Characterization of alanine and malate dehydrogenases from a marine psychrophile strain PA-43

Alanine dehydrogenase (AlaDH: EC 1.4.1.1), malate dehydrogenase (MDH: EC 1. 1.1.37), and glutamate dehydrogenase (EC 1.4.1.2), all NAD(+) dependent, we re detected in extracts from a psychrophilic bacterium, strain PA-43, isola ted from a sea urchin off the Icelandic coast. Characterization tests sugge sted that the strain had a close relationship to Vibrio, but sequencing of part of the 16S rDNA gene placed the bacterium among Shewanella species in a constructed phylogenetic tree. The bacterium had an optimum growth temper ature of 16.5 degreesC, and maximum dehydrogenase expression was obtained i n a rich medium supplemented with NaCl. Both AlaDH and MDH were purified to homogeneity. AlaDH is a hexamer, with an approximate relative molecular ma ss of 260,000, whereas MDH is dimeric, with an apparent relative molecular mass of approximately 70,000, Both enzymes were thermolabile, and the optim um temperatures for activity were shifted toward lower temperatures than th ose found in the same enzymes from mesophiles, 37 degreesC for MDH and appr oximately 47 degreesC for AlaDH. The pH optima for AlaDH in the forward and reverse reactions were 10.5 and 9, respectively, whereas those for MDH wer e 10-10.2 and 8.8, respectively. Partial amino acid sequences, comprising a pproximately 30% of the total sequences from each enzyme, were determined f or N-terminal, tryptic, and chymotryptic fragments of the enzymes. The AlaD H showed the highest similarity to AlaDHs from the psychrotroph Shewanella Ac10 and the mesophile Vibrio proteolyticus, whereas MDH was most similar t o the MDHs from the mesophiles Escherichia coli and Haemophilus influenzae, with lower identity to the psychrophilic malate dehydrogenases from Vibrio 5710 and Photobacterium SS9.