NADK2 (NAD kinase 2, mitochondrial) — review notes

UniProt: Q4G0N4 (NADK2_HUMAN). HGNC:26404. Synonyms: C5orf33, MNADK, NADKD1.
442 aa precursor; residues 1–62 mitochondrial transit peptide, chain 63–442.
EC 2.7.1.23. Belongs to the NAD kinase family.

Core biology

NADK2 is the mitochondrial NAD(+) kinase — the enzyme that generates the
mitochondrial NADP(+) pool. It phosphorylates NAD(+) using ATP (or, in vitro,
inorganic polyphosphate) to yield NADP(+):

NAD(+) + ATP = ADP + NADP(+) + H(+) (EC 2.7.1.23, RHEA:18629)
[file:human/NADK2/NADK2-uniprot.txt "Reaction=NAD(+) + ATP = ADP + NADP(+) + H(+); Xref=Rhea:RHEA:18629"]

• UniProt FUNCTION: "Mitochondrial NAD(+) kinase that phosphorylates NAD(+) to yield
  NADP(+). Can use both ATP or inorganic polyphosphate as the phosphoryl donor. Also
  has weak NADH kinase activity in vitro; however NADH kinase activity is much weaker
  than the NAD(+) kinase activity and may not be relevant in vivo."
  [file:human/NADK2/NADK2-uniprot.txt]
• Discovered as C5orf33, the "missing" source of human mitochondrial NADP(+):
  "NAD kinase is the sole NADP(+) biosynthetic enzyme... no mitochondrial NAD kinase
  has been identified in human, and the source of human mitochondrial NADP(+) remains
  elusive. Here we present evidence demonstrating that a human protein of unknown
  function, C5orf33, is a human mitochondrial NAD kinase"
  PMID:23212377

Experimental evidence (PMID:23212377, Ohashi/Kawai/Murata 2012, Nat Commun; full text available)

• Catalytic activity (IDA, MF GO:0003951): purified recombinant Δ62 protein forms
  NADP+ from NAD+ and ADP from ATP by TLC.
  PMID:23212377
• Kinetics: Km(NAD+) 0.022 mM, Km(ATP) 1.7 mM; NADHK activity only ~10% of NADK
  activity; inhibited by NADH, NADPH and NADP+.
  [file:human/NADK2/NADK2-uniprot.txt "Inhibited by NADH, NADPH and NADP(+)."]
• Subcellular location (IDA, CC mitochondrion GO:0005739): FLAG-tagged full-length
  protein localizes to mitochondria in HEK293A cells; Δ62 (no transit peptide) does not.
  PMID:23212377
• Also confirmed by cell fractionation: mitochondrial, not cytosolic.
  PMID:23212377
• Homodimer (IDA, MF GO:0042803 protein homodimerization activity): UniProt SUBUNIT
  = Homodimer (ECO:0000305|PubMed:23212377). Reactome names the "NADK2 dimer".
  [file:human/NADK2/NADK2-uniprot.txt "SUBUNIT: Homodimer."]
  Note: this is a structural/oligomerization property, not the catalytic function.
• Physiological role (NAD+ metabolic process; ROS defense): siRNA knockdown reduced
  mitochondrial NADK activity to 47% and increased intracellular ROS.
  PMID:23212377
  PMID:23212377
• Widely/ubiquitously expressed; mRNA more abundant than cytosolic NADK in most tissues.
  PMID:23212377

Localization corroboration

• HPA immunofluorescence (IDA, GO_REF:0000052): mitochondrion.
• High-throughput mitochondrial proteome (HTP, PMID:34800366, Morgenstern et al. 2021,
  Cell Metab): NADK2 in the high-confidence human mitochondrial proteome.
• UniProt SubCell mapping (IEA, SL-0173 → mitochondrion) and ARBA (mitochondrial matrix).
• Reactome TAS (R-HSA-8955030 NADK2 reaction; R-HSA-9838081/9838093 LONP1 substrate
  entries): mitochondrial matrix. The matrix is the correct submitochondrial compartment
  for this soluble matrix enzyme (transit peptide, no TM segments).

Downstream role of the mitochondrial NADP(H) pool

NADK2 supplies the NADPH used by NADP-dependent mitochondrial oxidoreductases. Loss of
NADK2 causes mitochondrial NADP(H) deficiency, secondarily crippling 2,4-dienoyl-CoA
reductase (DECR1, PUFA β-oxidation) and the first step of lysine degradation (AASS/DHTKD1
axis), producing DECR deficiency with hyperlysinemia. NADPH also feeds proline
biosynthesis via ALDH18A1/P5CS and antioxidant/one-carbon pathways.

According to PubMed (Houten et al. 2014, Hum Mol Genet
DOI, PMID:24847004):

"NADK2 encodes the mitochondrial NAD kinase, which is crucial for NADP biosynthesis
evidenced by decreased mitochondrial NADP(H) levels in patient fibroblasts. DECR and
also the first step in lysine degradation are performed by NADP-dependent
oxidoreductases explaining their in vivo deficiency."

UniProt DISEASE:

"2,4-dienoyl-CoA reductase deficiency (DECRD) [MIM:616034]: A rare, autosomal
recessive, inborn error of polyunsaturated fatty acids and lysine metabolism,
resulting in mitochondrial dysfunction. Affected individuals have a severe
encephalopathy... increased C10:2 carnitine levels and hyperlysinemia."
[file:human/NADK2/NADK2-uniprot.txt]

Annotation review decisions (summary)

Core MF: GO:0003951 NAD+ kinase activity (IDA, PMID:23212377) + GO:0005524 ATP binding
(phosphoryl-donor substrate; UniProt KW, not in GOA experimental set but real).
Core BP: GO:0019674 NAD+ metabolic process (IDA); the informative/precise BP is
GO:0006741 NADP+ biosynthetic process (UniProt DR InterPro; the actual anabolic role).
Core CC: GO:0005759 mitochondrial matrix (soluble matrix enzyme).

• All GO:0003951 calls (IDA, IBA, IEA) → the catalytic function is the core function; keep.
• Mitochondrion CC calls (IDA, IBA, IEA, HPA IDA, HTP) → correct; matrix is more precise.
• GO:0019674 NAD+ metabolic process → accept (IDA); note the anabolic/product side is
  NADP+ biosynthesis (GO:0006741), which is more informative and is proposed as a new term.
• GO:0042803 protein homodimerization activity (IDA) → real (homodimer) but it is an
  oligomerization property, not the catalytic MF; keep as non-core.
• LONP1 Reactome TAS matrix entries (R-HSA-9838081/9838093) → NADK2 is a LONP1 protease
  substrate; the CC (matrix) is correct, but these do not describe NADK2's own function;
  keep as non-core matrix localization.