PDBsum entry 1jcl

Lyase

PDB id: 1jcl

Contents

Protein chains

252 a.a. *

Ligands

HPD ×2

Waters ×708

* Residue conservation analysis

PDB id:

1jcl

Name:

Lyase

Title:

Observation of covalent intermediates in an enzyme mechanism at atomic resolution

Structure:

Deoxyribose-phosphate aldolase. Chain: a, b. Synonym: phosphodeoxyriboaldolase, deoxyriboaldolase. Engineered: yes

Source:

Escherichia coli. Organism_taxid: 562. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

1.05Å R-factor: 0.143 R-free: 0.169

Authors:

A.Heine,G.Desantis,J.G.Luz,M.Mitchell,C.-H.Wong,I.A.Wilson

Key ref:

A.Heine et al. (2001). Observation of covalent intermediates in an enzyme mechanism at atomic resolution. Science, 294, 369-374. PubMed id: 11598300 DOI: 10.1126/science.1063601

Date:

09-Jun-01 Release date: 31-Oct-01

Protein chains

P0A6L0  (DEOC_ECOLI) -  Deoxyribose-phosphate aldolase from Escherichia coli (strain K12)

Seq: 259 a.a.

Struc: 252 a.a.

Enzyme reactions

• Enzyme class: E.C.4.1.2.4 - deoxyribose-phosphate aldolase.
• Pathway: Deoxyribose-phosphate aldolase
• Reaction: 2-deoxy-D-ribose 5-phosphate = D-glyceraldehyde 3-phosphate + acetaldehyde

2-deoxy-D-ribose 5-phosphate (Bound ligand (Het Group name = HPD) corresponds exactly) = D-glyceraldehyde 3-phosphate + acetaldehyde

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1126/science.1063601

Science 294:369-374 (2001)

PubMed id: 11598300

Observation of covalent intermediates in an enzyme mechanism at atomic resolution.

A.Heine, G.DeSantis, J.G.Luz, M.Mitchell, C.H.Wong, I.A.Wilson.

ABSTRACT

In classical enzymology, intermediates and transition states in a catalytic mechanism are usually inferred from a series of biochemical experiments. Here, we derive an enzyme mechanism from true atomic-resolution x-ray structures of reaction intermediates. Two ultra-high resolution structures of wild-type and mutant d-2-deoxyribose-5-phosphate (DRP) aldolase complexes with DRP at 1.05 and 1.10 angstroms unambiguously identify the postulated covalent carbinolamine and Schiff base intermediates in the aldolase mechanism. In combination with site-directed mutagenesis and (1)H nuclear magnetic resonance, we can now propose how the heretofore elusive C-2 proton abstraction step and the overall stereochemical course are accomplished. A proton relay system appears to activate a conserved active-site water that functions as the critical mediator for proton transfer.

Selected figure(s)

Figure 1.
Fig. 1. DERA reaction. In vivo, DERA catalyzes the aldol reaction between the acetaldehyde donor and D-glyceraldehyde-3-phosphate acceptor to generate DRP.

Figure 4.
Fig. 4. Catalytic mechanism for DERA. (A) 1H NMR experiment. DERA-catalyzed exchange of C2 proton of (R)-2-deuteropropanaldehyde and (S)-2-deuteropropanaldehyde in D[2]O was established by 1H NMR. Incubation of (R)-2-deuteropropanaldehyde with DERA in D[2]O effects complete exchange of the -proton to a deuteron, as observed by the collapse of the C3 doublet resonances of the aldehyde and acetal to singlets (left). By contrast, incubation of (S)-2-deuteropropanaldehyde with DERA in D[2]O does not effect proton exchange, as observed by retention of the doublet resonances (right). (B) Proposed catalytic mechanism for DERA. The proposed mechanism is consistent with all of our ultra-high resolution structural, modeling, site-directed mutagenesis, and 1H NMR data. Lys167 is identified as the Schiff base-forming residue. After the enamine is formed, the system is poised for nucleophilic attack onto the carbonyl-carbon of the acceptor aldehyde D-glyceraldehyde-3-phosphate. A proton relay system between Asp102, Lys201, and an active site water molecule is responsible for shuffling a proton between C2 of the acetaldehyde imine and enamine and subsequent C3 hydroxyl protonation. The double arrows in green indicate rapid proton shuffling between Lys201 and Asp102. Crystallographically observed reaction intermediates are boxed (left, carbinolamine; right, Schiff base). (C) Stereochemical course of aldol reaction. When propanal is substituted for acetaldehyde as the donor, the pro-S proton is removed, and thus the aldol reaction proceeds with retention of configuration at C2, with the Si face of the resulting enamine approaching the Re face of the acceptor carbonyl. A:H, general acid; B, general base.

The above figures are reprinted by permission from the AAAs: Science (2001, 294, 369-374) copyright 2001.

Figures were selected by an automated process.