed with of the total volume of 0.02 M sodium acetate, pH 4.8. Some proteins were removed by precipitation by gently swirling on a shaker at 4 °C; the precipitate was removed by centrifugation as above, and the supernatant (containing beta-lactamase) was batch-loaded onto a CM-Sepharose (CL-6B from Sigma) column previously pre-equilibrated with the same sodium acetate buffer. The nonbound proteins were washed with the same buffer for several hours, and the bound proteins eluted with a linear gradient of 0-0.4 M NaCl. Fractions containing beta-lactamase activity were pooled and exchanged via ultrafiltration with an Amicon YM10 filter into 0.025 M imidazole buffer, pH 7.0. This protein solution was loaded onto a chromatofocusing column (PBE94 from Pharmacia) using the same buffer as start buffer and eluted with a pH gradient of 7 to 4 using the pH 4 Polybuffer 74-HCl from Pharmacia. Fractions containing beta-lactamase activity were pooled and exchanged into 0.05 M potassium phosphate buffer, pH 7.0, by ultrafiltration as above. All proteins were purified to more than 90% homogeneity as determined by SDS-PAGE (^1)and isoelectric focusing-PAGE using the Pharmacia PhastSystem.

Kinetics

Enzyme activities were determined from complete progress curves, fitted to the Michaelis-Menten equation using substrate concentrations at least five times the K(m). The data were analyzed on a Macintosh computer using the Kaleidagraph program (Abelbeck Software). All points were determined at least in triplicate. With cefuroxime and cefotaxime the catalytic parameters were obtained by initial velocities using 10-15 different substrate concentrations bracketing the K(m) and the data fitted to the Michaelis-Menten equation using the Kaleidagraph program. With some cephalosporin substrates, because the K(m) was too high, only k/K(m) could be determined using measurements of the first-order rate of hydrolysis under conditions of substrate concentrations much less than K(m). Substrate hydrolysis was measured at 30 °C with a Perkin-Elmer 320 spectrophotometer according to the method of Waley(1974). The reactions were carried out in 50 mM phosphate buffer, pH 7.0, using 0.1, 0.4, or 1 cm path length cuvette as needed. The wavelengths for monitoring substrates were as follows: 240 nm for penicillins; 300 nm for cephaloridine, 262 nm for cefuroxime, and 282 nm for cefotaxime. The concentration of beta-lactamase was determined by using alpha = 29,400 M cm.

Computational Procedures

The active site of the wild-type or mutant TEM beta-lactamases, defined by all the amino acid residues and water molecules present within 10 Å radius around the Ser Calpha, was optimized by energy minimization using the all-atom consistence valence force field (Biosym) parameters and charges. The initial binding interactions of benzylpenicillin and cefotaxime with the several enzymes were studied by manual docking of the substrate to the active site followed by unconstrained energy minimization of the final complex. The substrates were manually fitted into the active site to position the beta-lactam carbonyl oxygen in the oxyanion hole and to maximize hydrogen bonding. The model substrate was adjusted manually to relieve obvious steric problems with the protein.


RESULTS

Isolation and Construction of Mutant Enzymes

Random combinatorial mutagenesis, using the strategy described by Merino et al.(1992) and selective screening in cefotaxime plates with antibiotic concentration higher than the reported minimum inhibitory concentration, resulted in the double mutant E104M/G238S described in this work. In order to study the individual effect of each mutation against several substrates, we constructed several other mutants in those positions. Using subcloning approaches we separated the double mutant into its individual parts. For comparative purposes we constructed other enzyme variants including the single mutant E104K and the double mutant E104K/G238S. These mutations are present in other broadened specificity mutants that arose in other TEM beta-lactamase contexts (Sougakoff et al., 1988).

The mutant E104M/G238S, and its single amino acid replacement counterparts E104M and G238S, were purified to greater than 90% homogeneity as determined by SDS-PAGE and isoelectric focusing-PAGE. Mutant enzymes with the G238S alteration were less stable than the others. Because of questions of stability, most studies were performed within a few days of the final purification step.

