Pcal_1699, an extremely thermostable malate dehydrogenase from hyperthermophilic archaeon Pyrobaculum calidifontis.
Two malate dehydrogenase homologs, Pcal_0564 and Pcal_1699, have been found in the genome of Pyrobaculum calidifontis. The gene encoding Pcal_1699 consisted of 927 nucleotides corresponding to a polypeptide of 309 amino acids. To examine the properties of Pcal_1699, the structural gene was cloned, expressed in Escherichia coli and the purified gene product was characterized. Pcal_1699 was NADH specific enzyme exhibiting a high malate dehydrogenase activity (886 U/mg) at optimal pH (10) and temperature (90 °C). Unfolding studies suggested that urea could not induce complete unfolding and inactivation of Pcal_1699 even at a final concentration of 8 M; however, in the presence of 4 M guanidine hydrochloride enzyme structure was unfolded with complete loss of enzyme activity. Thermostability experiments revealed that Pcal_1699 is the most thermostable malate dehydrogenase, reported to date, retaining more than 90 % residual activity even after heating for 6 h in boiling water.
School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore, 54590, Pakistan.
Ghazaleh
G
Gharib
School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore, 54590, Pakistan. naeemrashid37@hotmail.com.
Naeem
N
Rashid
School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore, 54590, Pakistan.
Qamar
Q
Bashir
School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore, 54590, Pakistan.
Qura-Tul Ann Afza
QT
Gardner
School of Biological Sciences, University of the Punjab, Quaid-e-Azam Campus, Lahore, 54590, Pakistan.|School of Biological Sciences, University of Southampton, Southampton, SO16 7PX, UK.
Muhammad
M
Akhtar
The Research Organization of Science & Technology, Ritsumeikan University, Kusatsu, Shiga, 525-8577, Japan.
Tadayuki
T
Imanaka
D000595:Amino Acid Sequence; D019843:Archaeal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004795:Enzyme Stability; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D008291:Malate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism; D058767:Protein Unfolding; D041543:Pyrobaculum / Q000201:enzymology* / Q000235:genetics
D019843:Archaeal Proteins; D009243:NAD; D008291:Malate Dehydrogenase
Hyperthermophile; Malate dehydrogenase; Pyrobaculum calidifontis; Structural stability; Thermostable; Unfolding