Changing glycine 21 for glutamic acid in the β-subunit of penicillin G acylase from Kluyvera citrophila prevents protein maturation

Priéto, Ignacio; Rodrı́guez, Marı́a del Carmen; Márquez, Gabriel; Pérez-Aranda, A.; Barbero, José Luís

doi:10.1007/bf00183245

Cited by 13 publications

(10 citation statements)

References 21 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…Until now, no direct evidence exists about all amino acids participating in the autocatalytic maturation process. Possible candidates, such as the N‐terminal SN sequence (Ser264, Asn265) of the B‐chain, and Gly284 [28], are fully conserved in both penicillin amidases (Fig. 1).…”

Section: Sequence Alignment and Comparison With E Coli Penicillin Ammentioning

confidence: 99%

“…Amino acid sequence in a single letter code 11-mer QAGTQDLAHVS 20-mer QAGTQDLAHVSSPVLATELE 29-mer QAGTQDLAHVSSPVLATELERQDKHWGGR 37-mer QAGTQDLAHVSSPVLATELERQDKHWGGRGPDFAPKA Possible candidates, such as the N-terminal SN sequence (Ser264, Asn265) of the B-chain, and Gly284 [28], are fully conserved in both penicillin amidases ( Fig. 1).…”

Section: Length Of the Oligopeptidementioning

confidence: 99%

See 1 more Smart Citation

Fragments of pro‐peptide activate mature penicillin amidase of Alcaligenes faecalis

Kasche

Galunsky

Ignatova

2003

European Journal of Biochemistry

View full text Add to dashboard Cite

Penicillin amidase from Alcaligenes faecalis is a recently identified N-terminal nucleophile hydrolase, which possesses the highest specificity constant (k cat /K m ) for the hydrolysis of benzylpenicillin compared with penicillin amidases from other sources. Similar to the Escherichia coli penicillin amidase, the A. faecalis penicillin amidase is maturated in vivo from an inactive precursor into the catalytically active enzyme, containing one tightly bound Ca 2+ ion, via a complex post-translational autocatalytic processing with a multi-step excision of a small internal pro-peptide. The function of the pro-region is so far unknown. In vitro addition of chemically synthesized fragments of the pro-peptide to purified mature A. faecalis penicillin amidase increased its specific activity up to 2.3-fold. Mutations were used to block various steps in the proteolytic processing of the pro-peptide to obtain stable mutants with covalently attached fragments of the pro-region to their A-chains. These extensions of the A-chain raised the activity up to 2.3-fold and increased the specificity constants for benzylpenicillin hydrolysis mainly by an increase of the turnover number (k cat ).

show abstract

Section: Sequence Alignment and Comparison With E Coli Penicillin Ammentioning

confidence: 99%

Section: Length Of the Oligopeptidementioning

confidence: 99%

Fragments of pro‐peptide activate mature penicillin amidase of Alcaligenes faecalis

Kasche

Galunsky

Ignatova

2003

European Journal of Biochemistry

View full text Add to dashboard Cite

show abstract

“…Rational protein engineering of penicillin acylases from Kluyvera citrophila, Escherichia coli , and Pseudomonas has been used to obtain insight into the function8–11 and the maturation pathway of this enzyme 11, 12. A hypermutagenic E. coli strain was used to expand the substrate specificity of the K. citrophila acylase 13.…”

Section: Enzymes As Biocatalystsmentioning

confidence: 99%

Directed Evolution and Biocatalysis

Powell

Ramer

Cardayré

et al. 2001

Angew. Chem. Int. Ed.

217

View full text Add to dashboard Cite

This review describes the current state of biocatalysis in the chemical industry. Although we recognize the advantages of chemical approaches, we suggest that the use of biological catalysis is about to expand dramatically because of the recent developments in the artificial evolution of genes that code for enzymes. For the first time it is possible to consider the rapid development of an enzyme that is designed for a specific chemical reaction. This technology offers the opportunity to adapt the enzyme to the needs of the process. We describe herein the development of enzyme evolution technology and particularly DNA shuffling. We also consider several classes of enzymes, their current applications, and the limitations that should be addressed. In a review of this length it is impossible to describe all the enzymes with potential for industrial exploitation; there are other classes, which given appropriate activity, selectivity, and robustness, could become useful tools for the industrial chemist. This is an exciting era for biocatalysis and we expect great progress in the future.

show abstract

“…Durch rationales Protein-Engineering der Penicillinacylasen von Kluyvera citrophila, Escherichia coli und Pseudomonas konnte man Einblicke in die Funktion [8±11] und den Reifungsprozess dieses Enzyms [11,12] gewinnen. Mit einem hypermutagenen E.-coli-Stamm wurde die Substratspezifität der K.-citrophila-Acylase erweitert.…”

Section: Acylierungsreaktionenunclassified

“…Mit einem hypermutagenen E.-coli-Stamm wurde die Substratspezifität der K.-citrophila-Acylase erweitert. [11,12] Anschlieûend wurden Wirt-Vektor-Systeme für eine verbesserte Acylaseproduktion entwickelt, diese jedoch verursachten die Bildung von Einschlusskörpern. Dabei ergaben sich kleine Verbesserungen der enzymatischen Aktivität.…”

Section: Acylierungsreaktionenunclassified