Flexible Loop That Is Novel Catalytic Machinery in a Ligase. Atomic Structure and Function of the Loopless Glutathione Synthetase

Kato, Hiroaki; Tanaka, Takuji; Yamaguchi, Hiroshi; Hara, Takehisa; Nishi­oka, Takaaki; Katsube, Yukiteru; Oda, Jun’ichi

doi:10.1021/bi00183a001

Cited by 36 publications

(28 citation statements)

References 21 publications

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“…Taken together, the data comply with the assumption of substrate-induced conformational changes similar to those seen in other amide bond-forming ligases (29,(31)(32)(33)(34)(35)(36)(37)(38)(39)(40)(41). Whereas the free enzyme is readily cleaved by trypsin or factor Xa at Arg-556 in the presumed flexible ⍀-loop, substrate binding induces a compact form, in which the ⍀-loop is no longer attacked by proteases.…”

Section: Role Of Arginines Located In the Putative ⍀-Loop And The Glysupporting

confidence: 69%

Trypanothione Synthesis in Crithidia Revisited

Comini

Menge

Wissing

et al. 2005

Journal of Biological Chemistry

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In Crithidia fasciculata the biosynthesis of trypanothione (N 1 ,N 8 -bis(glutathionyl)spermidine; reduced trypanothione), a redox mediator unique to and essential for pathogenic trypanosomatids, was assumed to be achieved by two distinct enzymes, glutathionylspermidine synthetase and trypanothione synthetase (TryS), and only the first one was adequately characterized. We here report that the TryS of C. fasciculata, like that of Trypanosoma species, catalyzes the entire synthesis of trypanothione, whereas its glutathionylspermidine synthetase appears to be specialized for Gsp synthesis. A gene (GenBank TM accession number AY603101) implicated in reduced trypanothione synthesis of C. fasciculata was isolated from genomic DNA and expressed in Escherichia coli as His-tagged or Nus fusion proteins. The expression product proved to be a trypanothione synthetase (Cf-TryS) that also displayed a glutathionylspermidine synthetase, an amidase, and marginal ATPase activity. The dual specificity of the Cf-TryS preparations was not altered by removal of the tags. Steady-state kinetic analysis of Cf-TryS yielded a pattern that was compatible with a concerted substitution mechanism, wherein the enzyme forms a ternary complex with Mg 2؉ -ATP and GSH to phosphorylate GSH and then ligates the glutathionyl residue to glutathionylspermidine. Limiting K m values for GSH, Mg 2؉ -ATP, and glutathionylspermidine were 407, 222, and 480 M, respectively, and the k cat was 8.7 s ؊1 for the TryS reaction. Mutating Arg-553 or Arg-613 to Lys, Leu, Gln, or Glu resulted in marked reduction or abrogation (R553E) of activity. Limited proteolysis with factor Xa or trypsin resulted in cleavage at Arg-556 that was accompanied by loss of activity. The presence of substrates, in particular of ATP and GSH alone or in combination, delayed proteolysis of wild-type Cf-TryS and Cf-TryS R553Q but not in Cf-TryS R613Q, which suggests dynamic interactions of remote domains in substrate binding and catalysis.

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Section: Role Of Arginines Located In the Putative ⍀-Loop And The Glysupporting

confidence: 69%

Trypanothione Synthesis in Crithidia Revisited

Comini

Menge

Wissing

et al. 2005

Journal of Biological Chemistry

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“…One non-regular secondary structural element that has been observed in several enzyme systems to have mobile features that are critical to catalytic function is the Ω-loop (5, 7, 8, 10, 28-32). In several systems that have been characterized, this type of loop undergoes a transition from a catalytically inactive, open conformation to a catalytically active, closed conformation as the enzyme binds substrates (2, 3, 7, 9, 29, 33). In the case of PEPCK this open-closed transition of the Ω -loop is also accompanied by a disorder to order transition as the open Ω-loop is found to be conformationally dynamic, interconverting between two or more conformational states while upon closure the Ω–loop adapts a single conformational state.…”

Section: Resultsmentioning

confidence: 99%

“…Biochemical and crystallographic studies of these enzymes resulted in widely differing roles for the ω-loop domain being proposed. Based upon study of these aforementioned enzymes, an ω-loop can sequester and protect reaction intermediates, recruit charged groups to the active site, aid in correctly positioning substrates and side chains, stabilize specific enzyme conformations and alter the active site environment to facilitate the required chemistry (3, 7-9, 12). Based on the available data on PEPCK, the role of the ω-loop is to aid in correctly positioning substrates in the active site, and to sequester and protect the reaction intermediate (14).…”