Antibiotic Susceptibility

Antibiotic susceptibility testings of E. coli strains (JM101) carrying the plasmid-borne gene for the wild-type and mutant enzymes are shown in Table 1. Minimum inhibitory concentration values indicate that the single variants G238S, E104M, and the double mutants with the change G238S, and Lys or Met in position 104 were the most active against cefotaxime.



Kinetic Analysis

The single mutants E104M and G238S, and the double mutant E104M/G238S were chosen for a more detailed study. The kinetic parameters of the wild-type beta-lactamase and its mutant derivatives are summarized in Table 2Table 3Table 4.







Hydrolysis rate studies for each of these antibiotics with the different enzymes resulted in the k values shown in Table 2. The single variant E104M produces an increase in k over wild-type values against all the substrates tested, whereas the G238S mutant shows an increase in k only for cefotaxime.

It is clear from Table 3that the G238S substitution increased the affinity of the enzyme for a variety of beta-lactam substrates, with the exception of cefuroxime and to a lesser extent to cefotaxime. The substitution E104M, on the other hand, had little effect on the affinity for substrates. It is interesting to note that with cefuroxime none of the mutations affected K(m), and with cefotaxime the major effect on K(m) occurred only in the double mutant.

When catalytic efficiencies of the beta-lactamases were compared, it became evident that all the mutants were better enzymes against penicillin substrates as compared with cephalosporin substrates (Table 4). However, for the double mutant E104M/G238S, cefotaxime and cephaloridine became better substrates than 6-aminopenicillanic acid. As shown in Table 4, the variant E104M is a better enzyme than the wild-type against all the substrates tested. The modification G238S was detrimental for the catalytic efficiency against all substrates tested except cefotaxime. It is important to note that the double mutant catalytic efficiency against cefotaxime is nearly 1000-fold greater than of the wild type.

Our results demonstrate the lack of predictability of a particular mutation on the expected modification of k or K(m) for a particular substrate. For instance, the single mutant G238S shows decreased k and improved (decreased) K(m) against all substrates tested, excepting cefotaxime (for which k is increased and K(m) remains similar to the wild-type value) and cefuroxime (K(m) remains similar to the wild-type value), whereas the E104M modification increases the k value against all substrates tested but had little effect on K(m). However, this same alteration, present in the G238S context, caused a significant decrease in K(m) for some substrates but little effect on others!

Energy-minimized Docking

As shown in Table 5, the calculated interaction distances between the side chains of the enzymes and benzylpenicillin have an excellent correlation with the activity results of the different enzymes against this substrate (Table 2Table 3Table 4). The distance between the beta-lactam carbonyl carbon atom and the oxygen of Ser for the enzyme mutants E104M and E104M/G238S is almost the same as that calculated for the wild type. These mutants showed a higher k value against benzylpenicillin as compared with the single mutant G238S. In the oxyanion hole, the hydrogen bond between the beta-lactam carbonyl and the backbone NH237 is stronger than the one with the backbone NH70, a result similar to that obtained by Juteau et al. (1991) when modeling substrate binding by the ROB-1 enzyme. It is clear from the contact distances in Table 5that the G238S and E104M/G238S mutant enzymes could be less efficient in neutralizing the charge developed in the beta-lactam carbonyl than the wild-type and E104M enzymes. This result correlates well with the data of k/K(m) for all the enzymes against benzylpenicillin. Unfortunately, the cefotaxime modeling results showed poorer correlation with the enzyme activity data. All our energy minimized dockings were calculated without modification to atomic charges on substrate or enzyme. It is possible that a more sophisticated modeling approach, taking into account the complex side chain of cefotaxime, and the likely development of partial charges on various enzyme and substrate atoms, may lead to better modeling of the cefotaxime complex.




DISCUSSION

In a manner similar to Carter et al.(1984), we decided to analyze our results using the mutant cycle displayed in Fig. 2a. If replacement of different side chains induces no structural changes in the enzyme or enzyme-substrate complex their effects will be independent, and the overall change in interaction energy of the enzyme-substrate transition state in double mutants will be the sum of the corresponding terms for the two single mutants.


