A Bound Water Molecule Is Crucial in Initiating Autocatalytic Precursor Activation in an N-terminal Hydrolase

Yoon, Jongchul; Oh, Bora; Kim, Kyunggon; Park, Jungeun; Han, Dohyun; Kim, Kyeong Kyu; Shin, Sun; Lee, Dongsoon; Kim, Youngsoo

doi:10.1074/jbc.m309281200

Cited by 19 publications

(19 citation statements)

References 27 publications

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“…To rectify the perplexing mechanistic implications suggested by these structures, we hypothesize a peptide flip places the reactive carbonyl into the conserved oxyanion hole, expels the "bound" water and positions the nucleophile for si-face attack. Our hypothesis is further supported by observations in CA, where the inactive F177P variant compresses the active site and expels the bound water (43). Instead of disrupting self-cleavage by removing an inappropriately polarized water molecule, the observed compression may prevent a peptide flip necessary for formation of a cleavage-competent state.…”

Section: Evidence For Hidden Conformational Rearrangement Preceding Ntnsupporting

confidence: 69%

Insights into cis-autoproteolysis reveal a reactive state formed through conformational rearrangement

Buller

Freeman

Wright

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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ThnT is a pantetheine hydrolase from the DmpA/OAT superfamily involved in the biosynthesis of the β-lactam antibiotic thienamycin. We performed a structural and mechanistic investigation into the cis-autoproteolytic activation of ThnT, a process that has not previously been subject to analysis within this superfamily of enzymes. Removal of the γ-methyl of the threonine nucleophile resulted in a rate deceleration that we attribute to a reduction in the population of the reactive rotamer. This phenomenon is broadly applicable and constitutes a rationale for the evolutionary selection of threonine nucleophiles in autoproteolytic systems. Conservative substitution of the nucleophile (T282C) allowed determination of a 1.6-Å proenzyme ThnT crystal structure, which revealed a level of structural flexibility not previously observed within an autoprocessing active site. We assigned the major conformer as a nonreactive state that is unable to populate a reactive rotamer. Our analysis shows the system is activated by a structural rearrangement that places the scissile amide into an oxyanion hole and forces the nucleophilic residue into a forbidden region of Ramachandran space. We propose that conformational strain may drive autoprocessing through the destabilization of nonproductive states. Comparison of our data with previous reports uncovered evidence that many inactivated structures display nonreactive conformations. For penicillin and cephalosporin acylases, this discrepancy between structure and function may be resolved by invoking the presence of a hidden conformational state, similar to that reported here for ThnT.crystallography | mechanism | reactive rotamer effect | self-cleavage

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Section: Evidence For Hidden Conformational Rearrangement Preceding Ntnsupporting

confidence: 69%

Insights into cis-autoproteolysis reveal a reactive state formed through conformational rearrangement

Buller

Freeman

Wright

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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“…The structure of the precursor GCA at 2.5 Å resolution suggests that a water molecule is the general base that activates the active site serine residue Yoon et al 2004). The water molecule is coordinated by the mainchain NHs of Thr69 and Gly168 and by the amide side chain of Asn244.…”

Section: Glutaryl 7-aminocephalosporanic Acid Acylase Precursor (Gca mentioning

confidence: 99%

Unconventional serine proteases: Variations on the catalytic Ser/His/Asp triad configuration

2008

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Serine proteases comprise nearly one-third of all known proteases identified to date and play crucial roles in a wide variety of cellular as well as extracellular functions, including the process of blood clotting, protein digestion, cell signaling, inflammation, and protein processing. Their hallmark is that they contain the so-called ''classical'' catalytic Ser/His/Asp triad. Although the classical serine proteases are the most widespread in nature, there exist a variety of ''nonclassical'' serine proteases where variations to the catalytic triad are observed. Such variations include the triads Ser/His/Glu, Ser/ His/His, and Ser/Glu/Asp, and include the dyads Ser/Lys and Ser/His. Other variations are seen with certain serine and threonine peptidases of the Ntn hydrolase superfamily that carry out catalysis with a single active site residue. This work discusses the structure and function of these novel serine proteases and threonine proteases and how their catalytic machinery differs from the prototypic serine protease class.

