Catalytic contribution of threonine 244 in human ALDH2

Lilian González-Segura, K. K. Ho, Samantha Perez-Miller, Henry Weiner, Thomas Hurley

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Amongst the numerous conserved residues in the aldehyde dehydrogenase superfamily, the precise role of Thr-244 remains enigmatic. Crystal structures show that this residue lies at the interface between the coenzyme-binding and substrate-binding sites with the side chain methyl substituent oriented toward the B-face of the nicotinamide ring of the NAD(P)+ coenzyme, when in position for hydride transfer. Site-directed mutagenesis in ALDH1A1 and GAPN has suggested a role for Thr-244 in stabilizing the nicotinamide ring for efficient hydride transfer. Additionally, these studies also revealed a negative effect on cofactor binding which is not fully explained by the interaction with the nicotinamide ring. However, it is suggestive that Thr-244 immediately precedes helix αG, which forms one-half of the primary binding interface for the coenzyme. Hence, in order to more fully investigate the role of this highly conserved residue, we generated valine, alanine, glycine and serine substitutions for Thr-244 in human ALDH2. All four substituted enzymes exhibited reduced catalytic efficiency toward substrate and coenzyme. We also determined the crystal structure of the T244A enzyme in the absence and presence of coenzyme. In the apo-enzyme, the alpha G helix, which is key to NAD binding, exhibits increased temperature factors accompanied by a small displacement toward the active site cysteine. This structural perturbation was reversed in the coenzyme-bound complex. Our studies confirm a role for the Thr-244 beta methyl in the accurate positioning of the nicotinamide ring for efficient catalysis. We also identify a new role for Thr-244 in the stabilization of the N-terminal end of helix αG. This suggests that Thr-244, although less critical than Glu-487, is also an important contributor toward coenzyme binding.

Original languageEnglish
Pages (from-to)32-40
Number of pages9
JournalChemico-Biological Interactions
Volume202
Issue number1-3
DOIs
StatePublished - Feb 25 2013

Fingerprint

Coenzymes
Threonine
Niacinamide
Hydrides
NAD
Enzymes
Crystal structure
Mutagenesis
Aldehyde Dehydrogenase
Valine
Substrates
Site-Directed Mutagenesis
Catalysis
Alanine
Glycine
Serine
Cysteine
Catalytic Domain
Substitution reactions
Stabilization

Keywords

  • Aldehyde dehydrogenase
  • Coenzyme binding
  • Disorder
  • Isomerization
  • NAD

ASJC Scopus subject areas

  • Toxicology

Cite this

Catalytic contribution of threonine 244 in human ALDH2. / González-Segura, Lilian; Ho, K. K.; Perez-Miller, Samantha; Weiner, Henry; Hurley, Thomas.

In: Chemico-Biological Interactions, Vol. 202, No. 1-3, 25.02.2013, p. 32-40.

Research output: Contribution to journalArticle

González-Segura, L, Ho, KK, Perez-Miller, S, Weiner, H & Hurley, T 2013, 'Catalytic contribution of threonine 244 in human ALDH2', Chemico-Biological Interactions, vol. 202, no. 1-3, pp. 32-40. https://doi.org/10.1016/j.cbi.2012.12.009
González-Segura, Lilian ; Ho, K. K. ; Perez-Miller, Samantha ; Weiner, Henry ; Hurley, Thomas. / Catalytic contribution of threonine 244 in human ALDH2. In: Chemico-Biological Interactions. 2013 ; Vol. 202, No. 1-3. pp. 32-40.
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AU - Ho, K. K.

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AU - Weiner, Henry

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AB - Amongst the numerous conserved residues in the aldehyde dehydrogenase superfamily, the precise role of Thr-244 remains enigmatic. Crystal structures show that this residue lies at the interface between the coenzyme-binding and substrate-binding sites with the side chain methyl substituent oriented toward the B-face of the nicotinamide ring of the NAD(P)+ coenzyme, when in position for hydride transfer. Site-directed mutagenesis in ALDH1A1 and GAPN has suggested a role for Thr-244 in stabilizing the nicotinamide ring for efficient hydride transfer. