Overproduction of bovine {beta}-casein in Escherichia coli and engineering of its main chymosin cleavage site

A cDNA clone containing the entire coding region for bovineß-casein A³ flanked by 53 base pairs of 5' non-codingand 358 base pairs of 3' non-coding sequences was isolated froma bovine mammary cDNA phagemid library. The coding segment formature ß-casein was subcloned into the T7 expressionsystem, in which the expression of recombinant ß-caseinwas controlled by the T7 gene 10 promoter and ribosome bindingsite. High level expression of Met-ß-casein to 20%of the total soluble proteins was obtained in Escherichia coliwithin 2 h after induction of T7 RNA-polymerase synthesis. Inan attempt to induce secretion the coding segment for matureß-casein was coupled to the ompA translations initiationsignal and signal peptide coding sequence but no secretion ofthe fusion protein and no processing of the signal peptide fromthe fusion protein was observed. Instead, the Met-ß-caseincould be isolated in asoluble form from E.coli cells after anosmotic shock, indicative of a periplasmic location. This proceduredid not lyse the cells. The protein was purified to homogeneityafter a pH 4.8 isoelectric precipitation followed by reversed-phasehigh-performance liquid chromatography. The ß-caseincDNA was altered to change the main chymosin cleavage siteinß-casein at position 192–193 in two ways, namelyfrom Leu–Tyr to Pro–Pro and to Leu–stop. Thesemutations were designed to prevent generation of the bitterpeptide ßcasein(193–209) by chymosin cleavage.The mutant Met-ß-caseins were expressed in E.colito the same level as wild-type Met-ß-casein. Purifiedmutant Met-ß-casein(Prol92– Prol93) was no longerhydrolysed by chymosin at the 192–193 bond.     

Expression and characterization of Lactobacillus 30a histidine decarboxylase in Escherichia coli

The genes coding for histidine decarboxylase from a wild-typestrain and an autoactivation mutant strain of Lactobacillus30a have been cloned and expressed in Escherichia coli. Themutant protein, G58D, has a single Asp for Gly substitutionat position 58. The cloned genes were placed under control ofthe ß-galactosidase promoter and the products arenatural length, not fusion proteins. The enzyme kinetics ofthe proteins isolated from E. coli are comparable to those isolatedfrom Lactobacillus 30a. At pH 4.8 the K_m of wild-type enzymeis 0.4 mM and the k_cat = 2800 min^–1; the correspondingvalues for G58D are 0.5 mM and 2750 min^–1. The wild-typeand G58D have autoactivation half-times of 21 and 9 h respectivelyunder pseudophysiological conditions of 150 mM K⁺ and pH 7.0.At pH 7.6 and 0.8 M K⁺ the half times are 4.9 and 2.9 h. Therelatively slow rate of autoactivation for purified proteinand the differences in cellular and non-cellular activationrates, coupled with the fact that wild-type protein is readilyactivated in wild-type Lactobacillus 30a but poorly activatedin E. coli, suggest that wild-type Lactobacillus 30a containsa factor, possibly an enzyme, that enhances the activation rate.     

In vivo copper- and cadmium-binding ability of mammalian metallothionein domain

The ß domain of mouse metallothionein 1 (ßMT) wassynthesized in Escherichia coli cells grown in the presenceof copper or cadmium. Homogenous preparations of Cu–ßMTand Cd–ßMT were used to characterize the correspondingin vivo-conformed metal-clusters, and to compare them with thespecies obtained in vitro by metal replacement to a canonicalZn₃–ßMT structure. The copper-containing ßMTclusters formed inside the cells were very stable. In contrast,the nascent ß peptide, although it showed cadmium bindingability, produced a highly unstable species, whose stoichiometrydepended upon culture conditions. The absence of ßMT proteinin E.coli protease-proficient hosts grown in cadmium-supplementedmedium pointed to drastic proteolysis of a poorly folded ßpeptide, somehow enhanced by the presence of cadmium. Possiblefunctional and evolutionary implications of the bioactivityof mammalian ßMT in the presence of monovalent and divalentmetal ions are discussed.     