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“…This is the main reason why several hypotheses concerning the autocatalytic mechanism of these enzymes have been proposed. For example, in glutaryl 7-aminocephalosporanic acid acylase, the cleavage occurs at the Gly 169 -Ser 170 bond (39 (40). In both mutated enzymes, some distortions of main-chain geometry were observed near the scissile bond, but the geometry of the scissile peptide bond has standard stereochemistry.…”

Section: Conformation Of Thr 179 -A Theoretically Modeled Thrmentioning

confidence: 99%

The Mechanism of Autocatalytic Activation of Plant-type L-Asparaginases

Michalska

Hernández-Santoyo

Jaskólski

2008

Journal of Biological Chemistry

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Plant L-asparaginases and their bacterial homologs, such as EcAIII found in Escherichia coli, form a subgroup of the N-terminal nucleophile (Ntn)-hydrolase family. In common with all Ntn-hydrolases, they are expressed as inactive precursors that undergo activation in an autocatalytic manner. The maturation process involves intramolecular hydrolysis of a single peptide bond, leading to the formation of two subunits (␣ and ␤) folded as one structural domain, with the nucleophilic Thr residue located at the freed N terminus of subunit ␤. The mechanism of the autocleavage reaction remains obscure. We have determined the crystal structure of an active site mutant of EcAIII, with the catalytic Thr residue substituted by Ala (T179A). The modification has led to a correctly folded but unprocessed molecule, revealing the geometry and molecular environment of the scissile peptide bond. The autocatalytic reaction is analyzed from the point of view of the Thr 179 side chain rotation, identification of a potential general base residue, and the architecture of the oxyanion hole.Posttranslational modifications of proteins can be divided into reactions leading to covalent attachment, usually at a side chain, of a specific chemical group, such as phosphate or carbohydrate, and into reactions that lead to cleavage or cleavage/ rearrangement of the polypeptide backbone. The latter processes can be enzyme-catalyzed or occur without an additional biocatalyst. An important class of backbone rearrangement is connected with autocatalytic processes in maturating proteins. Notable examples in this category include intein splicing and simple backbone cleavage. Protein splicing requires cleavage of two peptide bonds that surround the so-called intein, followed by ligation of the flanking polypeptides, the N-and C-exteins. The process consists of four steps: acyl rearrangement, transesterification, cyclization of an asparagine residue, and a second acyl rearrangement (1). In maturation following the autocleavage pathway, only a single peptide cleavage occurs, and the mechanism includes only acylation and water-dependent deacylation. Although the mechanism of protein splicing has been extensively studied and seems to be well understood, the autoproteolysis reactions are rather obscure. The known examples of autoproteolytic proteins include a large group of N-terminal nucleophile (Ntn) 2 -hydrolases (2). In Ntn enzymes, a cleavage of a precursor molecule is required to generate a catalytic residue at the newly formed N terminus, which can be a threonine, serine, or cysteine. The family of Ntn-hydrolases includes such enzymes as aspartylglucosaminidases (3, 4), penicillin acylases (5-7), taspase1 (8), and plant-type L-asparaginases (9 -12). Sequence similarity within the Ntn-hydrolase family is very limited, but despite the variation of primary structure, the proteins share a common sandwich-like ␣␤␤␣ fold created by two ␤-sheets surrounded by two layers of ␣-helices (13). The autocatalyzed maturation of Ntn-hydrolases involves either the remo...

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A Bound Water Molecule Is Crucial in Initiating Autocatalytic Precursor Activation in an N-terminal Hydrolase

Cited by 19 publications

References 27 publications

Insights into cis-autoproteolysis reveal a reactive state formed through conformational rearrangement

Insights into cis-autoproteolysis reveal a reactive state formed through conformational rearrangement

Unconventional serine proteases: Variations on the catalytic Ser/His/Asp triad configuration

The Mechanism of Autocatalytic Activation of Plant-type L-Asparaginases

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