Precursor Structure of Cephalosporin Acylase: Insights into Auto-Proteolytic Activation in a New N-Terminal Hydrolase Family

Kim, Youngsoo; Kim, Sanggu; Earnest, Thomas; Hól, Wim G. J.

doi:10.1100/tsw.2002.54

Cited by 15 publications

(30 citation statements)

References 3 publications

(6 reference statements)

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“…Mechanistic analysis is more difficult because these residues lack the added constraint of a γ-methyl. Nonetheless, the structurally characterized systems, PA and CA, display an active site that bears several striking similarities to the inactive state of ThnT (17,18,42). The protein backbone flanking the scissile bond is comparable to the inactive conformation identified in state B of ThnT.…”

Section: Evidence For Hidden Conformational Rearrangement Preceding Ntnmentioning

confidence: 93%

“…Once described as belonging to the Ntn superfamily, the autoactivation of the less numerous DmpA/OAT (D/O) superfamily has not been studied (14). Understanding the autoproteolytic protein scaffold is of significant practical interest because enzymes from the D/O or Ntn superfamiles are implicated in the biosynthesis of all classes of the naturally occurring bicyclic β-lactam antibiotics (15)(16)(17)(18)(19).…”

mentioning

confidence: 99%

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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 Ntnmentioning

confidence: 93%

mentioning

confidence: 99%

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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“…It has in the case of the pyruvoyl-dependent enzymes therefore been the origin of debate-the initial reports found no evidence of an oxyanion hole, 33,34,55 but later research argued for the existence of a conserved oxyanion hole in the pyruvate-dependent enzymes as well as in the inteins, hedgehog proteins, and Ntn glycosylasparaginases. 35 Work on the precursor structures of the inteins 36,37 and Ntn hydrolases [39][40][41][42][43][44] do indeed also argue for the presence of an oxyanion hole, as does the work done on the Nup98 precursor. 27 However, as mentioned above, in the fully cleaved MUC1 SEA structure, no such stabilization of the tetrahedral intermediate is evident.…”

Section: Intramolecular Autoproteolysis In Other Proteinsmentioning

confidence: 96%

“…Several precursor structures are available, and there are evidence both of direct strain (in glycosylasparaginase 39 ) and indirect strain in the precursor peptide (in cephalosporin acylase 40,41 ). However, there are also evidence of very little strain (in penicillin acylase 42 and cephalosporin acylase 43 ) and the complete lack thereof (in the proteasome β subunit 44 ). There is no precursor structure of the hedgehog, GPS, and SEA domains, although the structural properties of the SEA precursor can be deduced from NMR in combination with molecular dynamics simulations.…”

Section: Intramolecular Autoproteolysis In Other Proteinsmentioning

confidence: 99%

“…4 In non-exposed autoproteolytic sites, an active protonation mechanism therefore appears necessary. A survey of the structures available indicates that such a protonation mechanism has been proposed for all enzymes (i.e., for the pyruvoyldependent enzymes, 33-35 the inteins, 36,37 the hedgehog domain, 21 and the Ntn hydrolases [39][40][41][42][43][44] ) except for the SEA domain 2,8 and the Nup98 27 structures, which both have autoproteolytic sites quite exposed to solvent. Yet, autoproteolysis in the wild-type MUC1 SEA domain still occurs readily at pH 10 (data not shown), thus providing further support for a shifting of the N → O acyl equilibrium towards the ester by conformational destabilization of the amide.…”

Section: Intramolecular Autoproteolysis In Other Proteinsmentioning

confidence: 99%

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SEA Domain Autoproteolysis Accelerated by Conformational Strain: Energetic Aspects

Sandberg

Johansson

Macao

et al. 2008

Journal of Molecular Biology

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A subclass of proteins with the SEA (sea urchin sperm protein, enterokinase, and agrin) domain fold exists as heterodimers generated by autoproteolytic cleavage within a characteristic G(-1)S+1VVV sequence. Autoproteolysis occurs by a nucleophilic attack of the serine hydroxyl on the vicinal glycine carbonyl followed by an N-->O acyl shift and hydrolysis of the resulting ester. The reaction has been suggested to be accelerated by the straining of the scissile peptide bond upon protein folding. In an accompanying article, we report the mechanism; in this article, we provide further key evidence and account for the energetics of coupled protein folding and autoproteolysis. Cleavage of the GPR116 domain and that of the MUC1 SEA domain occur with half-life (t((1/2))) values of 12 and 18 min, respectively, with lowering of the free energy of the activation barrier by approximately 10 kcal mol(-1) compared with uncatalyzed hydrolysis. The free energies of unfolding of the GPR116 and MUC1 SEA domains were measured to approximately 11 and approximately 15 kcal mol(-1), respectively, but approximately 7 kcal mol(-1) of conformational energy is partitioned as strain over the scissile peptide bond in the precursor to catalyze autoproteolysis by substrate destabilization. A straining energy of approximately 7 kcal mol(-1) was measured by using both a pre-equilibrium model to analyze stability and cleavage kinetics data obtained with the GPR116 SEA domain destabilized by core mutations or urea addition, as well as the difference in thermodynamic stabilities of the MUC1 SEA precursor mutant S1098A (with a G(-1)A+1VVV motif) and the wild-type protein. The results imply that cleavage by N-->O acyl shift alone would proceed with a t((1/2)) of approximately 2.3 years, which is too slow to be biochemically effective. A subsequent review of structural data on other self-cleaving proteins suggests that conformational strain of the scissile peptide bond may be a common mechanism of autoproteolysis.

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The Crystal Structure of MCAT from Mycobacterium tuberculosis Reveals Three New Catalytic Models

Huang

et al. 2007

Journal of Molecular Biology

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Precursor Structure of Cephalosporin Acylase: Insights into Auto-Proteolytic Activation in a New N-Terminal Hydrolase Family

Cited by 15 publications

References 3 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

SEA Domain Autoproteolysis Accelerated by Conformational Strain: Energetic Aspects

The Crystal Structure of MCAT from Mycobacterium tuberculosis Reveals Three New Catalytic Models

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