Active-site serine mutants of the <i>Streptomyces albus</i> G <i>β</i>-lactamase

Jacob, F; Joris, Bernard; Frère, Jean‐Marie

doi:10.1042/bj2770647

Cited by 22 publications

(16 citation statements)

References 33 publications

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“…To investigate, variants of the clinically important OXA‐48 and KPC‐2 SBLs were prepared in which the nucleophilic serine was replaced with a cysteine. Consistent with previous reports on the cysteine variants of SBLs, OXA‐48 S70C ( k cat / K M 0.098±0.017 μ m −1 s −1 with the fluorogenic cephem substrate FC5) was not as catalytically active as the wild‐type enzyme ( k cat / K M 0.36±0.06 μ m −1 s −1 with FC5; Supporting Information, Table S1). However, the impact of this substitution on the enzymatic mechanism of the SBLs has not been previously described.…”

Section: Figuresupporting

confidence: 90%

Non‐Hydrolytic β‐Lactam Antibiotic Fragmentation by l,d‐Transpeptidases and Serine β‐Lactamase Cysteine Variants

et al. 2019

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Enzymes often use nucleophilic serine,t hreonine, and cysteine residues to achieve the same type of reaction;the underlying reasons for this are not understood. While bacterial d,d-transpeptidases (penicillin-binding proteins) employ an ucleophilic serine, l,d-transpeptidases use an ucleophilic cysteine.T he covalent complexes formed by l,d-transpeptidases with some b-lactam antibiotics undergo non-hydrolytic fragmentation. This is not usually observed for penicillinbinding proteins,o rf or the related serine b-lactamases. Replacement of the nucleophilic serine of serine b-lactamases with cysteine yields enzymes whichf ragment b-lactams via as imilar mechanism as the l,d-transpeptidases,i mplying the different reaction outcomes are principally due to the formation of thioester versus ester intermediates.The results highlight fundamental differences in the reactivity of nucleophilic serine and cysteine enzymes,a nd imply new possibilities for the inhibition of nucleophilic enzymes.Nature employs enzymes with nucleophilic serine,t hreonine,orcysteine residues to catalyze closely related reactions. Well-known examples of this are the serine and cysteine proteases,i nw hich these different nucleophilic residues are used to cleave peptide bonds.A na nalogous situation exists for the transpeptidase enzymes involved in bacterial peptidoglycan biosynthesis;the d,d-transpeptidases (or penicillinbinding proteins;P BPs) employ an ucleophilic serine, whereas the l,d-transpeptidases (Ldts) employ anucleophilic cysteine.T he reasons for the use of ap articular nucleophilic residue in ap articular enzyme context are not understood.

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Section: Figuresupporting

confidence: 90%

Non‐Hydrolytic β‐Lactam Antibiotic Fragmentation by l,d‐Transpeptidases and Serine β‐Lactamase Cysteine Variants

et al. 2019

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“…Evidence presented indicated that an A/G wobble with a seryl-tRNA at the first position of the codon-anticodon interaction was responsible for a limited amount of mistranslation, resulting in production of a small pool of wildtype serine-containing enzyme. A similar phenomenon has been noted by other authors (15). The data presented here are strongly suggestive of a similar mechanism for production of LPase activity in E. coli IT41 and of the Ts growth and preprotein processing phenotype, although it is likely that mistranslation occurs at a rate higher than 0.1% in this case, as the wild-type levels of LPase in E. coli have been estimated to be in the region of 1,000 molecules per cell (40).…”

Section: Discussionmentioning

confidence: 42%

Molecular cloning and expression of the spsB gene encoding an essential type I signal peptidase from Staphylococcus aureus

1996

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The gene, spsB, encoding a type I signal peptidase has been cloned from the gram-positive eubacterium Staphylococcus aureus. The gene encodes a protein of 191 amino acid residues with a calculated molecular mass of 21,692 Da. Comparison of the protein sequence with those of known type I signal peptidases indicates conservation of amino acid residues known to be important or essential for catalytic activity. The enzyme has been expressed to high levels in Escherichia coli and has been demonstrated to possess enzymatic activity against E. coli preproteins in vivo. Experiments whereby the spsB gene was transferred to a plasmid that is temperature sensitive for replication indicate that spsB is an essential gene. We identified an open reading frame immediately upstream of the spsB gene which encodes a type I signal peptidase homolog of 174 amino acid residues with a calculated molecular mass of 20,146 Da that is predicted to be devoid of catalytic activity.

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“…This scenario has been supported by structural analysis of the S70G mutant of TEM-1 (46), and has been observed in the mechanistically-related serine proteases (47). In several cases, it was shown that residual activity is the result of a small sub-population of wild-type enzyme produced through the mistranslation of the mutant codon (43, 48). This is not likely the case for OXA-1 S67G, however, as the nitrocefinase activity we observe displays a different K m compared to that seen with wild-type (49).…”

Section: Discussionmentioning

confidence: 99%

Mutation of the Active Site Carboxy-Lysine (K70) of OXA-1 β-Lactamase Results in a Deacylation-Deficient Enzyme

et al. 2009

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Class D β-lactamases hydrolyze β-lactam antibiotics by using an active site serine nucleophile to form a covalent acyl-enzyme intermediate, and subsequently employ water to deacylate the β-lactam and release product. Class D β-lactamases are carboxylated on the ε-amino group of an active site lysine, with the resulting carbamate functional group serving as a general base. We discovered that substitutions of the active site serine and lysine in OXA-1 β-lactamase, a monomeric class D enzyme, significantly disrupt catalytic turnover. Substitution of glycine for the nucleophilic serine (S67G) results in an enzyme that can still bind substrate but is unable to form a covalent acyl-enzyme intermediate. Substitution of the carboxylated lysine (K70), on the other hand, results in enzyme that can be acylated by substrate, but is impaired for deacylation. We employed the fluorescent penicillin BOCILLIN FL™ to show that three different substitutions for K70 (alanine, aspartate and glutamate) accumulate significant acyl-enzyme intermediate. Interestingly, BOCILLIN FL™ deacylation rates vary depending on the identity of the substituting residue, from t1/2 ≈ 60 min for K70A to undetectable deacylation for K70D. Tryptophan fluorescence spectroscopy was used to confirm that these results are applicable to natural (i.e. non-fluorescent) substrates. Deacylation by K70A, but not K70D or K70E, can be partially restored by the addition of short-chain carboxylic acid mimetics of the lysine carbamate. In conclusion, we establish the functional role of the carboxylated lysine in OXA-1 and highlight its specific role in acylation and deacylation.

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Active-site serine mutants of the Streptomyces albus G β-lactamase

Cited by 22 publications

References 33 publications

Non‐Hydrolytic β‐Lactam Antibiotic Fragmentation by l,d‐Transpeptidases and Serine β‐Lactamase Cysteine Variants

Non‐Hydrolytic β‐Lactam Antibiotic Fragmentation by l,d‐Transpeptidases and Serine β‐Lactamase Cysteine Variants

Molecular cloning and expression of the spsB gene encoding an essential type I signal peptidase from Staphylococcus aureus

Mutation of the Active Site Carboxy-Lysine (K70) of OXA-1 β-Lactamase Results in a Deacylation-Deficient Enzyme

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