Fields Peter A, Rudomin Emily L, Somero George N
Biology Department, Franklin and Marshall College, Lancaster, PA 17604-3003, USA.
J Exp Biol. 2006 Feb;209(Pt 4):656-67. doi: 10.1242/jeb.02036.
The blue mussel Mytilus galloprovincialis, a native of the Mediterranean Sea, has invaded the west coast of North America in the past century, displacing the native blue mussel, Mytilus trossulus, from most of its former habitats in central and southern California. The invasive success of M. galloprovincialis is conjectured to be due, in part, to physiological adaptations that enable it to outperform M. trossulus at high temperatures. We have examined the structure and function of the enzyme cytosolic malate dehydrogenase (cMDH) from these species, as well as from the more distantly related ribbed mussel, Mytilus californianus, to characterize the effects of temperature on kinetic properties thought to exhibit thermal adaptation. The M. trossulus cMDH ortholog differs from the other cMDHs in a direction consistent with cold adaptation, as evidenced by a higher and more temperature-sensitive Michaelis-Menten constant for the cofactor NADH (Km(NADH)). This difference results from minor changes in sequence: the M. trossulus ortholog differs from the M. galloprovincialis ortholog by only two substitutions in the 334 amino acid monomer, and the M. californianus and M. trossulus orthologs differ by five substitutions. In each case, only one of these substitutions is non-conservative. To test the effects of individual substitutions on kinetic properties, we used site-directed mutagenesis to create recombinant cMDHs. Recombinant wild-type M. trossulus cMDH (rWT) has high Km(NADH) compared with mutants incorporating the non-conservative substitutions found in M. californianus and M. galloprovincialis - V114H and V114N, respectively - demonstrating that these mutations are responsible for the differences found in substrate affinity. Turnover number (kcat) is also higher in rWT compared with the two mutants, consistent with cold adaptation in the M. trossulus ortholog. Conversely, rWT and V114H appear more thermostable than V114N. Based on a comparison of Km(NADH) and kcat values among the orthologs, we propose that immersion temperatures are of greater selective importance in adapting kinetic properties than the more extreme temperatures that occur during emersion. The relative warm adaptation of M. galloprovincialis cMDH may be one of a suite of physiological characters that enhance the competitive ability of this invasive species in warm habitats.
原产于地中海的蓝贻贝（Mytilus galloprovincialis）在过去一个世纪入侵了北美西海岸，在加利福尼亚州中部和南部的大部分原栖息地取代了本地蓝贻贝（Mytilus trossulus）。据推测，M. galloprovincialis的入侵成功部分归因于其生理适应性，使其能够在高温下胜过M. trossulus。我们研究了这些物种以及亲缘关系更远的加州贻贝（Mytilus californianus）的胞质苹果酸脱氢酶（cMDH）的结构和功能，以表征温度对被认为表现出热适应性的动力学特性的影响。M. trossulus的cMDH直系同源物与其他cMDH的差异方向与冷适应性一致，辅因子NADH的米氏常数（Km(NADH)）更高且对温度更敏感就证明了这一点。这种差异源于序列的微小变化：M. trossulus的直系同源物与M. galloprovincialis的直系同源物在334个氨基酸单体中仅存在两个替换差异，而M. californianus和M. trossulus的直系同源物存在五个替换差异。在每种情况下，这些替换中只有一个是非保守的。为了测试单个替换对动力学特性的影响，我们使用定点诱变来创建重组cMDH。与分别包含在M. californianus和M. galloprovincialis中发现的非保守替换——V114H和V114N——的突变体相比，重组野生型M. trossulus cMDH（rWT）具有较高的Km(NADH)，这表明这些突变是底物亲和力差异的原因。与这两个突变体相比，rWT的周转数（kcat）也更高，这与M. trossulus直系同源物的冷适应性一致。相反，rWT和V114H似乎比V114N更耐热。基于直系同源物之间Km(NADH)和kcat值的比较，我们提出，在适应动力学特性方面，浸没温度比出露期间出现的更极端温度具有更大的选择重要性。M. galloprovincialis cMDH的相对热适应性可能是增强这种入侵物种在温暖栖息地竞争能力的一系列生理特征之一。
