Autocatalytic Activation And Characterization Of A Thermophilic Glutamyl Endopeptidase From Thermoactinomyces Sp.CDF

Glutamyl endopeptidases (GSEs) specifically hydrolyze peptide bonds formed by a-carboxyl groups of Glu and Asp residues, and are related to chymotrypsin-like serine proteases. GSEs are widely distributed in bacteria, and the GSEs from pathogenic bacteria are important virulence factors. Because of their narrow substrate specificities, GSEs are very useful tools for protein structure analysis, MS-based proteomics, and site-specific cleavage of recombinant fusion peptides/proteins. Meanwhile, GSEs can be used in industrial processes such as food processing and peptide synthesis. However, most known GSEs are from mesophilic bacteria and have relatively low thermostability, which limits their applications. Identification and characterization of GSEs with high thermostability, such as those from thermophiles, will not provide insight into temperature adaptation mechanisms of microorganisms, but also broaden the application area of GSEs.In this study, we have obtained a draft genome sequence of Thermoactinomyces sp. CDF. The Thermoactinomyces sp. CDF genome encodes at least 58 protease genes, including a gene encoding a GSE (named TS-GSE). Sequence analysis of the TS-GSE gene reveals that TS-GSE is synthesized as a precursor composed of a signal peptide (30 residues), an N-terminal propeptide (61 residues), and a mature domain (218 residues). The DNA fragment encoding the TS-GSE proform, which contains the N-terminal propeptide and the mature domain, was cloned and then introduced into Escherichia coli BL21(DE3) for expression. The mature form of recombinant TS-GSE was purified and characterized. In the presence of 10 mM CaCl2, the mature TS-GSE displayed a temperature optimum of 85? and retained approximately 90% of its original activity after incubation at 70? for 6 h. In the absence of CaCl2 the enzyme retained only 20% of the original activity after incubation at 70? for 1 h, indicating that Ca2+ contributes to the thermostability of TS-GSE. Furthermore, mutational analysis showed that the disulfide bonds, Cys32-Cys48 and Cys180-Cys183, cumulatively contribute to the thermostability of TS-GSE, and Cys180-Cys183 plays a more important role in stabilizing the enzyme. Mass spectrometry analysis of the hydrolysates of oxidized insulin B-chain by TS-GSE revealed that the cleavage occurred at Glu-Xaa bonds, confirming that TS-GSE is indeed a glutamyl endopeptidase. These results demonstrate that TS-GSE represents the most thermostable GSE reported to date.We found that the TS-GSE proform possessed two cis-processing sites and was capable of autocatalytic activation via multiple pathways. The N-terminal propeptide could be cis-processed at the Glu-1 -Ser1 bond to directly generate the mature enzyme. It could also be cis-processed at the Glu-12-Lys-11 bond to yield an intermediate, which was then converted into the mature form after removal of the remaining part (11 residues) of the propeptide. The segment surrounding the two processing sites was flexible, which allowed the proform and the intermediate form to be trans-processed into the mature form by active TS-GSE that matured earlier. In addition, the flexible segment in the N-terminal propeptide of TS-GSE proform is sensitive not only to active TS-GSE but also to trypsin, proteinase K, and protease C2, which has been previously characterized to be the major extracellular protease produced by Thermoactinomyces sp. CDF. Therefore, the maturation of TS-GSE can also occur hetero-catalytically. Deletion analysis revealed that the N-terminal propeptide is important for correct folding and maturation of TS-GSE. The propeptide, even its last 11-amino acid peptide segment, could inhibit the activity of its cognate mature domain.The capacity of TS-GST to digest protein substrates was compared with that of V8 protease, a commercial GSE. The digestion pattern of ?-casein cleaved by TS-GSE was generally similar to that by V8 protease;however, compared with V8 protease, TS-GSE had an increased hydrolysis rate. The substrates BSA and ovalbumin were hardly degraded by V8 protease at 37?, most likely because the potential cleavage sites in the two globular proteins were inaccessible to the enzyme at this temperature. V8 protease was inactivated when the reaction temperature was increased to 70?, and was thus unable to cleave the two protein substrates. In contrast, TS-GSE efficiently hydrolyzed BSA and ovalbumin at 70?, implying that the thermal-induced denaturation/structural change of the substrates facilitates access of the thermostable enzyme to the cleavage sites. These results suggest that TS-GSE is a glutamyl endopeptidase capable of hydrolyzing proteins at high temperatures.