The nucleotide sequence of dthA
was analyzed using BLAST (1
) and found to be weakly related to a putative x-prolyl dipeptidyl peptidase from Delftia acidovorans
SPH-1 (36% identity). Blast and PSI-BLAST (1
) analysis also yielded similarity to four proteins of known function belonging to the α/β hydrolase fold family of enzymes. This diverse family includes proteases, esterases, and lipases, among other functions (9
). An alignment of the deduced amino acid sequence of dthA
with these four sequences was performed using Clustal X version 1.83 (15
) and is shown in Fig. . The results suggested the presence of conserved residues in DthA which are characteristic of α/β hydrolases and which form part of a Ser-His-Asp catalytic triad in previously characterized proteins (9
). Residues S166 and D292 from DthA aligned with the serine and aspartic acid residues that have been identified, respectively, as the nucleophile and the acidic residues that form part of the catalytic triad in the homologous proteins (2
). No histidine residues from DthA aligned with the catalytic histidine residues in the Clustal X alignment. However, four iterations of PSI-BLAST produced alignments where H320 from DthA aligned with the catalytic histidine of its homologues (data not shown).
FIG. 3. Partial multiple sequence alignment of DthA with glutaryl 7-aminocephalosporanic acid acylase from Brevibacillus laterosporus J1 (BlGAA), cocaine esterase from Rhodococcus sp. strain MB1 (RhCOCE), and amino acid ester hydrolases from Xanthomonas citri (more ...)
To investigate the involvement of a nucleophilic serine in catalysis, the effect of three serine protease inhibitors was investigated with cell extracts of E. coli pGEM-dthA. The extracts were incubated in phosphate buffer with the inhibitors aprotinin (15.3 mM), PMSF (5 mM), and AEBSF (2 mM) for 15 min at 30°C. DEET hydrolysis activity was then assayed as described above and compared to that of a control without inhibitors.
Aprotinin (15.3 mM) did not have a significant effect on DEET hydrolysis. PMSF, which acts by sulfonylating the serine residue in the active site of serine hydrolases, decreased activity by only 36 ± 9%. Interestingly, PMSF has also been reported to cause weak or no inhibition of other hydrolases that share homology with DthA (2
). AEBSF, however, which has a mechanism of inhibition similar to that of PMSF, completely inhibited DEET hydrolysis by DthA. It is not clear why AEBSF completely inhibited activity in DthA whereas PMSF did not.
Although the inhibition by AEBSF is consistent with the presence of a serine nucleophile in the active site and the in silico results are suggestive of a Ser-His-Asp catalytic triad, a more in-depth investigation of the catalytic residues in DthA using site-directed mutagenesis is needed in order to confirm the role of these residues in DEET hydrolysis.