Figure 2: a, the changes in interaction energy of enzyme and transition state are represented by the DeltaG terms. For example, DeltaG is the difference in Gibbs-free energy of binding of the transition state to the enzyme forms Glu, Gly (wild-type) Glu, Ser (single mutant). DeltaG(1) = -RTln((k/K)/(k/K)). b-g, the energy of interaction of each side chain with the transition state (Kcal/mol) in the hydrolytic reaction. Calculations shown are from k/K terms (units are s mM) obtained for all the substrates tested.



The kinetics of the purified enzymes, with ampicillin as substrate, suggest that the individual mutations are independent (Fig. 2b). Thus, the loss in interaction energy of the double mutant with the transition state is close to the algebraic sum of the free energies of the single mutants. It is interesting to note that the K(m) value for the double mutant is mostly accounted for by the mutation G238S (Table 3). If K(m) is a good measure of the affinity for the substrate, then this result suggests that the second alteration E104M has little effect on the conformation of the active site, at least for the interaction with this substrate.

In contrast, with cefuroxime as substrate, the catalytic properties of the mutants are accounted for only by the modification E104M (Table 2). When the change G238S is added, in the context of the single mutant E104M, no additional effect is observed (Fig. 2c). Unexpectedly, the favorable effect shown by the E104M single mutant in the wild-type context is even higher when this alteration is added in the G238S context. The gain in interaction energy observed for the double mutant contrasts with the lower value expected from independent single mutants. The analysis of the K(m) values of the single and double mutants with this substrate reveals that the binding of this substrate with the active site is very similar to that of the wild type. This result is in contrast with the proposed structural effect for the mutation G238S (Huletsky et al., 1993) and observed in this work with the penicillin substrates tested and with cephaloridine. This fact, and others (see below), point to the difficulties in predicting the effect of some mutations on the interaction with different substrates.

With other substrates, the analysis of the individual contributions of the single mutants is more complicated. With some substrates (6-aminopenicillanic acid, cephaloridine, and benzylpenicillin), a relatively independent behavior of the single mutations is observed (Fig. 2, d-f). The loss in interaction energy of the double mutant with the transition state deviates little from the value expected from independent effects of each mutation. With benzylpenicillin as a substrate, the results show that there are no effects of adding the modification E104M in the G238S context (Fig. 2f). This result might suggest more severe alterations (as sensed by the substrate benzylpenicillin) of the active site in response to the modification G238S. With all the above substrates, there are additional variations in K(m) shown by the single mutants as compared with the double mutant (Table 3).

With cefotaxime as substrate neither single mutant significantly alters the K(m) when compared with the reported wild-type value. However, there is a decrease in the K(m) in the double mutant. The analysis of the individual contributions, using k/K(m) values, shows that the gain in interaction energy of the double mutant is a little more than the value expected from independent mutants (Fig. 2g). It is also clear from Fig. 2g that adding either mutation in the context of the other produces an identical and more favorable improvement in the k/K(m) parameter as compared with the effect of the single mutations in the wild-type context. This seems to indicate some kind of interdependence of both mutations that might explain the small increase in interaction energy shown by the double mutant.

Based on the lowered K(m) for most beta-lactams, Huletsky et al.(1993) suggested that the single mutation G238S has a structural role. Our results show a similar trend. However, the present study contributes some significant additional information, namely the G238S mutation does not lower K(m) against second and third generation cephalosporins as shown by the results obtained with the antibiotics cefuroxime and cefotaxime. Interestingly, the double mutant E104M/G238S also shows a decrease in K(m) for cefotaxime, in spite of the fact that the single substitutions have no effect on K(m). With cefuroxime, the K(m) of the double mutant is the same as that observed with the single variants. The G238S variant shows drastically reduced k values against all substrates tested except cefotaxime, where a distinct increase was observed. With this latter substrate it is clear that the ability of the single mutants to hydrolyze it is due to an improvement in the catalytic parameters, but the combination of both single mutants results in an improvement of the binding parameter as well. The ability to catalyze hydrolysis of the second generation cephalosporin cefuroxime is provided entirely by the modification E104M. In contrast, the change G238S is detrimental in the wild-type context decreasing the k and k/K(m) parameters against this same substrate. Strikingly, the single variant E104M and the double mutant show the same improvement in the ability to hydrolyze cefuroxime.

