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, were detected in extracts from a psychrophilic bacterium, strain PA-43, isolated from a sea urchin off the Icelandic coast. Characterization tests suggested 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 temperature of 16.5 degrees C, and maximum dehydrogenase expression was obtained in a rich medium supplemented with NaCl. Both AlaDH and MDH were purified to homogeneity. AlaDH is a hexamer, with an approximate relative molecular mass of 260,000, whereas MDH is dimeric, with an apparent relative molecular mass of approximately 70,000. Both enzymes were thermolabile, and the optimum temperatures for activity were shifted toward lower temperatures than those found in the same enzymes from mesophiles, 37 degrees C for MDH and approximately 47 degrees C for AlaDH. The pH optima for AlaDH in the forward and reverse reactions were 10.5 and 9, respectively, whereas those for MDH were 10-10.2 and 8.8, respectively. Partial amino acid sequences, comprising approximately 30% of the total sequences from each enzyme, were determined for N-terminal, tryptic, and chymotryptic fragments of the enzymes. The AlaDH showed the highest similarity to AlaDHs from the psychrotroph Shewanella Ac10 and the mesophile Vibrio proteolyticus, whereas MDH was most similar to the MDHs from the mesophiles Escherichia coli and Haemophilus influenzae, with lower identity to the psychrophilic malate dehydrogenases from Vibrio 5710 and Photobacterium SS9.
Irwin
J A
JA
Department of Biochemistry, University College Dublin, Belfield, Ireland.
Gudmundsson
H M
HM
Marteinsson
V T
VT
Hreggvidsson
G O
GO
Lanzetti
A J
AJ
Alfredsson
G A
GA
Engel
P C
PC
D050788:Alanine Dehydrogenase; D000594:Amino Acid Oxidoreductases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D003080:Cold Temperature; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008291:Malate Dehydrogenase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010802:Phylogeny; D012617:Sea Urchins / Q000382:microbiology; D017386:Sequence Homology, Amino Acid; D020592:Shewanella / Q000201:enzymology / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification; D014733:Vibrio / Q000201:enzymology / Q000235:genetics
D008291:Malate Dehydrogenase; D000594:Amino Acid Oxidoreductases; D050788:Alanine Dehydrogenase