Importance of lysine-286 at the NADP site of glutamate dehydrogenase from Salmonella typhimurium

Abstract

Affinity labeling studies of NADP(+)-glutamate dehydrogenase from Salmonella typhimurium have shown that the peptide Leu-282-Lys-286 is located near the coenzyme site [Haeffner-Gormley et al. (1991) J. Biol. Chem. 266, 5388-5394]. The present study was undertaken to evaluate the role of lysine-286. The mutant enzymes K286R, K286Q, and K286E were prepared by site-directed mutagenesis, expressed in Escherichia coli, and purified. The Vmax values (micromoles of NADPH per minute per milligram of protein) were similar for WT (270), K286R (529), K296Q (409), and K286E (382) enzymes. As measured at pH 7.9, the Km value for NADPH was much greater for K286E (280 microM) than for WT (9.8 microM), K286R (30 microM), or K286Q (66 microM) enzymes. The efficiencies (kcat/Km) of the WT and K286R mutant were similar (1.2 x 10(3) min-1 microM-1 and 1.0 x 10(3) min-1 microM-1, respectively) while those of K286Q (0.30 x 10(3) min-1 microM-1) and K286E (0.07 x 10(3) min-1 microM-1) were greatly reduced. The decreased efficiency of the K286E mutant results from the increase in Km-NADPH, consistent with a role for a basic residue at position 286 which enhances the binding of NADPH. Plots of Vmax vs pH showed the pH optima to be 8.1-8.3 for all enzymes at saturating NADPH concentrations. A 40-fold increase in Km-NADPH for K286E was observed as the pH increased from 5.98 to 8.08, from which a unique pKe of 6.5 was calculated.(ABSTRACT TRUNCATED AT 250 WORDS)

A highly active decarboxylating dehydrogenase with rationally inverted coenzyme specificity.

“…Dehydrogenases discriminate among nicotinamide coenzymes through interactions established between the protein and the 2'-phosphate of NADP and the 2'-and 3'-hydroxyls of NAD. Engineering dihydrolipoamide and malate dehydrogenases demonstrates that changing the preference of an NADdependent enzyme can be achieved by introducing positively charged residues to neutralize the negatively charged 2'-phosphate of NADP (1,2). Yet, as earlier attempts to invert the preference of glutathione reductase and glutamate dehydrogenase illustrate, engineering the preference of an NADPdependent enzyme toward NAD is more troublesome (3,24). Perhaps, the strict reliance on homology as a criterion for replacing amino acids is insufficient to optimize directional interactions, such as hydrogen bonds to the 2'-and 3'-hydroxyls of NAD.…”

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“…Such a higher complexity of the signature for the dinucleotide-binding motif makes it possible to discriminate more precisely between the three isoforms: (i) the hexapeptide GAGNVA is found for GDH4 from Viridiplantae ; (ii) the hexapeptide GSGNVA is the signature for GDH4 from not Viridiplantae (subset G). The finding of the same motif for subsets G, I2 and I3, together with high percentage of identity between them (86% between G and I2, 70% between G and I3), suggest that GDHs not EC classified from subsets I2 and I3 are also GDH4; (iii) the hexapeptides GFGNVG or GFGNAG are found for subsets A, D, H ( Viridiplantae ) and for subsets B, E, I1 ( not Viridiplantae ); (iv) a very different heptapeptide G(Q) [V/T] [D/G] [M/P] [S/D]G, sharing the first and the third conserved Gly, is found for large GDHsfrom subsets C, K and L. The K m NADPH values for the wild-type GDH4 from Salmonella typhimurium and for the mutant GDH K 286 E are 9.8 μM and 280 μM, respectively [23]. This indicates that the side chain of the equivalent Lys residue for plant GDH4 (Lys 360 of Ref , Fig.…”

Purification, Characterization, and Submitochondrial Localization of the 32-Kilodalton NADH Dehydrogenase from Maize

“…However, the Arum 32-kD NADH DH was shown to oxidize both NADH and NADPH (Chauveau and Lance, 1991), and thus the cofactor specificity is likely dependent on the species. It has been shown with other enzymes that single amino acid changes can change the cofactor specificity (Feeney et al, 1990;Haeffner-Gormley et al, 1992); therefore, speciesdependent cofactor specificity is not surprising. The 32-kD NADH DH was found to effectively utilize deamino-NADH as a substrate.…”