Insights into substrate specificity of archaeal Entner-Doudoroff aldolases

DOI: 10.1021/acs.biochem.8b00535

Authors: Viatcheslav Zaitsev (University of St Andrews) , Ulrike Johnsen (Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität) , Matthias Reher (Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität) , Marius Ortjohann (Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität) , Garry L. Taylor (University of St Andrews) , Michael J. Danson (University of Bath) , Peter Schönheit (Institut für Allgemeine Mikrobiologie, Christian-Albrechts-Universität) , Susan Crennell (University of Bath)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Biochemistry

State: Published (Approved)
Published: May 2018

Abstract: The thermoacidophilic archaea Picrophilus torridus and Sulfolobus solfataricus catabolise glucose via a non-phosphorylative Entner-Doudoroff pathway and a branched Entner-Doudoroff pathway, respectively. Key enzymes for these Entner-Doudoroff pathways are the aldolases, 2-keto-3-deoxygluconate aldolase (KDG-aldolase) and 2-keto-3-deoxy-6-phosphogluconate aldolase (KD(P)G-aldolase). KDG-aldolase from P. torridus (Pt-KDG-aldolase) is highly specific for the non-phosphorylated substrate, 2-keto-3-deoxygluconate (KDG), whereas KD(P)G-aldolase from S. solfataricus (Ss-KD(P)G-aldolase) is an enzyme catalyzing the cleavage of both KDG and 2-keto-3-deoxy-6-phosphogluconate (KDPG), with a preference for KDPG. The structural basis for the high specificity of Pt-KDG-aldolase for KDG as compared to the more promiscuous Ss-KD(P)G-aldolase has not been analysed before. In the current paper we report the elucidation of the structure of Ss-KD(P)G-aldolase in complex with KDPG at 2.35Å and that of KDG-aldolase from P. torridus at 2.50 Å resolution. By superimposition of the active sites of the two enzymes, and subsequent site-directed mutagenesis studies, a network of four amino acids, namely Arg106, Tyr132, Arg237 and Ser241, was identified in Ss-KD(P)G-aldolase that interact with the negatively-charged phosphate group of KDPG, thereby raising the affinity of the enzyme for KDPG. This KDPG-binding network is absent in Pt-KDG-aldolase, which explains the low catalytic efficiency of KDPG cleavage.

Journal Keywords: Peptides and proteins; Monomers; Chemical structure; Chemical specificity; Phosphates

Diamond Keywords: Archaea; Enzymes

Subject Areas: Biology and Bio-materials, Chemistry


Instruments: I04-Macromolecular Crystallography

Added On: 06/06/2018 15:33

Discipline Tags:

Biochemistry Catalysis Chemistry Structural biology Life Sciences & Biotech

Technical Tags:

Diffraction Macromolecular Crystallography (MX)