Structural and Functional Properties of Isocitrate Dehydrogenase from the Psychrophilic Bacterium Desulfotalea psychrophila Reveal a Cold-active Enzyme with an Unusual High Thermal Stability

• Published in: Journal of molecular biology Vol. 372; no. 1; pp. 130 - 149
• Main Authors: Fedøy, Anita-Elin, Yang, Nannan, Martinez, Aurora, Leiros, Hanna-Kirsti S., Steen, Ida Helene
• Format: Journal Article
• Language: English
• Published: England Elsevier Ltd 07-09-2007
• Subjects: Amino Acid Sequence; Bacteria; Binding Sites; cold adaptation; Cold Temperature; crystal structure; Desulfitobacterium hafniense; Desulfotalea psychrophila; Enzyme Stability; Gram-Negative Anaerobic Bacteria - enzymology; Hot Temperature; isocitrate dehydrogenase; Isocitrate Dehydrogenase - chemistry; Isocitrate Dehydrogenase - metabolism; Isocitrate Dehydrogenase - physiology; Isocitrates - metabolism; Models, Biological; Models, Molecular; Molecular Sequence Data; Protein Binding; Protein Structure, Secondary; psychrophilic; Sequence Homology, Amino Acid; thermal stability; Thermotoga maritima; Transition Temperature; T m; psychrophilic; isocitrate dehydrogenase; Cg; Dh; Af; thermal stability; Dp; IDH; Ap; ASA; cold adaptation; Bs; Pc; Av; DSC; Pf; TK; crystal structure; Tm; Hc; Ec
• ISSN: 0022-2836; 1089-8638
• DOI: 10.1016/j.jmb.2007.06.040

Isocitrate dehydrogenase (IDH) has been studied extensively due to its central role in the Krebs cycle, catalyzing the oxidative NAD(P)
+-dependent decarboxylation of isocitrate to α-ketoglutarate and CO
2. Here, we present the first crystal structure of IDH from a psychrophilic bacterium,
Desulfotalea psychrophila (
DpIDH). The structural information is combined with a detailed biochemical characterization and a comparative study with IDHs from the mesophilic bacterium
Desulfitobacterium hafniense (
DhIDH), porcine (
PcIDH), human cytosolic (
HcIDH) and the hyperthermophilic
Thermotoga maritima (
TmIDH).
DpIDH was found to have a higher melting temperature (
T
m
=
66.9 °C) than its mesophilic homologues and a suboptimal catalytic efficiency at low temperatures. The thermodynamic activation parameters indicated a disordered active site, as seen also for the drastic increase in
K
m for isocitrate at elevated temperatures. A methionine cluster situated at the dimeric interface between the two active sites and a cluster of destabilizing charged amino acids in a region close to the active site might explain the poor isocitrate affinity. On the other hand,
DpIDH was optimized for interacting with NADP
+ and the crystal structure revealed unique interactions with the cofactor. The highly acidic surface, destabilizing charged residues, fewer ion pairs and reduced size of ionic networks in
DpIDH suggest a flexible global structure. However, strategic placement of ionic interactions stabilizing the N and C termini, and additional ionic interactions in the clasp domain as well as two enlarged aromatic clusters might counteract the destabilizing interactions and promote the increased thermal stability. The structure analysis of
DpIDH illustrates how psychrophilic enzymes can adjust their flexibility in dynamic regions during their catalytic cycle without compromising the global stability of the protein.