The data obtained for the E104M variant are more difficult to interpret in structural terms. Sowek et al.(1991) suggested that the hydrolysis of ceftazidime by the variant E104K could be explained by an interaction between the new positive charge of the Lys and the carboxylate of the aminothiazole oxime side chain of the substrate. However, because the same variant presented an enhanced activity against substrates with no carboxyl group on the side chain, such as cefotaxime, the authors proposed the formation of new favorable hydrogen bond interactions between the side chain of Lys and the substrate side chain. With our variant, because the long hydrophobic side chain of M104 cannot form hydrogen bonds, it is clear that alternative explanations are necessary in order to understand the better hydrolytic capabilities of this mutant with all the substrates tested when compared both with the wild-type enzyme and with the E104K variant (see Table VI of Sowek et al.(1991)). As can be seen in Fig. 3, Glu of TEM beta-lactamase (Strynadka et al., 1992; coordinates kindly provided by Dr. M. N. G. James) could be playing a role in the replenishment of the hydrolytic water molecule in a similar way as proposed for Asn in the Bacillus licheniformis beta-lactamase (Knox and Moews, 1991). As shown in Fig. 3, Asn may be able to interact with water molecules 565 and 667. Strikingly, the substitution Met for Glu in position 104 produces a better enzyme than the wild type against all substrates tested. This results might suggest a lesser importance of the outer rim in the replenishment mechanism as compared with Asn in the central position of the observed train of water molecules. Alternatively, the substitution E104M could provide a hydrophobic character to the active site making more favorable interactions with the hydrophobic tip of the beta-lactam side chain substituent as suggested for the Staphylococcus aureus enzyme (Herzberg and Moult, 1987). The fact that K(m) is not altered in the interaction of the E104M mutant against all substrates tested suggests that the hydrophobic interactions stabilize preferentially the transition state as compared with the ground state of the substrate. The modeling results of the mutants E104M and E104M/G238S with benzylpenicillin docked in the active site show that the calculated interaction distances between the beta-lactam carbonyl carbon atom and the oxygen of Ser and the distances between the beta-lactam carbonyl and the oxyanion hole are closer to the calculated wild-type values than those shown by the less active mutant G238S (Table 5).


Figure 3: Stereoview of some active site residues (TEM coordinates kindly provided by Dr. M.N.G. James). Some crystallographic water molecules are included as solid spheres. The potential water molecules contacting residue 104 are indicated. Penicillin G (BPE) is positioned for alpha-face acylation by reactive Ser as modeled by Moews et al.(1990).



In summary, our results show that the disturbance of a complex set of interactions between different residues and the substrate, probably together with subtle conformational changes, can result in unpredictable specificity alterations. The specificity changes could be the result of the improvement of binding interactions in some regions of the active site (such as the modification G238S) or in the improvement of the transition state stabilization due to alterations in other region of the active site (such as the modification E104M). Our results add to a data base that should prove useful once structural data of these mutants is obtained. This, together with detailed computer simulations may help clarify the molecular basis of beta-lactamase substrate interaction.


FOOTNOTES

*
This work was supported in part by grants from the National Science Foundation (to A. L. F.) and from Dirección General de Asuntos del Personal Académico/Universidad Nacional Autónoma de México (to X. S.).

§
Supported by a fellowship from the Dirección Gereral de Intercambio Académico/Universidad Nacional Autónoma de México.

To whom correspondence should be addressed: Apartado postal 510-3 Cuernavaca, Morelos 62271 México. Tel.: 52-73-114900; Fax: 52-73-172388; soberon{at}pbr322.ceingebi.unam.mx.

(^1)
The abbreviation used is: PAGE, polyacrylamide gel electrophoresis.


ACKNOWLEDGEMENTS

We thank Paul Gaytán and Eugenio López for the oligonucleotides and Maria Elena Munguía for excellent technical assistance. We also thank Evan Lewis for suggestions and help in the experimental work at Santa Cruz. We acknowledge time on the CRAY-YMP computer from DGSCA/UNAM.


