scholarly journals Structural characterization of tartrate dehydrogenase: a versatile enzyme catalyzing multiple reactions

2010 ◽  
Vol 66 (6) ◽  
pp. 673-684 ◽  
Author(s):  
Radhika Malik ◽  
Ronald E. Viola

The first structure of an NAD-dependent tartrate dehydrogenase (TDH) has been solved to 2 Å resolution by single anomalous diffraction (SAD) phasing as a complex with the intermediate analog oxalate, Mg2+and NADH. This TDH structure fromPseudomonas putidahas a similar overall fold and domain organization to other structurally characterized members of the hydroxy-acid dehydrogenase family. However, there are considerable differences between TDH and these functionally related enzymes in the regions connecting the core secondary structure and in the relative positioning of important loops and helices. The active site in these complexes is highly ordered, allowing the identification of the substrate-binding and cofactor-binding groups and the ligands to the metal ions. Residues from the adjacent subunit are involved in both the substrate and divalent metal ion binding sites, establishing a dimer as the functional unit and providing structural support for an alternating-site reaction mechanism. The divalent metal ion plays a prominent role in substrate binding and orientation, together with several active-site arginines. Functional groups from both subunits form the cofactor-binding site and the ammonium ion aids in the orientation of the nicotinamide ring of the cofactor. A lysyl amino group (Lys192) is the base responsible for the water-mediated proton abstraction from the C2 hydroxyl group of the substrate that begins the catalytic reaction, followed by hydride transfer to NAD. A tyrosyl hydroxyl group (Tyr141) functions as a general acid to protonate the enolate intermediate. Each substrate undergoes the initial hydride transfer, but differences in substrate orientation are proposed to account for the different reactions catalyzed by TDH.

2010 ◽  
Vol 132 (20) ◽  
pp. 7049-7054 ◽  
Author(s):  
Nataša Mitić ◽  
Kieran S. Hadler ◽  
Lawrence R. Gahan ◽  
Alvan C. Hengge ◽  
Gerhard Schenk

Biochemistry ◽  
2015 ◽  
Vol 54 (11) ◽  
pp. 2032-2039 ◽  
Author(s):  
Dennis K. Hansen ◽  
Helen Webb ◽  
Jonas Willum Nielsen ◽  
Pernille Harris ◽  
Jakob R. Winther ◽  
...  

2014 ◽  
Vol 19 (7) ◽  
pp. 1149-1164 ◽  
Author(s):  
Mireia Tomas ◽  
María Ayelen Pagani ◽  
Carlos S. Andreo ◽  
Mercè Capdevila ◽  
Roger Bofill ◽  
...  

Author(s):  
V. V. Andrushchenko ◽  
S. V. Kornilova ◽  
L. E. Kapinos ◽  
E. V. Hackl ◽  
Yu P. Blagoi

2020 ◽  
Vol 202 (8) ◽  
Author(s):  
Sasha A. Weiditch ◽  
Sarah C. Bickers ◽  
Diane Bona ◽  
Karen L. Maxwell ◽  
Voula Kanelis

ABSTRACT The last gene in the genome of the bacteriophage HK97 encodes gp74, an HNH endonuclease. HNH motifs contain two conserved His residues and an invariant Asn residue, and they adopt a ββα structure. gp74 is essential for phage head morphogenesis, likely because gp74 enhances the specific endonuclease activity of the HK97 terminase complex. Notably, the ability of gp74 to enhance the terminase-mediated cleavage of the phage cos site requires an intact HNH motif in gp74. Mutation of H82, the conserved metal-binding His residue in the HNH motif, to Ala abrogates gp74-mediated stimulation of terminase activity. Here, we present nuclear magnetic resonance (NMR) studies demonstrating that gp74 contains an α-helical insertion in the Ω-loop, which connects the two β-strands of the ββα fold, and a disordered C-terminal tail. NMR data indicate that the Ω-loop insert makes contacts to the ββα fold and influences the ability of gp74 to bind divalent metal ions. Further, the Ω-loop insert and C-terminal tail contribute to gp74-mediated DNA digestion and to gp74 activity in phage morphogenesis. The data presented here enrich our molecular-level understanding of how HNH endonucleases enhance terminase-mediated digestion of the cos site and contribute to the phage replication cycle. IMPORTANCE This study demonstrates that residues outside the canonical ββα fold, namely, the Ω-loop α-helical insert and a disordered C-terminal tail, regulate the activity of the HNH endonuclease gp74. The increased divalent metal ion binding when the Ω-loop insert is removed compared to reduced cos site digestion and phage formation indicates that the Ω-loop insert plays multiple regulatory roles. The data presented here provide insights into the molecular basis of the involvement of HNH proteins in phage DNA packing.


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