NAD-specific glutamate dehydrogenase from Thermus thermophilus HB8: purification and enzymatic properties

Abstract

An NAD-specific glutamate dehydrogenase from Thermus thermophilus HB8 was purified 350-fold by a several-step procedure involving Blue-Sepharose chromatography. The native protein had a molecular mass of approximately 289 kDa, and consisted of six subunits with a molecular mass of 48 kDa each. The optimum pH for the deaminating reaction was 8.0. The optimum temperature was around 85^90³C. NAD-glutamate dehydrogenase showed a high coenzyme specificity, catalysed preferentially glutamate catabolism and presented K m values for NAD and L-glutamate of 0.27 þ 0.03 mM and 49 þ 10 mM, respectively. No activity was detected with NADH or NADPH, 2-oxoglutarate and ammonia. Enzyme activity was found to be very stable at 72³C. Differential scanning calorimetry revealed a t m of 95³C for denaturation. z 1998 Federation of European Microbiological Societies. Published by Elsevier Science B.V.

Hetero-oligomeric glutamate dehydrogenase from Thermus thermophilus

“…Ruiz and colleagues have reported that native GDH partially purified from T. thermophilus HB8 is only active in oxidative deamination, and not in reductive amination (Ruiz et al, 1998(Ruiz et al, , 2003. Their results and ours are consistent in that GDH exhibits higher oxidative deamination activity than reductive amination activity.…”

“…Native GDH from T. thermophilus HB8 was partially purified and characterized by Ruiz et al (1998Ruiz et al ( , 2003. The purified GDH preparation contained two major bands that showed similar mobility on SDS-PAGE.…”

“…Although GDH has been purified from T. thermophilus HB8 and characterized (Ruiz et al, 1998(Ruiz et al, , 2003, it is not clear which isozyme was purified and characterized. Very recently, Bolivar and colleagues reported that recombinant GDH from T. thermophilus HB27, named GdhB in our present study, has GDH activity (Bolivar et al, 2008); however, the regulatory mechanism remains to be elucidated.…”

Expression of a Heterologous Glutamate Dehydrogenase Gene in Lactococcus lactis Highly Improves the Conversion of Amino Acids to Aroma Compounds

“…NAD ϩ -dependent GDHs are either tetrameric or hexameric and are mainly involved in glutamate catabolism, while NADP ϩ -dependent GDHs are hexameric and are mainly involved in ammonia assimilation and hence in glutamate synthesis. Catabolic NAD ϩ -dependent GDHs have been found in several microorganisms, including Saccharomyces cerevisiae (34), Candida utilis (9), Neurospora crassa (2,8), Streptomyces fradiae (17), Peptostreptococcus asaccharolyticus (28), Halobacterium halobium (3), Thermus thermophilus (23), Clostridium symbiosum (32), and Clostridium difficile (16), but only a few of these enzymes have been genetically and biochemically characterized (the C. symbiosum, C. difficile, and P. asaccharolyticus enzymes). Of the known gdh genes, the gdh gene of P. asaccharolyticus seems to be suitable for expression in L. lactis, since it is a gene from a gram-positive coccus that is phylogenetically closely related to L. lactis.…”

Structure, function, and regulation of enzymes involved in amino acid metabolism of bacteria and archaea

“…T. thermophilus possesses two putative gdh genes (gdhA and gdhB) in its genome, both of which encode GDH sharing approximately 46% identity in amino acid sequence. Although GDH has been purified from T. thermophilus HB8 and characterized, 10) it was not clear which isozyme was purified and characterized. Boliver and colleagues reported that recombinant GDH from T. thermophilus HB27, named GdhB in our present study, has GDH activity, 11) however, the function of GdhA remains to be elucidated.…”