Substitutions of Thr-103-Ile and Trp-138-Gly in Amidase from Pseudomonas aeruginosa Are Responsible for Altered Kinetic Properties and Enzyme Instability

Abstract

Pseudomonas aeruginosa Ph1 is a mutant strain derived from strain AI3. The strain AI3 is able to use acetanilide as a carbon source through a mutation (T103I) in the amiE gene that encodes an aliphatic amidase (EC 3.5.1.4). The mutations in the amiE gene have been identified (Thr103Ile and Trp138Gly) by direct sequencing of PCR-amplified mutant gene from strain Ph1 and confirmed by sequencing the cloned PCR-amplified gene. Site-directed mutagenesis was used to alter the wild-type amidase gene at position 138 for Gly. The wild-type and mutant amidase genes (W138G, T103I-W138G, and T103I) were cloned into an expression vector and these enzymes were purified by affinity chromatography on epoxy-activated Sepharose 6B-acetamide/phenylacetamide followed by gel filtration chromatography. Altered amidases revealed several differences in kinetic properties, namely, in substrate specificity, sensitivity to urea, optimum pH, and enzyme stability, compared with the wild-type enzyme. The W138G enzyme acted on acetamide, acrylamide, phenylacetamide, and p-nitrophenylacetamide, whereas the double mutant (W138G and T103I) amidase acted only on p-nitrophenylacetamide and phenylacetamide. On the other hand, the T103I enzyme acted on p-nitroacetanilide and acetamide. The heat stability of altered enzymes revealed that they were less thermostable than the wild-type enzyme, as the mutant (W138G and W138G-T103I) enzymes exhibited t1/2 values of 7.0 and 1.5 min at 55 degrees C, respectively. The double substitution T103I and W138G on the amidase molecule was responsible for increased instability due to a conformational change in the enzyme molecule as detected by monoclonal antibodies. This conformational change in altered amidase did not alter its M(r) value and monoclonal antibodies reacted differently with the active and inactive T103I-W138G amidase.

Structure of Amidase from Pseudomonas aeruginosa Showing a Trapped Acyl Transfer Reaction Intermediate State

“…aeruginosa is a mesophilic bacterium with optimal growth at 37°C (2). However, wild-type amidase exhibits a very high heat stability at 55°C (19), making this enzyme unusually thermostable for this class of bacteria. From an industrial point of view, thermozymes offer major biotechnological advances over mesophilic enzymes (59), and a detailed understanding of the underlying interactions that define protein stability is essential to design more stable proteins.…”

“…Thermostability seems to depend on several structural features, such as the hydrophobic effect, H-bonding and salt bridges, surface charged residue distribution, and packing effects and amino acid composition in the protein interior (59,60). The unusually high stability of the homo-hexameric amidase particles (19) has raised the hypothesis that this property might have resulted from putative inter-molecular disulfide bridges. However, no disulfide bridge was detected in the three-dimensional model and, furthermore, the distribution of the cystein residues precludes any inter-molecular disulfide bond, as the minimal distance between inter-molecular cysteine sulfur atoms is 10.5 Å. Additionally, the closest intra-molecular distance between cysteine C␣s (residues 166 and 198) is about 6 Å, but their side chains point away from each other.…”

“…Site-directed mutagenesis studies have identified amino acid residues involved in the enzyme activity or in substrate specificity (13,19,20). Single point mutations in P. aeruginosa amidase enabled the hydrolysis of longer aliphatic amides such as butyramide and valeramide (2,21) or aromatic amides like phenylacetamide (22) and acetanilide (23).…”

Crystallization, diffraction data collection and preliminary crystallographic analysis of hexagonal crystals ofPseudomonas aeruginosaamidase

“…The wild-type amidase from P. aeruginosa was extracted from cells and purified by a twostep procedure involving affinity and gel-filtration chromatography as described previously (Domingos et al, 1989;Karmali et al, 2001;Martins et al, 2005) with some modifications. The elution of amidase activity from the affinity column (epoxy-activated Sepharose 6B acetamide) was carried out with a linear gradient of acetamide and hydroxylamine (0-40 mM) in 20 mM Tris-HCl buffer pH 7.2 containing 1 mM -mercaptoethanol, 1 mM EDTA, 10%(v/v) glycerol and 1 mM benzamidine (TMEGB).…”

“…Although the detailed catalytic mechanism is still not totally understood (Findlater & Orsi, 1973;Woods et al, 1979;Novo et al, 2002), this enzyme has attracted great attention because of the variations in its substrate and inhibitor specificities that can be generated by single-point mutations (Tata et al, 1994;Karmali et al, 2000Karmali et al, , 2001Gregoriou & Brown, 1979, 1980. Microbial amidases with altered substrate specificities can be used in several industrial applications such as the detoxification of industrial effluents containing toxic amides and the production of hydroxamic acids and other organic acids (Fournand et al, 1998).…”