Enzyme and Microbial Technology 40 (2007) 1389–1397 A tryptophan residue is identified in the substrate binding of penicillin G acylase from Kluyvera citrophila R. Suresh Kumar a , A.A. Prabhune a , A.V. Pundle a , M. Karthikeyan b , C.G. Suresh a,∗ a Division of Biochemical Sciences, National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411008, India b Information Division, National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411008, India Received 31 July 2006; received in revised form 22 September 2006; accepted 11 October 2006 Abstract Penicillin acylases are important enzymes in pharmaceutical industry for the production of semi-synthetic -lactam antibiotics via the key intermediate 6-aminopenicillanic acid. The penicillin G acylase purified from Kluyvera citrophila (KcPGA) on modification with tryptophan- specific reagents such as N-bromo succinamide (NBS) and 2-hydroxy 5-nitrobenzylbromide (HNBB) showed partial loss of activity and substrate protection. Various solute quenchers and substrate were used to probe the microenvironment of the putative reactive tryptophan through fluorescence quenching. Homology modeling of KcPGA structure has been carried out. Docking substrate on this modeled KcPGA structure identifies the tryptophan residue that is directly influenced by substrate binding. To confirm the biological significance of this particular tryptophan, we did a sequence comparison of PGAs from various organisms. The sequence alignment clustered the matches into two sets, those closer to (>40% identical) KcPGA and had the tryptophan of interest present in them formed the first set, while those less identical (<30%) to KcPGA and the particular tryptophan absent in them formed the second set. It is clear from the reported kinetic parameters of representative members of these two sets that the affinity for penicillin G (penG) of the former class is several times better. Thus, based on our studies we suggest that the tryptophan residue in the identified position is important for binding substrate penG by the acylases. © 2006 Elsevier Inc. All rights reserved. Keywords: K. citrophila; Penicillin G acylase; Tryptophan modification; Fluorescence measurement; Substrate-docking; Sequence alignment 1. Introduction The microbial enzymes such as penicillin G acylases (peni- cillin amidohydrolases, PGAs, EC 3.5.1.11) have a high impact on the pharmaceutical industry by their application in the pro- duction of antibiotics. They are employed in the deacylation of benzyl penicillin to 6-aminopenicillianic acid (6-APA), the precursor molecule for production of semi-synthetic penicillins [1–3]. Their high efficiency has resulted in the replacement of conventional chemical processes in favor of enzymatic ones by the industry [4]. The penicillin G acylases (PGAs) have been purified and characterized from various sources [5–11]. The PGA from Kluyvera citrophila (KcPGA) has attracted attention due to its better and more suitable features for industrial applications as compared to PGA from Escherichia coli (EcPGA). It is compar- atively easier to immobilize KcPGA and shows more stability ∗ Corresponding author. Tel.: +91 20 25902236; fax: +91 20 25902648. E-mail address: cg.suresh@ncl.res.in (C.G. Suresh). towards extreme conditions of temperature, pH, and presence of organic solvents [12–15]. The stabilization of the enzyme by immobilization is also reported [16]. KcPGA is translated as a single-chain precursor consisting of 844 amino acid residues in the cytoplasm. Subsequent processing by removal of a 26 residue signal peptide and a 54 residue spacer peptide produces in periplasm the mature enzyme in the form of a heterodimer consisting of an - and a -chain of 209 and 555 amino acid residues, respectively [17]. It has been reported that altering amino acid residue Gly 21 affects protein maturation [18]. The serine residue with a newly generated free -amino group at the N-terminus of -subunit acts both as nucleophile and as base in catalysis. Thus, KcPGA can be placed in the Ntn hydro- lase family, since having Ser at the N-terminus of -chain and the modeled three-dimensional structure showing characteris- tic Ntn-hydrolase ‘–––’ tertiary fold [19,20]. There have been reports of this enzyme being cloned and overexpressed in E. coli [21,22]. The active site of the enzyme has drawn special attention due to its importance in understanding the catalytic mechanism and substrate specificity for application in protein engineering 0141-0229/$ – see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.enzmictec.2006.10.022