Divergence of AMP Deaminase in the Ice Worm Mesenchytraeus solifugus (Annelida, Clitellata, Enchytraeidae)

Abstract

Glacier ice worms, Mesenchytraeus solifugus and related species, are the largest glacially obligate metazoans. As one component of cold temperature adaptation, ice worms maintain atypically high energy levels in an apparent mechanism to offset cold temperature-induced lethargy and death. To explore this observation at a mechanistic level, we considered the putative contribution of 5' adenosine monophosphate deaminase (AMPD), a key regulator of energy metabolism in eukaryotes. We cloned cDNAs encoding ice worm AMPD, generating a fragment encoding 543 amino acids that included a short N-terminal region and complete C-terminal catalytic domain. The predicted ice worm AMPD amino acid sequence displayed conservation with homologues from other mesophilic eukaryotes with notable exceptions. In particular, an ice worm-specific K188E substitution proximal to the AMP binding site likely alters the architecture of the active site and negatively affects the enzyme's activity. Paradoxically, this would contribute to elevated intracellular ATP levels, which appears to be a signature of cold adapted taxa.

Figure 1

Amino acid alignment of AMP deaminase homologues. The full-length ice worm (Mesenchytraeus solifugus) AMPD fragment obtained in this study is represented (543 amino acids). N-terminal domains are shaded dark-gray; C-catalytic domain is shaded light gray. Periods represent conserved residues with respect to M. solifugus AMPD. Dashes indicate gaps in the linear amino acid sequence. Gray boxes represent ice worm-specific amino acid substitutions, and black boxes represent unique ice worm substitutions. Secondary structures are represented as α-helices (boxes) or β-sheets (arrows). Residues denoted by colored spots are explained in the legend at the bottom of the alignment. Ice worm-specific amino acid loops (V8-D24 and T129-G131, see text) are highlighted in red. GenBank accession numbers are Arabidopsis thaliana (NP 565886); Homo sapiens (NP 000027); Drosophila melanogaster (AAF48329); Caenorhabditis elegans (NP 001040752); Mesenchytraeus solifugus (EU624492); Enchytraeus albidus (EU624493), Lumbriculus variegatus (EU624494).

Figure 2

Ribbon diagram of the predicted ice worm AMPD protein. (a) The M. solifugus AMPD ribbon diagram (green) is superimposed to the A. thaliana-crystal ribbon structure [30] (blue). Double arrowheads point to the large N-terminal transmembrane domain characterizing A. thaliana AMPD; single arrowhead points to the ice worm-specific 16 amino acid loop; the arrow points to the ice worm-specific loop connecting strands β2 and β3. The catalytic zinc is represented by a yellow sphere, the coformycin 5′-phosphate and phosphate ion are depicted as stick models. (b) A. thaliana-crystal backbone structure [30] depicting 16 ice worm nonconservative species-specific amino acid substitutions, represented by space filling models.

Figure 3

Conformation near the catalytic zinc atom and the AMP-binding pocket, and the phosphate effector site. (a) The zinc atom shows a trigonal bipyramidal conformation ligating coformycin 5′-phosphate, three histidines (H97, H99 and H389), and one aspartic acid (D445). In yellow (space filling model) the specific ice worm substitution F304L. Phe170 and Tyr174 displace the ribose ring of coformycin 5′-phosphate, and Lys169, Lys173, Arg182, Asp444 and Glu448 are able to stabilize the phosphate group of coformycin 5′-phosphate by generating a hydrophilic pocket around this moiety. In yellow (space filling model) the nonconservative ice-worm substitution K188E. Arrows identify residues shifted with respect to those in A. thaliana AMPD. The catalytic zinc is represented by a yellow sphere, the coformycin 5′-phosphate and phosphate ion as stick models. (b) Interaction of the phosphate ion with neighboring amino acid residues (Lys213, Arg92 and Arg86). In yellow (space filling model) is the single specific N216K ice worm substitution.

Figure 4

Four sterically constraining residues (K173, Y174, D444 and D445) define the surface area of the AMPD substrate binding plane as ~38 Ų in A. thaliana (a), ~36 Ų in A. thaliana K188E (b), ~36 Ų in the ice worm E188 (c), and predicted loss of structure in ice worm E188K (d). Position 188 is visible to the right in each panel.