The twin arginine translocation (tat) machinery (Ignatova et al., 2002) followed by autocatalytic intramolecular peptide-bond cleavage. This autocatalytic processing removes a 26-residue signal peptide anddoi:10.1107/S174430911301943X# 2013 International Union of Crystallography All rights reservedActa Cryst. (2013). F69, 925crystallization communicationsa 54-residue linker peptide and results in the formation of active enzyme in the periplasm, which is a heterodimer of and chains of 209 and 557 amino-acid residues, respectively (Choi et al., 1992; Bock et al., 1983; Oh et al., 1987). Basically identified as a member of the Ntn hydrolase superfamily (Brannigan et al., 1995), KcPGA, like EcPGA, is translated as an inactive precursor (pre-pro-PGA). The overall sequence identity between EcPGA and KcPGA is 87 ( chain, 84.2 ; chain, 87.6 ; spacer peptide, 90.7 ). Cleavage of the Thr289 er290 bond leads to the unveiling of the primary amine group of Ser 1 (Ser290 of the precursor), creating the active centre in mature PGA. The rate-limiting step in the production of active enzyme is the intramolecular autoproteolytic processing of the precursor molecule and the final removal of the linker peptide (Kasche et al., 1999; Hewitt et al., 2000; Done et al., 1998). Lee et al. (2000) showed that in vitro processing of the precursor PGA from E. coli was analogous to that observed in in vivo studies and depended on the pH in the same manner, with an optimum processing pH in the physiological range 6.4.0. A sequence alignment of precursor proteins from four Gram-negative and two Grampositive bacteria identified a conserved lysine residue (Lys299). Sitedirected mutation of this Lys, which is sequentially close to the -chain N-terminal serine residue (Ser290), and study using GSTprecursor PGA fusion protein further confirmed that the Lys residue is the most probable candidate responsible for the pH-dependent activation. Therefore, activation may involve Lys299 and Ser290 as critical residues for autocatalytic processing of the PGA precursor (Lee et al., 2000). These residues are also conserved in KcPGA. The same mechanism, pH and temperature dependence of precursor autocatalytic processing to yield a processed form is known in other enzymes (Bron et al., 1998; Little, 1993; Guan et al., 1998). Understanding the three-dimensional structure of the precursor and processing intermediates may unravel the mechanism of action and the post-translational processing of the industrially useful KcPGA enzyme. 1986; accession No. M15418). Cleavage sites for the restriction endonucleases NdeI and XhoI, shown in bold, were included in the sense (50 -CAAGAGGATCATATGAAAAATAGAAATCGTATGATCGTG-30 ) and antisense (50 -GCCGAACTCGAGGCGCTGTACCTGCAGCACTT-30 ) primer sequences, respectively.AMPC The PCR products were digested using the corresponding restriction enzymes, purified by gel electrophoresis and inserted into the plasmid pET26b(+) (EMD Biosciences/Novagen, USA).Fmoc-Pro-OH The ligation products were used to transform NovaBlue competent cells resistant to kanamycin.PMID:24120168 Recombinant plasmids were isolated and their sequencing confirmed the success of the cloning experiment. This plasmid pET26-KcPGA was then used as a template for the preparation of the mutant Ser290Gly (Ser 1Gly) using the QuikChange site-directed mutagenesis kit (Stratagene, USA). Forward sense (50 -CTACCCGACCACTGGCAATATGTGGGTG-30 ) and reverse antisense (50 -CACCCACATATTGCCAGTGGTCGGGTAG-30 ) primers were used for mut.