Protein engineering of penicillin acylase - PubMed (original) (raw)

Protein engineering of penicillin acylase

V I Tishkov et al. Acta Naturae. 2010 Jul.

Abstract

Penicillin acylases (PA) are widely used for the production of semi-synthetic β-lactam antibiotics and chiral compounds. In this review, the latest achievements in the production of recombinant enzymes are discussed, as well as the results of PA type G protein engineering.

Keywords: E.coli; expression; penicillin acylase; protein engineering; structure.

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Figures

Fig. 1

Fig. 1

Modern classification of β-lactam acylases according to the type of substrate [7]. 7-ACA – 7-aminocephalosporanic acid

Fig. 2

Fig. 2

Alignment of amino acid sequences of penicillin acylases G from different sources. Catalytic βSer1 residue and residues from the oxyanion hole are marked by “*” and “#”, respectively. Sequences of the signal peptide and intersubunit spacer are underlined in bold italic, and underlined, respectively.

Fig. 3

Fig. 3

Phylogenetic tree of penicillin acylases G.

Fig. 4

Fig. 4

Structure of the active heterodimer of PA-G from E.coli (pdb 1pnk) [24]. α- and β-subunits are shown in yellow and dark blue, respectively. Catalytic βSer1 residue and Ca 2+ ion are shown in red and green, respectively.

Fig. 5

Fig. 5

Structure of the active heterodimer of PA-G from A .faecalis (pdb 3K3W). α- and β-subunits are shown in yellow and dark blue, respectively. Catalytic βSer1 residue and Ca 2+ ion are shown in red and green, respectively. Disulfide bond in β-subunit between residues Cys492 and Cys525 in wild-type enzyme is shown in magenta. Insert in right part of figure shows fixation of N-terminus of α-subunit and C-terminus of β-subunit due to creation of new disulfide bond (shown in orange) after double mutation αQ3C/βP751C.

Fig. 6

Fig. 6

Main amino acid residues in the active site of PA from E. coli (structure PDB 1AI4). Catalytic βSer1 residue and residue βGln23 are shown in red and orange, respectively. Residues from oxyanion hole are presented in magenta. Residues from substrate-binding domain are shown in green and yellow for subdomains S1 and S2, respectively. Residue αPhe146 belonging to both subdomains is in blue.

Scheme. 1

Scheme. 1

A minimal scheme for half-synthetic β-lactam antibiotics preparation by nucleophilic substitution. E, S, and ES are the enzyme, substrate (donor of acyl moiety), and the enzyme-substrate complex, respectively. EA, Nu, and EANu are the acyl-enzyme, nucleophile, and the double complex of acyl and nucleophile. K s , K n, and K p - are binding constants of substrate free enzyme E, the nucleophile with acyl-enzyme EA, and the product with free enzyme E, respectively.

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