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First published online January 31, 2006
Journal of Experimental Biology 209, 656-667 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02036

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Temperature sensitivities of cytosolic malate dehydrogenases from native and invasive species of marine mussels (genus Mytilus): sequence-function linkages and correlations with biogeographic distribution

Peter A. Fields^1,*, Emily L. Rudomin¹ and George N. Somero²

¹ Biology Department, Franklin and Marshall College, Lancaster, PA 17604-3003, USA
² Hopkins Marine Station of Stanford University, Pacific Grove, CA 93950, USA

View larger version (45K): [in a new window]   Fig. 1. Nucleotide sequences of the coding regions of cmdh cDNAs from Mytilus trossulus (M. t.), M. galloprovincialis (M. g.) and M. californianus (M. c.). Dashes represent identity with the M. trossulus sequence. The codons for the non-conservative substitutions at residue 114 in each ortholog are highlighted. In a number of positions, nucleotide ambiguities (i.e. double peaks) were noted in the source electropherograms: M=A or C; R=A or G; Y=C or T; W=A or T; all ambiguities are synonymous in the deduced amino acid sequence.

View larger version (15K): [in a new window]   Fig. 2. Amino acid sequences of cMDHs from Mytilus trossulus (M. t.), M. galloprovincialis (M. g.) and M. californianus (M. c.), deduced from the nucleotide sequences given in Fig. 1. Dashes represent identity with the M. trossulus sequence. The single non-conservative substitution at position 114 between M. trossulus and the other two congeners is indicated by the arrows. The catalytic loop region of cMDH (see Discussion) is highlighted in black.

View larger version (18K): [in a new window]   Fig. 3. Michaelis-Menten constants for the cofactor NADH of cMDHs either purified from muscle tissue or expressed recombinantly. (A) KmNADH values for cMDHs purified from muscle tissue of M. trossulus, M. galloprovincialis and M. californianus, measured from 5 to 45°C [* indicates a significant difference (=0.05) between M. trossulus and M. galloprovincialis cMDHs; ** between M. trossulus and M. californianus; and *** between M. californianus and M. galloprovincialis]. (B) KmNADH values of recombinant M. trossulus cMDH (rWT) and mutants of M. trossulus cMDH replacing the residue at position 114 with that found in M. galloprovincialis (V114N) or M. californianus (V114H), measured from 5 to 45°C [* indicates a significant difference (=0.05) between rWT and V114N cMDHs, ** between rWT and V114H; and *** between V114H and V114N]. (C) A comparison of KmNADH values measured at 35°C between M. trossulus (M. t.) cMDH and rWT, M. galloprovincialis (M. g.) cMDH and mutant V114N, and M. californianus (M. c.) cMDH and mutant V114H. KmNADH values sharing a letter designation (X,Y,Z) are not significantly different (=0.05); all other comparisons are significantly different.

View larger version (23K): [in a new window]   Fig. 4. Arrhenius activation energies (Ea) of M. trossulus, M. galloprovincialis and M. californianus cMDHs, as well as rWT M. trossulus cMDH, mutants V114H and V114N. Vmax values were standardized to 1.00 at 0°C before loge-transformation, to aid in visualizing differences among the slopes. Ea is proportional to the negative slope of the regression relating loge Vmax to the reciprocal of absolute temperature. An analysis of covariance indicates that none of the Ea values are significantly different (=0.05).

View larger version (16K): [in a new window]   Fig. 5. Turnover numbers (kcat) of rWT M. trossulus cMDH and the mutants V114N and V114H from 5 to 35°C. Mutations V114N and V114H significantly reduce the catalytic rate relative to rWT at all temperatures except 5°C for V114H. Solid bars indicate the physiological immersion temperature ranges of M. trossulus and M. galloprovincialis, where rWT and V114N exhibit similar catalytic rates.

View larger version (16K): [in a new window]   Fig. 6. Thermal denaturation profiles of rWT M. trossulus cMDH and the mutants V114H and V114N. Samples were held at 42.5°C for the indicated time before residual activity was measured in triplicate. The rWT M. trossulus and V114H mutants are not significantly different in stability, but the V114N mutant denatures at a significantly higher rate than the other two forms.

View larger version (27K): [in a new window]   Fig. 7. (A) A model of one monomer of M. trossulus cMDH, based on the structure of the pig ortholog (PDB accession number 4MDH). All residues that differ between the M. trossulus and the M. galloprovincialis or M. californianus orthologs are labeled; the non-conservative mutation at position 114 is shown in dark spacefill. Representative active site residues are shown in light spacefill, and the highly mobile catalytic loop, which must move by10 Å during catalysis, is black. (B) A magnified view of mutant V114N, showing the relationship of that residue to the catalytic loop (black), as well as the hydrogen bond that may form between the amide nitrogen of the asparagine side chain and the carbonyl oxygen of 143Y on the neighboring helix 1F. Models were visualized with VMD (Humphrey et al., 1996).