Structure of human NADK2 reveals atypical assembly and regulation of NAD kinases from animal mitochondria

Abstract

All kingdoms of life produce essential nicotinamide dinucleotide NADP(H) using NAD kinases (NADKs). A panel of published NADK structures from bacteria, eukaryotic cytosol, and yeast mitochondria revealed similar tetrameric enzymes. Here, we present the 2.8-Å structure of the human mitochondrial kinase NADK2 with a bound substrate, which is an exception to this uniformity, diverging both structurally and biochemically from NADKs. We show that NADK2 harbors a unique tetramer disruptor/dimerization e lement, which is conserved in m itochondrial k inases of a nimals (EMKA) and absent from other NADKs. EMKA stabilizes the NADK2 dimer but prevents further NADK2 oligomerization by blocking the tetramerization interface. This structural change bears functional consequences and alters the activation mechanism of the enzyme. Whereas tetrameric NADKs undergo cooperative activation via oligomerization, NADK2 is a constitutively active noncooperative dimer. Thus, our data point to a unique regulation of NADP(H) synthesis in animal mitochondria achieved via structural adaptation of the NADK2 kinase.

The mitochondrial NAD kinase functions as a major metabolic regulator upon increased energy demand

“…( B ) 3-D structure of human MNADK. NAD + (sticks) binds to the catalytic pocket (PDB code: 7N29 ) [ 65 ]. Red denotes the conserved region RSEGHLCLPVRYT, where the Cysteine residue that undergoes SNO is highlighted with green.…”

Molecular properties and regulation of NAD+ kinase (NADK)

“…Furthermore, NADPH allosterically inhibits NADK by enhancing the tetramer formation [ 59 ]. NADK2 possesses a unique tetramer disruptor/dimerization element conserved in mitochondrial kinases of animals (EMKA), which prevents NADK2 oligomerization, promotes dimerization and maintains NADK2 in a constitutively active conformation [ 60 ]. Thus, mammalian NADKs are regulated by phosphorylation, acetylation and protein-protein interaction ( Fig.…”

Mitochondrial NAD kinase in health and disease

“…Recently, Du et al examined NADK2 activity using a direct method, which monitors the formation of the radiolabeled product by incubating NADK2 with radiolabeled ATP. The authors demonstrated that NADK2 can use both NAD + and NADH as equivalent substrates with comparable catalytic activity [ 25 ]. Although the exact degree of catalytic efficiency using substrates NAD + vs. NADH by NADK2 varies in different studies, they all consistently show NADK2's NADH kinase activity [ 13 , 25 ].…”

“…The authors demonstrated that NADK2 can use both NAD + and NADH as equivalent substrates with comparable catalytic activity [ 25 ]. Although the exact degree of catalytic efficiency using substrates NAD + vs. NADH by NADK2 varies in different studies, they all consistently show NADK2's NADH kinase activity [ 13 , 25 ]. This result is significant, because it shows that NADK2 generates not only NADP + , but also NADPH, representing a novel pathway for NADPH generation.…”