Intramolecular Processing of Prothermolysin

Abstract

Thermolysin, an extracellular zinc endopeptidase from Bacillus thermoproteolyticus, is synthesized as a pre-proenzyme and the prosequence has been shown to assist the refolding of the denatured enzyme in vitro and to inhibit enzyme activity (O'Donohue, M. J., and Beaumont, A. (1996) J. Biol. Chem. 271, 26477-26481). To determine whether prosequence cleavage from the mature enzyme is autocatalytic and if so, whether it is an intermolecular or intramolecular process, N-terminal histidine-tagged prothermolysin was expressed in Escherichia coli. Although partial processing to mature enzyme occurred, most of the proenzyme was recovered intact from inclusion bodies. This was then solubilized in guanidinium hydrochloride, immobilized on a cobaltcontaining resin, and after dialysis against renaturation buffer, was quantitatively transformed to mature enzyme. However, when a mutation was introduced into the mature sequence to inactivate thermolysin, the proenzyme was not processed either in vivo or in vitro. In addition, mutated prothermolysin was not processed by exogenous thermolysin under a variety of experimental conditions. The results demonstrate that thermolysin maturation can proceed via an autocatalytic intramolecular pathway.Thermolysin (EC 3.4.24.27), a 34.6 kDa extracellular neutral protease produced by the Gram-negative Bacillus thermoproteolyticus, is the prototype member of the M4 family of zinc endopeptidases (1). This thermostable enzyme was the first zinc endopeptidase to be crystallized, and subsequent crystallographic studies have led to a mechanism of action being proposed for the zinc metallopeptidases in general (reviewed in Ref. 2). An active site model of thermolysin has also been used as a template for the design of inhibitors for several mammalian zinc metallopeptidases, for which structural data is lacking, such as neutral endopeptidase-24.11 (EC 3.4.24.11), angiotensin-converting enzyme (EC 3.4.15.1) and endothelinconverting enzyme (reviewed in Refs. 3 and 4). The M4 enzymes are synthesized as pre-proproteins (5, 6), with thermolysin itself having a 27-residue N-terminal pre-sequence, followed by a 204-residue prosequence and the 316-residue mature enzyme (7). As compared with other enzyme classes (reviewed in Refs. 8 and 9), there have been comparatively few studies on the roles of prosequences of bacterial zinc peptidases, although they have been shown to be essential for the production of active enzyme in vivo (10 -13) probably by facilitating folding (14). The prosequences, as independent polypeptides, can also act as inhibitors of their respective mature enzymes (14,15).From site-directed mutagenesis studies of the M4 enzymes, it has been inferred that prosequence processing is autocatalytic, as mutations of active site residues often result in low or negligible yields of mature enzyme (16 -19). In addition, certain mutations in the active site of thermolysin that change specificity lead to the production of more than one enzyme species differing by their N-terminals.1 For these reasons ...

Structure, function and latency regulation of a bacterial enterotoxin potentially derived from a mammalian adamalysin/ADAM xenolog

“…S2D). Taken together, all these results indicate that directly expressed fragilysin-3 is unstable and strongly point to a role for the PD, in addition to latency maintenance, as a chaperone that assists in the folding and stabilization of CD, as previously reported for other MP zymogens (14,15). In the absence of this chaperone, fragilysin-3 CD is not able to fold correctly in the environment provided by the bacterial expression host.…”

Unconventional Activation Mechanisms of MMP-26, a Human Matrix Metalloproteinase with a Unique PHCG XXD Cysteine-switch Motif

“…It is likely that similarly to bacterial metalloproteinases (e.g. prothermolysin), which do not exhibit the cysteine-switch motif, proMMP-26 can naturally mature via an autocatalytic intramolecular pathway (25). Furthermore, it is tempting to hypothesize that if the fold of proMMP-26 is relatively similar to that of other MMPs, the four histidine residues existing in proMMP-26 (the three from the active site domain and the one from the cysteine-switch motif) may all directly interact with the zinc atom similarly to those observed in the Zn 2ϩ finger motifs.…”

Structural basis for the autoprocessing of zinc metalloproteases in the thermolysin family

“…How it functions, however, is still unknown. Biochemical studies have revealed that the maturation of TLPs occurs through autoprocessing and that this autoprocessing pathway is mediated by the propeptide (14,15). The propeptide in TLPs and in some serine proteases is also referred to as an intramolecular chaperone (IMC), as it is essential for the folding of the catalytic domain but is not required for its function (16).…”