The extreme durability of polyethylene terephthalate (PET) debris has rendered it a long-term environmental burden. At the same time, current recycling efforts still lack sustainability. Two recently discovered bacterial enzymes that specifically degrade PET represent a promising solution. First, Ideonella sakaiensis PETase, a structurally well-characterized consensus α/β-hydrolase fold enzyme, converts PET to mono-(2-hydroxyethyl) terephthalate (MHET). MHETase, the second key enzyme, hydrolyzes MHET to the PET educts terephthalate and ethylene glycol. Here, we report the crystal structures of active ligand-free MHETase and MHETase bound to a nonhydrolyzable MHET analog. MHETase, which is reminiscent of feruloyl esterases, possesses a classic α/β-hydrolase domain and a lid domain conferring substrate specificity. In the light of structure-based mapping of the active site, activity assays, mutagenesis studies and a first structure-guided alteration of substrate specificity towards bis-(2-hydroxyethyl) terephthalate (BHET) reported here, we anticipate MHETase to be a valuable resource to further advance enzymatic plastic degradation.
"Proton-collecting antenna" are conjectured to consist of several carboxylates within hydrogen-bond (HB) networks on the surface of proteins, which funnel protons to the orifice of an internal proton wire leading to the protein's active site. Yet such constructions were never directly visualized. Here we report an X-ray structure of green fluorescent protein (GFP) of the highest resolution to date (0.9 A). It allows the identification of some pivotal hydrogen atoms pertinent to uncertainties concerning the protonation state of the chromophore. Applying a computer algorithm for mapping proton wires in proteins reveals the previously observed "active site wire" connecting Glu222 with the surface carboxylate Glu5. In addition, it is now possible to identify what appears to be a proton-collecting apparatus of GFP. It consists of a negative surface patch containing carboxylates, threonines, and water molecules, connected by a HB network to Glu5. Furthermore, we detect exit points via Asn146 and His148 to a hydrophobic surface region. The more extensive HB network of the present structure, as compared with earlier GFP structures, is not accidental. A systematic investigation of over 100 mutants shows a clear correlation between the observed water content of GFP X-ray structures and their resolution. With increasing water content, the proton wires become progressively larger. These findings corroborate the scenario in which the photodissociated proton from wild-type GFP can leak outside, whereafter another proton is recruited via the proton-collecting apparatus reported herein.
Escherichia coli asparaginase |I catalyzes the hydrol-) sis of L-asparagine to L-aspartate via a threonine-bound acylenzyme intermediate. A nearly inactive mutant in which one of the active site threonines, Thr-89, was replaced by valine was constructed, expressed, and crystallized. Its structure, solved at 2.2 A resolution, shows high overall similarity to the wild-type enzyme, but an aspartyl moiety is covalently bound to Thr-12, resembling a reaction intermediate. Kinetic analysis confirms the deacylation deficiency, which is also explained on a structural basis. The previously identified oxyanion hole is described in more detail.:£ey words'." Asparaginase II; Acyl-enzyme intermediate; !'hreonine amidohydrolase; Enzymatic mechanism !. IntroductionAsparaginases catalyze the hydrolysis of L-asparagine to L~spartate and ammonia. The enzymes isolated from Escheri-'hia coli (EcA) and Erwinia chrysanthemi (ERA) have been ltilized as anti-leukemia drugs for many years [1]. Treatment vith asparaginases is often accompanied by severe side effects, vhich are partially attributed to the glutaminase activity of hese enzymes [2]. In order to understand the enzyme specifi-:ity and to ultimately eliminate the glutaminase activity, the nechanism of action must be elucidated. Several members of t larger family of homologous L-asparaginases have thus been horoughly investigated over many years.Results of kinetic measurements [3,4] indicated that the en-,ymatic reaction proceeds via a covalent intermediate, probtbly a ~-aspartyl enzyme (Fig. 1). This mechanism was con-,irmed by NMR studies with aspartate through the oxygen ~xchange reaction with bulk solvent at the side chain carboxdate [5]. These experiments showed that at pH < 5 L-aspartate ~ith a protonated side chain binds to the enzyme as tightly as ~-asparagine and it can also form an acyl-enzyme intermediate, subsequently hydrolyzed to L-aspartate. Thus, at low pH 3oth L-aspartate and L-asparagine can function as substrates. l'he nature and identity of the primary nucleophile of the mzyme participating in the formation of the acyl-enzyme in-*Corresponding author. Fax: (1) . Each has the same tetrameric quaternary structure as it has in solution, a homotetramer of approx. 4x330 residues. The tetramer is more accurately described as a dimer of dimers. Each of two identical active sites in each dimer is formed by both monomers. In the structure the active sites have been observed with and without aspartate as ligand. Part of the active site is covered by a flexible loop that contains the two important residues Thr-12 and Tyr-25. The hydroxyl groups of Thr-12 and Thr-89 are closest to the side chain carboxylate of an aspartate bound in the active site. These residues are the most likely candidates for the primary nucleophile. Bacterial L-asparaginases were the first threonine amidohydrolases described in the literature. Only recently, two other threonine amidohydrolases, 20S proteasome [14] and aspartylglucosaminidase [15], have been reported. Both enzymes belong t...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.