Enzymatic amidation of recombinant (Leu27) growth hormone releasing hormone-Gly45

By chemoenzymatic synthesis the gene for a (Leu²⁷) analogueof human growth hormone releasing hormone-Gly⁴⁵ [(Leu²⁷GHRH-Gly⁴⁵]was constructed, cloned and expressed in Escherichia coli asa fusion protein with ß-galactosidase under the controlof the lac promoter and operator. Upon induction with isopropyl-D-thio-ß-galactopyranosidethe fusion protein accumulated to a yield of 15–20% ofthe total cellular protein. After cyanogen bromide deavage ofthe fusion protein the precursor peptide (Leu²⁷)hGHRH-Gly⁴⁵was separated by extraction and purified by ion exchange andh.p.l.c.-RP18 chromatography. The purified peptide was analysedby sequencing, isolectric focusing, amino acid analysis andamino acid analysis after V8 protease digestion. The carboxy-terminalglydne was subsequently amidated by PAM (peptidylglycine--amidating-monooxygenase),an enzyme which was isolated and characterized from fresh bovinepituitaries. Correct amidatlon of the penultimate amino acid,leucine, was verified by peptide sequencing with an authenticleucine amide reference.     

The structure of E.coli soluble inorganic pyrophosphatase at 2.7 A resolution

Hie structure of E.coli soluble inorganic pyrophosphatase hasbeen refined at 2.7 resolution to an R-factor of 20.9. Theoverall fold of the molecule is essentially the same as yeastpyrophosphatase, except that yeast pyrophosphatase is longerat both the N- and C-termini. Escherichia coli pyrophosphataseis a mixed +ß protein with a complicated topology.The active site cavity, which is also very similar to the yeastenzyme, is formed by seven ß-strands and an -helixand has a rather asymmetric distribution of charged residues.Our structure-based alignment extends and improves upon earliersequence alignment studies; it shows that probably no more than14, not 15–17 charged and polar residues are part of theconserved enzyme mechanism of pyrophosphatases. Six of theseconserved residues, at the bottom of the active site cavity,form a tight group centred on Asp70 and probably bind the twoessential Mg⁺ ions. The others, more spreadout and more positivelycharged, presumably bind substrate. Escherichia coli pyrophosphatasehas an extra aspartate residue in the active site cavity, whichmay explain why the two enzymes bind divalent cation differently.Based on the structure, we have identified a sequence motifthat seems to occur only in soluble inorganic pyrophosphatases.     

Weak points of antiparallel {beta}-sheets. How are they filled up in globular proteins?

A lack of knowledge about the construction of tight packingis now the main obstacle for a successful design of artificialproteins. In this paper we examine a way of close packing antiparallelß-sandwihes. We show that there are some ‘weakpoints’ at the surfaces of ß-sheets, which cannotbe filled by the surrounding aliphatic side chains that arethe most abundant. Theoretically, these ‘weak points‘can be filled either by aromatic side chains of the same sheetor by the residues of the other parts of the protein molecule.The analysis of protein structures shows that both possibilitiesare used by nature and that there are many cases when these‘weak points’ are not filled by any atom. They remainfree and form a majority of the defects of close packing inprotein globules.     

Expression of synthetic genes coding for completely new, nutritionally rich, artificial proteins

Synthetic genes (A, AB and AHB) constructed and cloned intopKK233-2 vector were recloned from the parent plasmid into thenew procaryotic expression vectors pGFY221N and pBIO52. GeneAF^–B (coding for all amino acids besides phenylalanine)was obtained by ‘cassette mutagenesis’ from geneAB. The plasmid pGFY221N was constructed from pGFY218L by replacingthe PstI by an NcoI site; plasmid pBIO52 was derived from pGFY221Nthrough replacing the 221-bp EoRl/NcoI fragment with a syntheticDNA segment of 52 bp representing the Escherichia coli atpEgene translational initiation region. The genes A, AB, AHB andAF^–B in the vector pGFY221N were expressed with a six-amino-acid-longleader sequence; in pBIO52 the genes were expressed directly.in vitro expression experiments were successful with all thegenes except with the AHB gene integrated into pGFY221N. Inthe E. coli minicell system expression was demonstrated withthe A gene in pGFY221N and the AF^–B and AHB genes in pBIO52.Complete translation of the expressed genes AB, AF^–B andAHB in either the in vitro or in vivo systems could be shownby using ³⁵S-labelled N-terminal methionine and C-terminal cysteine.Both amino acids occur only once in the peptide sequences.     

