Structural and kinetic studies on ligand binding in wild-type and active-site mutants of penicillin acylase

Abstract

Penicillin acylase catalyses the condensation of Calpha-substituted phenylacetic acids with beta-lactam nucleophiles, producing semi-synthetic beta-lactam antibiotics. For efficient synthesis a low affinity for phenylacetic acid and a high affinity for Calpha-substituted phenylacetic acid derivatives is desirable. We made three active site mutants, alphaF146Y, betaF24A and alphaF146Y/betaF24A, which all had a 2- to 10-fold higher affinity for Calpha-substituted compounds than wild-type enzyme. In addition, betaF24A had a 20-fold reduced affinity for phenylacetic acid. The molecular basis of the improved properties was investigated by X-ray crystallography. These studies showed that the higher affinity of alphaF146Y for (R)-alpha-methylphenylacetic acid can be explained by van der Waals interactions between alphaY146:OH and the Calpha-substituent. The betaF24A mutation causes an opening of the phenylacetic acid binding site. Only (R)-alpha-methylphenylacetic acid, but not phenylacetic acid, induces a conformation with the ligand tightly bound, explaining the weak binding of phenylacetic acid. A comparison of the betaF24A structure with other open conformations of penicillin acylase showed that betaF24 has a fixed position, whereas alphaF146 acts as a flexible lid on the binding site and reorients its position to achieve optimal substrate binding.

Improving the diastereoselectivity of penicillin G acylase for ampicillin synthesis from racemic substrates

“…2. Their aromatic rings are known to interact with the phenylacetyl side chain of substrates and to limit the size of the C a -substituent that can bind in phenylacetic acid derivatives (Done et al, 1998;Alkema et al, 2004). bPhe71 is known to undergo a change in conformation upon binding with large substrates and forms a stacked conformation with thiazolidine ring of the b-lactam nucleus.…”

“…Additionally, bPhe71 and aPhe146 are known to be structurally linked via a calcium ion (Alkema et al, 2000;McVey et al, 2001). bPro22 is known to be a part of the hydrophobic substratebinding pocket (Alkema et al, 2004).…”

“…Svedas, et al completed an extensive study on the substrate specificity of wild-type PGA and found that PGA did not have any significant stereospecificity for substrates with the polar groups -OH or -NH 2 at the C a position (Svedas et al, 1996). Alkema et al studied the selectivity of wild-type PGA and the PGA variants bPhe24Ala, aPhe146Tyr and the double variant bPhe24Ala/aPhe146Tyr with various C a -substituted substrates, though the selectivity with an amino group substituent was not studied (Alkema et al, 2004). The selectivity of wild-type PGA was found to be dependent on the size and polarity of the substituent.…”

Protein Engineering of Penicillin Acylase

“…Domain S2 consists of the following residues: αArg145, αPhe146, βPhe71, and βArg263 (marked yellow in Fig. 6) [76-80]. …”

Development of immobilized systems with penicillin acylase from Esherichia coli for production of semisyntetic penicillins

“…Neki od njih su bili sledeći: kristalizacija proizvoda u integrisanim reaktorima, primena pH gradijenta u toku šaržnog procesa, modulacija svojstava enzima kovalentnom imobilizacijom, povećanje afiniteta enzima prema acil donorima sa C-substituentima preko mutageneze usmerene položajem, povećanje enantioselektivnosti PAC iz različitih mikroorganizama pomoću imobilizacije, primena membranskih neizotermnih reaktora, korišćenje dvofaznih sistema, ko-rastvarači, korastvarači i niske temperature ili čak primena zamrznutih medijuma [24,57,104,152,154,171,173,176,[188][189][190][191][192][193][194][195].…”