Alanine dehydrogenase from the psychrophilic bacterium strain PA-43: overexpression, molecular characterization, and sequence analysis.
The gene encoding alanine dehydrogenase (AlaDH; EC 1.4.1.1) from the marine psychrophilic bacterium strain PA-43 was cloned, sequenced, and overexpressed in Escherichia coli. The primary structure was deduced on the basis of the nucleotide sequence. The enzyme subunit contains 371 amino acid residues, and the sequence is 90% and 77% identical, respectively, to AlaDHs from Shewanella Ac10 and Vibrio proteolyticus. The half-life of PA-43 AlaDH at 52 degrees C is 9 min, and it is thus more thermolabile than the AlaDH from Shewanella Ac10 or V. proteolyticus. The enzyme showed strong specificity for NAD(+) and l-alanine as substrates. The apparent K(m) for NAD(+) was temperature dependent (0.04 mM-0.23 mM from 15 degrees C to 55 degrees C). A comparison of the PA-43 deduced amino acid sequence to the solved three-dimensional structure of Phormidium lapideum AlaDH showed that there were likely to be fewer salt bridges in the PA-43 enzyme, which would increase enzyme flexibility and decrease thermostability. The hydrophobic surface character of the PA-43 enzyme was greater than that of P. lapideum AlaDH, by six residues. However, no particular modification or suite of modifications emerged as being clearly responsible for the psychrophilic character of PA-43 AlaDH.
Irwin
Jane A
JA
Department of Biochemistry and Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Belfield, Dublin 4, Ireland.
Lynch
Susan V
SV
Coughlan
Suzie
S
Baker
Patrick J
PJ
Gudmundsson
Haflidi M
HM
Alfredsson
Gudni A
GA
Rice
David W
DW
Engel
Paul C
PC
D000064:Acclimatization; D050788:Alanine Dehydrogenase; D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D003080:Cold Temperature; D004269:DNA, Bacterial / Q000235:genetics; D004789:Enzyme Activation / Q000187:drug effects; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006090:Gram-Negative Bacteria / Q000201:enzymology* / Q000235:genetics*; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013816:Thermodynamics
D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D000594:Amino Acid Oxidoreductases; D050788:Alanine Dehydrogenase