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confidence: 99%

The Ω-Loop Lid Domain of Phosphoenolpyruvate Carboxykinase Is Essential for Catalytic Function

Johnson

Holyoak

2012

Biochemistry

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Phosphoenolpyruvate carboxykinase (PEPCK) is an essential metabolic enzyme operating in the gluconeogenesis and glyceroneogenesis pathways. Recent studies have demonstrated that the enzyme contains a mobile active site lid domain that transitions between an open/disorded conformation to a closed/ordered conformation as the enzyme progresses through the catalytic cycle. The understanding of how this mobile domain functions in catalysis is incomplete. Previous studies show that the closure of the lid domain stabilizes the reaction intermediate and protects the reactive intermediate from spurious protonation and thus contributes to the fidelity of the enzyme. In order to more fully investigate the roles of the lid domain in PEPCK function we created three mutations that replaced the 11-residue lid domain with one, two or three glycine residues. Kinetic analysis of the mutant enzymes demonstrates that none of the enzyme constructs exhibit any measurable kinetic activity resulting in a decrease in the catalytic parameters by at least 106. Structural characterization of the mutants in complexes representing the catalytic cycle suggest that the inactivity is due to a role for the lid domain in the formation of the fully closed state of the enzyme that is required for catalytic function. In the absence of the lid domain, the enzyme is unable to achieve the fully closed state and is rendered inactive despite possessing all of the residues and substrates required for catalytic function. This work demonstrates how enzyme catalytic function can be abolished through the alteration of conformational equilibria despite all elements required for chemical conversion of substrates to products remaining intact.

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“…Because the switch from C to A addition requires a structural rearrangement of the amino acid template (6,13,19), it is conceivable that such a flexible region is required as a hinge for reorganization of the NTP binding pocket during CCA synthesis. Similar loops connecting individual movable regions are described as essential elements of many proteins interacting with specific ligands (20,21).…”

Section: Discussion the Absence Of The Flexible Loop Region Is A Commmentioning

confidence: 99%

Evolution of tRNA nucleotidyltransferases: A small deletion generated CC-adding enzymes

Neuenfeldt

Just

Betat

et al. 2008

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

133

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CCA-adding enzymes are specialized polymerases that add a specific sequence (C-C-A) to tRNA 3 ends without requiring a nucleic acid template. In some organisms, CCA synthesis is accomplished by the collaboration of evolutionary closely related enzymes with partial activities (CC and A addition). These enzymes carry all known motifs of the catalytic core found in CCA-adding enzymes. Therefore, it is a mystery why these polymerases are restricted in their activity and do not synthesize a complete CCA terminus. Here, a region located outside of the conserved motifs was identified that is missing in CC-adding enzymes. When recombinantly introduced from a CCA-adding enzyme, the region restores full CCAadding activity in the resulting chimera. Correspondingly, deleting the region in a CCA-adding enzyme abolishes the A-incorporating activity, also leading to CC addition. The presence of the deletion was used to predict the CC-adding activity of putative bacterial tRNA nucleotidyltransferases. Indeed, two such enzymes were experimentally identified as CC-adding enzymes, indicating that the existence of the deletion is a hallmark for this activity. Furthermore, phylogenetic analysis of identified and putative CCadding enzymes indicates that this type of tRNA nucleotidyltransferases emerged several times during evolution. Obviously, these enzymes descend from CCA-adding enzymes, where the occurrence of the deletion led to the restricted activity of CC addition. A-adding enzymes, however, seem to represent a monophyletic group that might also be ancestral to CCA-adding enzymes. Yet, experimental data indicate that it is possible that A-adding activities also evolved from CCA-adding enzymes by the occurrence of individual point mutations.tRNA maturation ͉ CCA-adding enzyme ͉ flexible loop

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Flexible Loop That Is Novel Catalytic Machinery in a Ligase. Atomic Structure and Function of the Loopless Glutathione Synthetase

Cited by 36 publications

References 21 publications

Trypanothione Synthesis in Crithidia Revisited

Trypanothione Synthesis in Crithidia Revisited

The Ω-Loop Lid Domain of Phosphoenolpyruvate Carboxykinase Is Essential for Catalytic Function

Evolution of tRNA nucleotidyltransferases: A small deletion generated CC-adding enzymes

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