Expression of deletion constructs of bovine {beta}-1, 4-galactosyltransferase in Escherichia coli: importance of Cysl34 for its activity

Bovine ß-1, 4-galactosyltransferase (ß-1,4-GT; EC 2.4.1.90 [EC] ) belongs to the glycosyltransferase familyand as such shares a general topology: an N-terminal cytoplasmictail, a signal anchor followed by a stem region and a catalyticdomain at the C-tenninal end of the protein. cDNA constructsof the N-terminal deleted forms of ß-1, 4-GT wereprepared in pGEX-2T vector and expressed in E.coli as glutathione-S-transferase(GST) fusion proteins. Recombinant proteins accumulated withininclusion bodies as insoluble aggregates that were solubilizedin 5 M guanidine HCl and required an ‘oxido-shuffling’reagent for regeneration of the enzyme activity. The recombinant(ß-1, 4-GT, devoid of the GST domain, has 30–85%of the sp. act. of bovine milk ß-1, 4-GT with apparentK_ms for N-acetylglucosamine and UDP-galactose similar to thoseof milk enzyme. Deletion analysesshow that both (ß-1,4-GT and lactose synthetase activities remain intact even inthe absence of the first 129 residues (pGT-dl29). The activitiesare lost when either deletions extend up to residue 142 (pGT-dl42)or Cysl34 is mutatedto Ser (pGT-dl29C134S). These results suggestthat the formation of a disulfide bond involving Cysl34 holdsthe protein in a conformation that is required for enzymaticactivity.     

Mutant forms of {beta}-galactosidase with an altered requirement for magnesium ions

By random approaches we have previously isolated many variantsof Escherichia coli ß-galactosidase within a shortcontiguous tract near the N-terminus (residues 8–12 ofwildtype enzyme), some of which have increased stability towardsheat and denaturants. The activity of these mutants was originallyanalysed and quantitated in situ in activity gels without theaddition of magnesium ions to the buffer system. We now showthat the improved stability is only observable under such conditionsof limiting magnesium ion concentrations or in the presenceof appropriate concentrations of a metal chelator. In the presenceof EDTA, purified preparations of one of these mutant enzymeswere much more resistant to denaturants than wild-type, butthis differential was completely nullified in the presence of1 mM Mg²⁺. However, the stability of this mutant enzyme in EDTAwas lower than that shown by it, or the wild-type enzyme, inthe presence of magnesium ions. In addition, certain alterationswithin another N-terminal tract (residues 27–31 of wild-type)resulted in enzymes with greater dependence on Mg²⁺ than naturalß-galactosidase. We conclude that a small number ofresidue changes in a large protein can profoundly modulate therequirement for metal ion stabilization, allowing partial abrogationof this need in certain cases. Thus, some enzymes which requiredivalent metal ions for structural purposes only may be engineeredtowards metal independence.     

The cystine knot of a squash-type protease inhibitor as a structural scaffold for Escherichia coli cell surface display of conformationally constrained peptides

The Ecballium elaterium trypsin inhibitor II (EETI-II), a memberof the squash family of protease inhibitors, is composed of28 amino acid residues and is a potent inhibitor of trypsin.Its compact structure is defined by a triple-stranded antiparallelß-sheet, which is held together by three intramoleculardisulfide bonds forming a cystine knot. In order to explorethe potential of the EETI-II peptide to serve as a structuralscaffold for the presentation of randomized oligopeptides, weconstructed two EETI-II derivatives, where the six-residue inhibitorloop was replaced by a 13-residue epitope of Sendai virus L-proteinand by a 17-residue epitope from human bone Gla-protein. EETI-IIand derived variants were produced via fusion to maltose bindingprotein MalE. By secretion of the fusion into the periplasmicspace, fully oxidized and correctly folded EETI-II was obtainedin high yield. EETI-II and derived variants could be presentedon the Escherichia coli outer membrane by fusion to truncatedLpp'–OmpA', which comprises the first nine residues ofmature lipoprotein plus the membrane spanning ß-strandfrom residues 46–66 of OmpA protein. Gene expression wasunder control of the strong and tightly regulated tetA promoter/operator.Cell viability was found to be drastically reduced by high levelexpression of Lpp'–OmpA'–EETI-II fusion protein.To restore cell viability, net accumulation of fusion proteinin the outer membrane was reduced to a tolerable level by introductionof an amber codon at position 9 of the lpp' sequence and utilizingan amber suppressor strain as expression host. Cells expressingEETI-II variants containing an epitope were shown to be surfacelabeled with the respective monoclonal antibody by indirectimmunofluorescence corroborating the cell surface exposure ofthe epitope sequences embedded in the EETI-II cystine knot scaffold.Cells displaying a particular epitope sequence could be enriched10⁷-fold by combining magnetic cell sorting with fluorescence-activatedcell sorting. These results demonstrate that E.coli cell surfacedisplay of conformationally constrained peptides tethered tothe EETI-II cystine knot scaffold has the potential to becomean effective technique for the rapid isolation of small peptidemolecules from combinatorial libraries that bind with high affinityto acceptor molecules.     

