Autoproteolytic Activation of ThnT Results in Structural Reorganization Necessary for Substrate Binding and Catalysis

Abstract

cis-Autoproteolysis is a post-translational modification necessary for the function of ThnT, an enzyme involved in the biosynthesis of the β-lactam antibiotic thienamycin. This modification generates an N-terminal threonine nucleophile that is used to hydrolyze the pantetheinyl moiety of its natural substrate. We determined the crystal structure of autoactivated ThnT to 1.8 Å through X-ray crystallography. Comparison to a mutationally inactivated precursor structure revealed several large conformational rearrangements near the active site. To probe the relevance of these transitions, we designed a pantetheine-like chloromethyl ketone (CMK) inactivator and co-crystallized it with ThnT. Although this class of inhibitor has been in use for several decades, the mode of inactivation had not been determined for an enzyme that uses an N-terminal nucleophile. The co-crystal structure revealed the CMK bound to the N-terminal nucleophile of ThnT through an ether linkage and analysis suggests inactivation through a direct displacement mechanism. More importantly, this inactivated complex shows three regions of ThnT that are critical to the formation of the substrate binding pocket undergo rearrangement upon autoproteolysis. Comparison of ThnT with other autoproteolytic enzymes of disparate evolutionary lineage revealed a high degree similarity within the pro-enzyme active site, reflecting shared chemical constraints. However, after autoproteolysis many enzymes, like ThnT, are observed to rearrange to accommodate their specific substrate. We propose this is a general phenomenon, whereby autoprocessing systems with shared chemistry may possess similar structural features that dissipate upon rearrangement into a mature state.

Intrinsic evolutionary constraints on protease structure, enzyme acylation, and the identity of the catalytic triad

“…5, the γ-methyl accelerates catalysis by intrinsically favoring the reactive g− rotamer. These rotamer distributions may be further shifted by interactions with residues proximal to the active site (48). This rationale explains the reduction in k cat observed upon Thr→Ser mutation of PβS (20) and glycosylasparaginase (44).…”

The solution of nitrogen inversion in amidases

“…A. An enzyme assisted hydrogen bond donated by the reacting NH group of the pantetheinylated carbapenem substrate and accepted by S320 has been suggested for the enzyme ThnT . ThnT is involved in antibiotic synthesis and contains an N‐terminal Thr dyad with a distinct fold compared with the proteasome β‐unit and penicillin acylase.…”

Exploring the Role of Conformational Heterogeneity in cis-Autoproteolytic Activation of ThnT

“…This unanticipated conformation was identical to that observed in the postautoproteolysis structure of wild-type ThnT and is necessary for substrate binding in the mature enzyme. 34 Stepwise inclusion of higher-resolution data during model refinement resulted in numerous peaks in the F o – F c map indicative of structural heterogeneity (Figure 3 C). Given that modeling of weak density results in a phase bias, we were concerned that modeling with multiple conformations would be impossible.…”

“…The trans isomer dominates in the structure of T282A and is the only one observed in the postautoproteolysis wild-type enzyme. 34 The cis isomer is more prevalent in the T282C structure, and we hypothesized that it facilitates efficient autoprocessing. Specifically, the data show that the backbone carbonyl of Gly77 in the cis isomer positions a water molecule to aid in the activation of the nucleophile for the N → O acyl shift.…”

“…Expression and purification of all ThnT proteins were similar to reported procedures. 34 Proteins were bound to a Ni-nitriloacetate column and eluted in 250 mM imidazole, 50 mM sodium phosphate (pH 7.5), 40 mM NaCl, and 10% glycerol. Protein was concentrated in a 10 KDa molecular weight cutoff Amicon filtration device (Bio-Rad) and buffer exchanged into 10 mM potassium phosphate (pH 7.5) with 10% glycerol (for kinetic experiments) or 20 mM potassium phosphate (pH 7.5) with 5% glycerol (for crystallography).…”