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First published online December 26, 2008
Journal of Experimental Biology 212, 169-177 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.024505
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Temperature adaptation of cytosolic malate dehydrogenases of limpets (genus Lottia): differences in stability and function due to minor changes in sequence correlate with biogeographic and vertical distributions

Yunwei Dong* and George N. Somero{dagger}

Hopkins Marine Station, Department of Biology, Stanford University, Pacific Grove, CA 93950, USA


Figure 1
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  		Fig. 1. Apparent Michaelis-Menten constant for the cofactor NADH (KmNADH) of cMDHs from foot muscle of Lottia gigantea, L. scabra, L. pelta and L. scutum. The asterisk indicates a significant difference in KmNADH between the two low- to mid-intertidal species (L. pelta and L. scutum) and the two mid- to high-intertidal species (L. gigantea and L. scabra; P<0.05). Inset: Arrhenius plot (ln KmNADH versus reciprocal temperature in Kelvins) of cMDH for L. gigantea, L. scabra, L. pelta and L. scutum. The slope of the relationship for cMDH of L. pelta was significantly steeper than that for L. scabra (P<0.001; ANCOVA). Statistics (means ± s.d.) for the KmNADH determinations are given in supplementary material Table 2.

 

Figure 2
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  		Fig. 2. Michaelis–Menten constant for cofactor NADH (KmNADH) of cMDHs from foot muscle of L. digitalis and L. austrodigitalis. Asterisks indicate the temperatures at which Km values were significantly different between species ({alpha}=0.05). Inset: Arrhenius plot (ln KmNADH versus reciprocal temperature in Kelvins) of cMDHs of L. digitalis and L. austrodigitalis. The slope of the relationship for cMDH of L. digitalis is significantly steeper than that for L. austrodigitalis (P<0.0001; ANCOVA).

 

Figure 3
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  		Fig. 3. Residual activities of cMDHs from Lottia limpets after heat treatment at 42.5°C for different times. (A) Residual activities of L. gigantea, L. scabra, L. pelta and L. scutum. The slopes of the regressions for cMDHs of L. pelta and L. scutum are significantly higher than those of the orthologs of L. gigantea and L. scabra (P<0.001). (B) Residual activities of cMDHs of L. digitalis and L. austrodigitalis. The denaturation rate of cMDH for L. digitalis is significantly higher than that of the L. austrodigitalis ortholog (P<0.001; ANCOVA).

 

Figure 4
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  		Fig. 4. Deduced amino acid sequence alignment of cMDHs from L. digitalis, L. austrodigitalis, L. pelta, L. scutum, L. gigantea and L. scabra. Above each segment of sequence is the designation for secondary structure in the cMDH of L. digitalis, which was calculated using the Swiss-Model software based on the pig cMDH [PDB, 5mdhA (Chapman et al., 1999Go)]. The cofactor binding residues, the residues functioning in catalysis and the residues involved in subunit–subunit interactions are shown in blue, red and green, respectively. The single substitution between L. digitalis and L. austrodigitalis (position 291) is shown in bold.

 

Figure 5
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  		Fig. 5. Three-dimensional model of a single cMDH monomer from (A) L. digitalis and (B) L. austrodigitalis. The positions of Gly-291/Ser-291 and amino acids within 4 Å of Gly-291/Ser-291 are indicated. Van der Waals radii are indicated for the atoms comprising Gly-291/Ser-291. The position of βL is also indicated. These models were produced using Swiss-Model software visualized with the Swiss-PDB Viewer (Guex and Peitsch, 1997Go).

 

Figure 6
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  		Fig. 6. A magnified view of the region near residue 291 (Gly/Ser) showing amino acids within 6 Å of Gly-291 (Ser-291) of cMDHs from (A) L. digitalis and (B) L. austrodigitalis. Hydrogen bonds were calculated using Swiss-PDB Viewer (Guex and Peitsch, 1997Go), and are indicated as dashed lines. Atoms that form hydrogen bonds in the ortholog from L. digitalis are marked as: 1, Thr-292 O; 2, Asp-293 OD1; 3, Thr-261 OG1; 4, Ile-290 N. Atoms that form hydrogen bonds in the ortholog from L. austrodigitalis are marked as: 5, Ser-291 O; 6, Arg-294 N; 7, Asp-293 OD1; 8, Arg-294 O; 9, Thr-261 OG1; 10, Ile-290 N; 11, Ser-291 N; 12, Val-295 O; 13, Thr-261 N; and 14, Ile-290 O.

 

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