A new protein conjugate that replaces the use of secondary antibodies engineered from the two staphylococcal enzymes protein A and 6-phospho-{beta}-galactosidase

The lacG gene encoding the 6-phospho-ß-galactosidase(E.C.3.2.1.85) of Staphylococcus aureus was fused to the proteinA gene in the plasmid pRIT2T. Escherichia coli cells containingthis plasmid produce a fusion protein with both IgG bindingand 6-phospho-ß-galactosidase activities after heatinduction. The recombinant gene was overexpressed and the hybridprotein was purified to homogeneity in high yield. The chimericprotein was shown to have almost identical enzymatic characteristicsto pure 6-phospho-ß-galactosidase. This result leadsto the conclusion that a free N-terminus of the 6-phospho-ß-galactosidaseis not required for biological activity. The hybrid proteinof protein A and 6-phospho-ß-galactosidase was usedas an enzyme conjugate in enzyme-linked immunosorbent assays(ELISA). The experiments presented demonstrate that the 6-phospho-ß-galactosidaseis a suitable fusion partner in various diagnostic applicationswhere an unique biological activity is required.     

6-Phospho-{beta}-galactosidases of Gram-positive and 6-phospho-{beta}-glucosidase B of Gram-negative bacteria: comparison of structure and function by kinetic and immunological methods and mutagenesis of the lacG gene of staphylococcus aureus

The 6-phospho-ß-galactosidase of Staphylococcus aureus,Lactococcus lactis and Lactobacillus casei and 6-phospho-ßglucosidaseB of Escherichia coli build a subfamily inside a greater enzymefamily, named the glycosal hydrolase family 1, which, hi addition,contains nine ß-glycosidases of different origins.Kinetic and immunological evidence is provided in this reportwhich strengthens the relationship of the four 6-phospho-ß-glycosidases.It is shown that the 6-phospho-ß-galactosidases and6-phospho-ß-glucosidase B are able to split aromaticß-galactoside phosphates and ß-glucosidephosphates. The turnover numbers of hydrolysis of substrateswith different epimerization at C-4 of the glycon vary up to15-fold only. Two polydonal antisera, one derived against thenative 6-phospho-ß-galactosidase from S.aureus andthe other derived against the 6-phospho-ß-glucosidaseB, cross-reacted with both enzymes. Peptides of the proteinswere separated by reverse phase HPLC. The cross-reacting peptideswere sequenced and shown to be localized at almost the sameposition in the aligned primary structures of both enzymes.An insertion of nine amino adds near these antigenic domainsis unique for the 6-phospho-ß-glycosidases and missingwithin the sequences of the ß-glycoside-specific membersof the family. The lacG gene of a 6-phospho-ß-galactosidasenegative S.aureus mutant was doned into E.coli and sequenced.In the totally inactive mutant protein only the glycine at position332 was changed to an arginine. This amino acid is part of thesequence insertion near the antigenic domain reacting with bothantisera. These data support the assumption that the regionis of great importance for the function of the enzymes and thatit is possible it determines the specificity of the phosphorylatedform of the substrates. In addition, the 6-phospho-ß-galactosidaseof S.aureus was modified by sitedirected mutagenesis of thecorresponding lacG gene hi order to replace residues Glul60and Glu375, which were suspected of being involved hi the generalacid catalysis of substrate hydrolysis, with glutamine residues.The mutant protein 160EQ retained some catalytic activity whilethe protein 375EQ was totally inactive. Glu375 is the activesite nudeophile of the 6-phospho-ß-galactosidase ofS.aureus. It is located in the sequence motif ENG where Glu358was identified as the catalytkally active nudeophile hi theß-glucosidase of Agrobacterium.     