REFERENCES

  1. Ambler, R. P., Coulson, A. F. W., Frére, J.-M., Ghuysen, J.-M., Joris, B., Forsman, M., Levesque, R. C., Tirab, G., and Waley, S. G. (1991) Biochem. J. 276, 269-272 [Medline] [Order article via Infotrieve]
  2. Carter, P. J., Winter, G., Wilkinson, A. J., and Fersht, A. R. (1984) Cell 38, 835-840 [Medline] [Order article via Infotrieve]
  3. Chen, C. C. H., and Herzberg, O. (1992) J. Mol. Biol. 224, 1103-1113 [Medline] [Order article via Infotrieve]
  4. Chen, C. C. H., Rahil, J., Pratt, R. F., and Herzberg, O. (1993) J. Mol. Biol. 234, 165-178 [CrossRef][Medline] [Order article via Infotrieve]
  5. Coutere, F., Lachapelle, J., and Levesque, R. C. (1992) Mol. Microbiol. 6, 1693-1705 [Medline] [Order article via Infotrieve]
  6. Herzberg, O. (1991) J. Mol. Biol. 217, 701-719 [Medline] [Order article via Infotrieve]
  7. Herzberg, O., and Moult, J. (1987) Science 236, 694-701 [Medline] [Order article via Infotrieve]
  8. Herzberg, O., Kapadia, G., Blanco, B., Smith, T. S., and Coulson, A. (1991) Biochemistry 30, 9503-9509 [Medline] [Order article via Infotrieve]
  9. Hooft van Huijsduijnen, R. A. M., Ayala, G., and DeLamarter, J. F. (1992) Nucleic Acids Res. 20, 919 [Medline] [Order article via Infotrieve]
  10. Huletsky, A., Knox, J. R., and Levesque, R. C. (1993) J. Biol. Chem. 268, 3690-3697 [Abstract/Free Full Text]
  11. Jacoby, G. A., and Medeiros, A. A. (1991) Antimicrob. Agents Chemother. 35, 1697-1704 [Medline] [Order article via Infotrieve]
  12. Jelsch, C., Mourey, L., Masson, J.-M., and Samama, J.-P. (1993) Proteins Struct. Funct. Genet. 16, 364-383 [Medline] [Order article via Infotrieve]
  13. Juteau, J.-M., Billings, E., Knox, J. R., and Levesque, R. C. (1992) Protein Eng. 5, 693-701 [Abstract]
  14. Knox, J. R., and Moews, P. C. (1991) J. Mol. Biol. 220, 435-455 [Medline] [Order article via Infotrieve]
  15. Knox, J. R., Moews, P. C., Escobar, W. A., and Fink, A. L. (1993) Protein Eng. 6, 11-18 [Abstract]
  16. Lee, K. Y., Hopkins, J. D., O'Brien, T. F., and Syvanan, M. (1991) Proteins Struct. Funct. Genet. 11, 45-51 [Medline] [Order article via Infotrieve]
  17. Merino, E., Osuna, J., Bolívar, F., and Soberón, X. (1992) BioTechniques 12, 508-510 [Medline] [Order article via Infotrieve]
  18. Moews, P. C., Knox, J. R., Dideberg, O.and Frére, J.-M. (1990) Proteins Struct. Funct. Genet. 7, 156-171 [Medline] [Order article via Infotrieve]
  19. Sougakoff, W., Goussard, S., and Courvalin, P. (1988) FEMS Microbiol. Lett. 56, 343-348 [CrossRef]
  20. Sowek, J. A., Singer, S. B., Ohringer, S., Malley, M. F., Dougherty, T. J., Gougoutas, J. Z., and Bush, K. (1991) Biochemistry 30, 3179-3188 [Medline] [Order article via Infotrieve]
  21. Strynadka, N. C. J., Adachi, H., Jensen, S. E., Johns, K., Sielecki, A., Betzel, C., Sutoh, K., and James, M. N. G. (1992) Nature 359, 700-705 [CrossRef][Medline] [Order article via Infotrieve]
  22. Waley, S. G. (1974) Biochem. J. 139, 789-790 [Medline] [Order article via Infotrieve]

©1995 by The American Society for Biochemistry and Molecular Biology, Inc.