A common precursor for the two subunits of the penicillin acylase from Escherichia coli ATCC11105

Oliver, G.; Valle, Fernando; Rosetti, F.; Gomez-Pedrozo, Marisa; Santamaría, Patricia; Gosset, Guillermo; Bolívar, Francisco

doi:10.1016/0378-1119(85)90018-6

Cited by 27 publications

(10 citation statements)

References 17 publications

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“…Some details of this processing have been inferred from the complete nucleotide sequence of the gene from E. coli ATCC 11105, together with partia1 amino acid sequencing (Schumacher et al, 1986): removal of a signal peptide (26 residues) and a spacer endopeptide (54 residues) leaves an NH2-terminal Msubunit (residues 27 -235) and a P-subunit (residues 290 ~ 846). This is supported by other partial (Bruns et al, 1985;Oliver et al, 1985) and complete (Oh et al, 1987;Guo et al, 1989) sequencing of DNA from the same or similar strains of E. coli. Similar processing has been reported for penicillin G acylase from other organisms (Daumy et al, 1985;Barber0 et al, 1986;Ohashi et al, 1988) and for cephalosporin acylases (Matsuda et al, 1987a, b) although a bacilliary penicillin V acylase appears to be an unrelated homotetramer (Olsson & U h l h , 1986;Olsson et al, 1985).…”

supporting

confidence: 66%

Site‐directed chemical conversion of serine to cysteine in penicillin acylase from Escherichia coli ATCC 11105

Slade¹,

Horrocks²,

Lindsay³

et al. 1991

European Journal of Biochemistry

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Penicillin acylase (EC 3.5.1.11) was completely inactivated with equimolar phenylmethane [35S]sulphonyl fluoride (PhMe35SO2F); the stability of the sulphonyl group in the modified protein was determined by measurement of the radioactivity in ultrafiltrates. In 8 M urea, the rate of loss of the sulphonyl group was similar to that observed in PhMeSO2F‐inactivated chymotrypsin [Gold, A. M. & Fahrney, D. (1964) Biochemistry 3, 783–791]. Incubation of the PhMeSO2F‐inactivated acylase with 0.7 M potassium thioacetate yielded an acetylthiol enzyme which was subsequently converted to a thiol‐enzyme during incubation with 10 mM 6‐aminopenicillanic acid. 4‐Pyridyl‐ethylcysteine was released by acid hydrolysis after reaction of the thiol‐protein with 4‐vinylpyridine. The rates of reaction of thiol‐penicillin acylase with iodoacetic acid and 2,2′‐dipyridyl disulphide were consistent with the presence of an incompletely accessible cysteinyl sidechain. After carboxymethylating the thiol‐enzyme with iodo[2‐3H]acetic acid, the label was shown by SDS/PAGE and sequencing analysis to be associated exclusively with the β‐chain NH2‐terminal residue, indicating conversion of Ser290 to S‐carboxymethyl‐cysteine. Near‐ultraviolet CD spectra showed the conformation of thiol‐penicillin acylase to be indistinguishable from that of the native protein but the catalytic activity was less than 0.02% of that of the normal enzyme. The possibility that Ser290 acts as a nucleophile in catalysis is discussed.

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supporting

confidence: 66%

Site‐directed chemical conversion of serine to cysteine in penicillin acylase from Escherichia coli ATCC 11105

Slade¹,

Horrocks²,

Lindsay³

et al. 1991

European Journal of Biochemistry

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“…In addition to this homology, other sequence boxes have been shown to be conserved in all Keck et al (1985). Bold letters represent the conserved amino acids among the different proteins compared according to Oliver et al (1985) these proteins. A putative penicillin binding site has been found in the PA sequence in accordance with the conditions of Joris et al (1988) and where the essential serine corresponds to Ser372 of the beta subunit in PA from K. citrophila.…”