Internalization and translocation of a new chimeric protein composed of Pseudomonas aeruginosa exotoxin A and mouse dihydrofolate reductase as a model system

In an attempt to introduce a large peptide that is not normallytranslocated across membranes into the cytosol of eukaryoticcells, we created a new chimeric protein termed CEDH betweenPseudomonas aeruginosa exotoxin A (ETA) and a variant enzymeof Mus musculus dihydrofolate reductase (DHFR) with reducedaffinity for antifolates, ETA^1–413.DHFR^1–187.ETA^609–613.We have defined, genetically constructed and expressed the chimericprotein in Escherichia coli. We showed that the CEDH chimericprotein, purified to homogeneity on an immunoaffinity resin,confers a methotrexate-resistant phenotype to Chinese hamsterovary cells. Furthermore, the chimeric protein allowed the growthof dihydrofolate reductase-deficient Chinese hamster ovary cellsin the absence of hypoxanthine and thymidine. These resultsdemonstrated that the chimeric protein exhibited enzyme activityand possessed the tightly folded native structure, and thatthe DHFR protein can be selectively internalized and translocatedvia domains of exotoxin A. These data show that the ETA systemis an efficient system for the delivery of a variety of largepolypeptides into the cytosol without stress to the target cells,and extends the use of this delivery system to proteins thatare not normally translocated across membranes.     

Replacing the ({beta}{alpha})-unit 8 of E.coli TIM with its chicken homologue leads to a stable and active hybrid enzyme

In order to investigate how structural modifications interferewith protein stability, we modified a (ß)-unit inE.coli triosephosphate isomerase (TIM), a typical (ß)-barrelprotein, assuming that the pseudosymmetrical ß-barrelcan be divided into eight successive loop/ß-strand/loop/-helixmotifs. We replaced the eighth (ß)-unit of E.coliTIM with the corresponding chicken (ß)-unit. The substitution,involving the replacement of 10 of the 23 residues of this (ß)-unit, was evaluated first by modelling, then experimentally.Modelling by bomology suggests how the amino add replacementsmight be accommodated in the hybrid E.coli/chicken TIM (ETCM8CHI).Both natural and hybrid recombinant TIMs, overproduced in E.coli,were purified to homogeneity and characterized as to their stabilityand kinetics. Our kinetic studies show that the modificationperformed here leads to an active enzyme. The stability studiesindicate that the stability of ETIM8CHI is comparable to thatof the wild type TIM.     

New secretory strategies for Kluyveromyces lactis {beta}-galactosidase

We examined several strategies for the secretion of Kluyveromyceslactis ß-galactosidase into the culture medium, inorder to facilitate the downstream processing and purificationof this intracellular enzyme of great industrial interest. Weconstructed plasmids by fusing the LAC4 gene or engineered variantsto the secretion signal of the K.lactis killer toxin or to thesecretion signal of the Saccharomyces cerevisiae -factor. Withthese plasmids we transformed strains of the yeasts K.lactisand S.cerevisiae, respectively and tested ß-galactosidaseextracellular activity in different culture media. We achievedpartial secretion of ß-galactosidase in the culturemedium since the high molecular weight and oligomeric natureof the enzyme, among other factors, preclude full secretion.The percentage of secretion was improved by directed mutagenesisof the N-terminus of the protein. We developed several deletionmutants which helped us to propose structure–functionrelationships by comparison with the available data on the homologousEscherichia coli ß-galactosidase. The influence ofthe culture conditions on heterologous ß-galactosidasesecretion was also studied.     

Synthesis of a novel histidine analogue and its efficient incorporation into a protein in vivo

Proteins containing unnatural amino acids have immense potentialin biotechnology and medicine. We prepared several histidineanalogues including a novel histidine analogue, ß-(1,2,3-triazol-4-yl)-DL-alanine.These histidine analogues were assayed for translational activityin histidine-auxotrophic Escherichia coli strain UTH780. Weobserved that several histidine analogues, including our novelhistidine analogue, were efficiently incorporated into the proteinin vivo; however, other analogues were rejected. These resultssuggest that the hydrogen atom at a specific position seriouslyaffects incorporation. Received April 10, 2003; revised June 20, 2003; accepted July 22, 2003.     

