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Mechanism-based inhibition of an aldolase at high concentrations of its natural substrate acetaldehyde: structural insights and protective strategies.

ABSTRACT: 2-Deoxy-d-ribose-5-phosphate aldolase (DERA) is used in organic synthesis for the enantioselective reaction between acetaldehyde and a broad range of other aldehydes as acceptor molecules. Nevertheless, its application is hampered by a poor tolerance towards high concentrations of acetaldehyde, its natural substrate. While numerous studies have been performed searching for new, more acetaldehyde-resistant DERAs, the mechanism underlying this deactivation process has remained elusive. By using NMR spectroscopy on both the protein and the small-molecule scale, we could show that a reaction product binds to the inner part of the enzyme, and that this effect can be partly reversed via heating. The crystal structure of DERA before and after acetaldehyde incubation was determined at high resolution, revealing a covalently bound reaction product bridging the catalytically active lysine (K167) to a nearby cysteine (C47) in the deactivated enzyme. A reaction mechanism is proposed where crotonaldehyde as the aldol product of two acetaldehyde molecules after water elimination forms a Schiff base with the lysine side chain, followed by Michael addition of the cysteine thiol group to the C? atom of the inhibitor. In support of this mechanism, direct incubation of DERA with crotonaldehyde results in a more than 100-fold stronger inhibition, compared to acetaldehyde, whereas mutation of C47 gives rise to a fully acetaldehyde-resistant DERA. Thus this variant appears perfectly suited for synthetic applications. A similar diagnostic and preventive strategy should be applicable to other biocatalysts suffering from mechanism-based inhibition by a reactive substrate, a condition that may be more common than currently appreciated in biotechnology.

SUBMITTER: Dick M

PROVIDER: S-EPMC6016325 | biostudies-literature | 2016 Jul

REPOSITORIES: biostudies-literature

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Mechanism-based inhibition of an aldolase at high concentrations of its natural substrate acetaldehyde: structural insights and protective strategies.

Dick Markus M Hartmann Rudolf R Weiergräber Oliver H OH Bisterfeld Carolin C Classen Thomas T Schwarten Melanie M Neudecker Philipp P Willbold Dieter D Pietruszka Jörg J

Chemical science 20160330 7

2-Deoxy-d-ribose-5-phosphate aldolase (DERA) is used in organic synthesis for the enantioselective reaction between acetaldehyde and a broad range of other aldehydes as acceptor molecules. Nevertheless, its application is hampered by a poor tolerance towards high concentrations of acetaldehyde, its natural substrate. While numerous studies have been performed searching for new, more acetaldehyde-resistant DERAs, the mechanism underlying this deactivation process has remained elusive. By using NM ...[more]

PMID: 30155096

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Project description:Giardia lamblia fructose-1,6-bisphosphate aldolase (FBPA) is a member of the class II zinc-dependent aldolase family that catalyzes the cleavage of d-fructose 1,6-bisphosphate (FBP) into dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate (G3P). In addition to the active site zinc, the catalytic apparatus of FBPA employs an aspartic acid, Asp83 in the G. lamblia enzyme, which when replaced with an alanine residue renders the enzyme inactive. A comparison of the crystal structures of D83A FBPA in complex with FBP and of wild-type FBPA in the unbound state revealed a substrate-induced conformational transition of loops in the vicinity of the active site and a shift in the location of Zn(2+). When FBP binds, the Zn(2+) shifts up to 4.6 A toward the catalytic Asp83, which brings the metal within coordination distance of the Asp83 carboxylate group. In addition, the structure of wild-type FBPA was determined in complex with the competitive inhibitor d-tagatose 1,6-bisphosphate (TBP), a FBP stereoisomer. In this structure, the zinc binds in a site close to that previously seen in the structure of FBPA in complex with phosphoglycolohydroxamate, an analogue of the postulated DHAP ene-diolate intermediate. Together, the ensemble of structures suggests that the zinc mobility is necessary to orient the Asp83 side chain and to polarize the substrate for proton transfer from the FBP C(4) hydroxyl group to the Asp83 carboxyl group. In the absence of FBP, the alternative zinc position is too remote for coordinating the Asp83. We propose a modification of the catalytic mechanism that incorporates the novel features observed in the FBPA-FBP structure. The mechanism invokes coordination and coplanarity of the Zn(2+) with the FBP's O-C(3)-C(4)-O group concomitant with coordination of the Asp83 carboxylic group. Catalysis is accompanied by movement of Zn(2+) to a site coplanar with the O-C(2)-C(3)-O group of the DHAP. glFBPA exhibits strict substrate specificity toward FBP and does not cleave TBP. The active sites of FBPAs contain an aspartate residue equivalent to Asp255 of glFBPA, whereas tagatose-1,6-bisphosphate aldolase contains an alanine in this position. We and others hypothesized that this aspartic acid is a likely determinant of FBP versus TBP specificity. Replacement of Asp255 with an alanine resulted in an enzyme that possesses double specificity, now cleaving TBP (albeit with low efficacy; k(cat)/K(m) = 80 M(-1) s(-1)) while maintaining activity toward FBP at a 50-fold lower catalytic efficacy compared with that of wild-type FBPA. The collection of structures and sequence analyses highlighted additional residues that may be involved in substrate discrimination.

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Project description:Aldosterone is a major mineralocorticoid hormone that plays a key role in the regulation of electrolyte balance and blood pressure. Excess aldosterone levels can arise from dysregulation of the renin-angiotensin-aldosterone system and are implicated in the pathogenesis of hypertension and heart failure. Aldosterone synthase (cytochrome P450 11B2, CYP11B2) is the sole enzyme responsible for the production of aldosterone in humans. Blocking of aldosterone synthesis by mediating aldosterone synthase activity is thus a recently emerging pharmacological therapy for hypertension, yet a lack of structural information has limited this approach. Here, we present the crystal structures of human aldosterone synthase in complex with a substrate deoxycorticosterone and an inhibitor fadrozole. The structures reveal a hydrophobic cavity with specific features associated with corticosteroid recognition. The substrate binding mode, along with biochemical data, explains the high 11β-hydroxylase activity of aldosterone synthase toward both gluco- and mineralocorticoid formation. The low processivity of aldosterone synthase with a high extent of intermediates release might be one of the mechanisms of controlled aldosterone production from deoxycorticosterone. Although the active site pocket is lined by identical residues between CYP11B isoforms, most of the divergent residues that confer additional 18-oxidase activity of aldosterone synthase are located in the I-helix (vicinity of the O(2) activation path) and loops around the H-helix (affecting an egress channel closure required for retaining intermediates in the active site). This intrinsic flexibility is also reflected in isoform-selective inhibitor binding. Fadrozole binds to aldosterone synthase in the R-configuration, using part of the active site cavity pointing toward the egress channel. The structural organization of aldosterone synthase provides critical insights into the molecular mechanism of catalysis and enables rational design of more specific antihypertensive agents.

Dataset Information

Mechanism-based inhibition of an aldolase at high concentrations of its natural substrate acetaldehyde: structural insights and protective strategies.

Publications

Mechanism-based inhibition of an aldolase at high concentrations of its natural substrate acetaldehyde: structural insights and protective strategies.

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