Section: Discussionmentioning

confidence: 99%

“…Williams and Zuzel (1985) showed that mutations in Met168 of PA from E. coli produced changes in substrate specificity. Moreover, Oliver et al (1985) have proposed an amino acid homology between E. coli penicillin binding proteins (PBPs) and a region around Met168 in the small subunit of K. citrophila PA. Assuming functional homology for this residue in E. coli and K. citrophila enzymes, we have mutated Met168 in PA from the latter.…”

Section: Introductionmentioning

confidence: 99%

Penicillin acylase mutants with altered site-directed activity from Kluyvera citrophila

Priéto¹,

Martín

Arche

et al. 1990

Appl Microbiol Biotechnol

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Oligonucleotide-directed mutagenesis has been used to obtain specific changes in the penicillin acylase gene from Kluyvera citrophila. Wild-type and mutant proteins were purified and the kinetic constants for different substrates were determined. Mutations in Met168 highly decreased the specificity constant of the enzyme for penicillin G, penicillin V and phenylacetyl-4-aminobenzoic acid and the catalytic constant kcat for phenylacetyl-4-aminobenzoic acid. Likewise, the phenylmethylsulphonyl-fluoride sensitivity was significantly decreased. It is concluded that the 168 residue is involved in binding by interaction with the acid moiety of the substrate. A putative penicillin-binding domain was located in penicillin acylase by sequence homology with other penicillin-recognizing enzymes. Lys374 and His481, the conserved amino acid residues that are essential for catalysis in these enzymes, can be changed in penicillin acylase with no changes to the kcat and phenylmethylsulphonyl fluoride reactivity, but change the Km. The likelihood of the existence of this proposed penicillin binding site is discussed. The reported results might be used to alter the substrate specificity of penicillin acylase in order to hydrolyse substrates of industrial significance other than penicillins.

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“…2 2 methods 2 2 1 Essay of pemcdhn amldase acttvtty Pemalhn amtdase acttvrty was routmely essayed on a synthetic substrate, 6-mtro, 3-phenylacetammobenzoic acid (NIPAB) The reaction was followed by measurmg the absorbance of the solutton at 405 nm [9] The acttvny umt of penicillin amidase (U) was determmed as the amount (nanomol) of product formed per ml during 1 mm Durmg immobihsatton of the enzyme on an msoluble support, the solutton was shaken and filtered before the absorbance was measured Correspondence address. N Burteau, Umverstte Cathohoue de Louvain, Umte de Blocmmse, Place Louis Pasteur, I, B-1348 LOuvmn-laNeuve, Belgium 2 2 2 Pemcdhn amtdase purification Abbrevratrons NIPAB, 6-mtro, 3-phenylacetammobenzotc acid, Abbrevmtlons.…”

Section: Methodsmentioning

confidence: 99%

Stabilisation and immobilisation of penicillin amidase

1989

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Pemcdlm amldase was coupled to a penodate-oxrdtsed dextran by reducttve alkylatron m the presence of sodmm cyanoborohydnde A loss of acttvtty (25%) was observed but the conIugate enzyme dextran was more thermostable than the native enzyme Native and dextran-conjugated pemctlhn am&se were rmmobthsed on ammo acttvated sthca (Promaxon, Spherostl, Aerostl) by a classical method usmg ~ut~aldehyde for the nattve enzyme and reducttve alkylatton for the mod&cd enzyme Good relanve acttvrty of the enzymes was obtained after ln~lubill~tion Immobthsatton of both native and mod&d enzymes resulted m the the~ostablhsatlon of the pemcdhn amtdase Enzyme stabrhsatton, Enzyme tmmobthsatton, Pemcdhn amrdase, (Escherrchm co/i)

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A common precursor for the two subunits of the penicillin acylase from Escherichia coli ATCC11105

Cited by 27 publications

References 17 publications

Site‐directed chemical conversion of serine to cysteine in penicillin acylase from Escherichia coli ATCC 11105

Site‐directed chemical conversion of serine to cysteine in penicillin acylase from Escherichia coli ATCC 11105

Penicillin acylase mutants with altered site-directed activity from Kluyvera citrophila

Stabilisation and immobilisation of penicillin amidase

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