Site-directed mutagenesis of pseudoazurin from Alcatigenes faecalis S-6; Pro80Ala mutant exhibits marked increase in reduction potential

Pseudoazurin (a blue copper protein or cupredoxin) of a denitrifyingbacterium Alcaligenes faecalis S-6 is a direct electron carrierfor a Cu-containing nitrite reductase (NIR) of the same organism.Site-directed mutagenesis of the pseudoazurin was carried outusing an Escherichia coli expression system. Replacement ofTyr74 by Phe to remove an internal hydrogen bond in the ß-barrelcaused a slight decrease in heat stability as well as a requirementfor a higher concentration of Cu²⁺ for production in the E.colihost. Exchange of Ala for Pro80 adjacent to His81, one of thefour ligands binding a type I Cu atom, caused a marked increasein reduction potential by 139 mV without change in the opticalabsorption spectrum. The ability of the pseudoazurin to transferelectrons to NIR was markedly diminished but the apparent K_mof NIR for pseudoazurin was not affected by the mutation. X-raydiffraction data collected on the oxidized and reduced formsof the Pro80Ala mutant show that a water molecule occupies thepocket created by the absent side chain. This observation suggeststhat the increase in reduction potential may be caused due tothe increased solvent accessibility to the Cu atom. The electrondensity difference maps on these structures (at 2.0 Å)show that this water moves during the change in oxidation state,and that there are small, but localized, conformational changes>6.5 Å from the copper site, as well as movement ofboth the Cu²⁺ and the cysteinate sulfur.     

Cloning and expression in E.coli of a synthetic gene for the bacteriocidal protein caltrin/seminalplasmin

A synthetic gene coding for the bacteriocidal protein caltrin/seminalplasminwas constructed and expressed in Escherichia coli as a fusionwith ß-galactosidase. The gene was designed with arecognition site for the plasma protease, Factor X_a, coded forimmediately prior to the N-terminus of caltrin. The ß-galactosidase-caltrinfusion protein was cleaved with Factor X_a to give caltrin, whichwas identified by its size on SDS-PAGE, its ability to reactwith an antiserum raised to the N-terminal nonapeptide of caltrinand its N-terminal amino acid sequence. After partial purification,synthetic caltrin was found to be active in an assay involvinginhibition of growth of E.coli.     

Structure-function discrimination of the N- and C- catalytic domains of human angiotensin-converting enzyme: implications for Cl- activation and peptide hydrolysis mechanisms

Human somatic angiotensin I-converting enzyme (sACE) has twoactive sites present in two sequence homologous protein domains(ACE_N and ACE_C) possessing several biochemical features thatdifferentiate the two active sites (i.e. chloride ion activation).Based on the recently solved X-ray structure of testis angiotensin-convertingenzyme (tACE), the 3D structure of ACE_N was modeled. Electrostaticpotential calculations reveal that the ACE_N binding grooveis significantly more positively charged than the ACE_C, whichprovides a first rationalization for their functional discrimination.The chloride ion pore for Cl2 (one of the two chloride ionsrevealed in the X-ray structure of tACE) that connects the externalsolution with the inner part of the protein was identified onthe basis of an extended network of water molecules. Comparisonof ACE_C with the X-ray structure of the prokaryotic ClC Cl^–channel from Salmonella enterica serovar typhimurium demonstratesa common molecular basis of anion selectivity. The criticalrole for Cl2 as an ionic switch is emphasized. Sequence andstructural comparison between ACE_N and ACE_C and of other proteinsof the gluzincin family highlights key residues that could beresponsible for the peptide hydrolysis mechanism. Currentlyavailable mutational and substrate hydrolysis data for bothdomains are evaluated and are consistent with the predictedmodel. Received May 23, 2003; revised October 16, 2003; accepted October 21, 2003     

An engineered Staphylococcus aureus PCI {beta}-lactamase that hydrolyses third-generation cephalosporins

The ß-lactamase from Staphylococcus aureus PCI hasbeen cloned into an Escherichia coli vector for site-directedmutagenesis and high-level protein expression. A mutant enzymehas been produced in which Ala238 is replaced by a serine, andIle239 is deleted (A238S:I239del). The engineered enzyme hydrolysesthird-generation cephalosporins substantially more rapidly thanthe parental enzyme does, while hydrolysis of benzylpenicillinis slower with the mutant than with the wild-type and nativeenzymes. The mutant P-lactamase has been crystallized and thestructure determined and refined at 2.8 A resolution. The dispositionof the ß-strand which forms the side of the activesite is altered in comparison with the native S.aureus ß-lactamasestructure, widening the active site cleft and providing spaceto accommodate the bulky side-chains of the third-generationcephalosporins.