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

Abstract

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...

Structural analysis of K+ dependence in l-asparaginases from Lotus japonicus

“…In all the initial models, the active centre (Fig. 4c) shows the same features as those from bacterial asparaginases (Michalska et al 2005(Michalska et al , 2008, excepting the sugar-binding site, which is occupied by the side chain of E195-the C terminus residue of the first ab monomer after cleavage-it could explain the reduced reactivity of plant asparaginases towards glycosylated asparagine.…”

“…-dependent asparaginase from Lotus japonicus, modelled from the X-ray structure of the plant K ? -independent enzyme , the E. coli enzyme precursor (Michalska et al 2008) and the enzyme bound to L-aspartate (Michalska et al 2005) as deposited at PDB (Berman et al 2000) (access codes 2GEZ, 2ZAK and 2ZAL, respectively). Sequence identities of LjNSE1 with respect to the targets were 56, 40 and 52%, respectively.…”

Role of asparaginase variable loop at the carboxyl terminal of the alpha subunit in the determination of substrate preference in plants

“…Thr 230 (residue 243 in LlA) acts as a general base, and the side chain of Asn 67 in the Na ? -binding loop (residue 66 in LlA) forms an oxyanion hole with a water molecule, stabilizing a negative charge forming on the oxygen atom of Gly 178 preceding the catalytic Thr 179 nucleophile in the cleavage intermediate (Michalska et al 2008). In this structure, lack of cleavage stabilized the linker region between a-and b-subunits, and up to 11 residues forming a loop could be visualized immediately preceding the cleavage site.…”

“…-activation was recently uncovered through a homology modeling and site-directed mutagenesis approach (Credali et al 2011). LjNSE1 and -2 were modeled against the coordinates of EcAIII and LlA (Michalska et al 2005(Michalska et al , 2008. Glu 248 , Asp 285 and Glu 286 in LjNSE1 were identified as determinants of K ?…”

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

“…Our analysis of uncleaved structures for γ-glutamyltranspeptidase (20,39), L-asparaginase EcAIII (40,41), Taspase1 (24), and GA (28) indicates that they have crystallized in inactive states (SI Appendix, Table S1). For gGTand EcaIII, the active site geometry was previously hypothesized to represent an active state that is comparable to state B of ThnT T282C and GA T152C.…”