Evidence that feedback inhibition of NAD kinase controls responses to oxidative stress

Abstract

Formation of NADP+ from NAD+ is catalyzed by NAD kinase (NadK; EC 2.7.1.23). Evidence is presented that NadK is the only NAD kinase of Salmonella enterica and is essential for growth. NadK is inhibited allosterically by NADPH and NADH. Without effectors, NadK exists as an equilibrium mixture of dimers and tetramers (KD = 1.0 +/- 0.8 mM) but is converted entirely to tetramers in the presence of the inhibitor NADPH. Comparison of NadK kinetic parameters with pool sizes of NADH and NADPH suggests that NadK is substantially inhibited during normal growth and, thus, can increase its activity greatly in response to temporary drops in the pools of inhibitory NADH and NADPH. The primary inhibitor is NADPH during aerobic growth and NADH during anaerobic growth. A model is proposed in which variation of NadK activity is central to the adjustment of pyridine nucleotide pools in response to changes in aeration, oxidative stress, and UV irradiation. It is suggested that each of these environmental factors causes a decrease in the level of reduced pyridine nucleotides, activates NadK, and increases production of NADP(H) at the expense of NAD(H). Activation of NadK may constitute a defensive response that resists loss of protective NADPH.

Fig. 1.

NadK is essential. (A) Segregation of a duplication with both nadK⁺ and nadK:Cm alleles. (B and C) Colonies of duplication strain carrying nadK⁺ and a nadK:Cm^R allele (TT23464) growing on rich medium wiith X-Gal (B) or the same medium with added chloramphenicol (C).

Fig. 2.

Evidence for a single NadK enzyme. Crude extracts were fractionated by native PAGE. Each gel was cut, and portions were stained separately for NadK activity or protein (see Materials and Methods). Lanes: 1, 50 mg of crude extract stained with the complete reaction mix; 2, 50 mg of crude extract stained with reaction mix lacking ATP; 3, 3 mg of purified Salmonella NadK-tagged protein stained with Coomassie blue. The assay mix included NAD⁺, ATP (to allow NADP production by kinase), and NADP-dependent G-6-PD to reduce kinase product to NADPH that, in turn, reduces phenazine methosulphate and iodonitrotetrazolium, producing a colored precipitate. The assay detects a few endogenous NAD-dependent reductases that produce NADH and can be detected even when ATP or G-6-PD is left out of the reaction mixture (data not shown). Only NAD kinase produces a ATP-dependent activity band (by providing NADP⁺ for included G-6-PD).

Fig. 3.

Sedimentation equilibrium analysis of styNadK protein. (Lower) Data for three protein concentrations (0.26, 0.13, and 0.065 mg/ml) with the corresponding calculated curve fit (solid line). The data fit a dimer-tetramer equilibrium with a K_D = 1.0 ± 0.9 μM. (Upper) The closeness of the fit is evident by the small random residuals.