Characterization of malate dehydrogenase from the hyperthermophilic archaeon Pyrobaculum islandicum
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Native and recombinant malate dehydrogenase (MDH) was characterized from the hyperthermophilic, facultatively autotrophic archaeon Pyrobaculum islandicum. The enzyme is a homotetramer with a subunit mass of 33 kDa. The activity kinetics of the native and recombinant proteins are the same. The apparent K m values of the recombinant protein for oxaloacetate (OAA) and NADH (at 80°C and pH 8.0) were 15 and 86 μM, respectively, with specific activity as high as 470 U mg−1. Activity decreased more than 90% when NADPH was used. The catalytic efficiency of OAA reduction by P. islandicum MDH using NADH was significantly higher than that reported for any other archaeal MDH. Unlike other archaeal MDHs, specific activity of the P. islandicum MDH back-reaction also decreased more than 90% when malate and NAD+ were used as substrates and was not detected with NADP+. A phylogenetic tree of 31 archaeal MDHs shows that they fall into 5 distinct groups separated largely along taxonomic lines suggesting minimal lateral mdh transfer between Archaea.
KeywordsMalate dehydrogenase Pyrobaculum islandicum Archaea Citric acid cycle Autotrophy
L. J. Yennaco and Y. Hu contributed equally to the design, execution, and interpretation of this research. We thank T. Lowe and for kindly providing the P. islandicum mdh sequence ahead of its public release, F. J. Jenney and M. W. W. Adams for providing the expression plasmid used in this study and for their suggestions regarding recombinant protein expression, and the Mass Spectrometry Facility at the University of Massachusetts for their MALDI-TOF analysis. This study was funded by grant MAS00897 from the US Department of Agriculture.
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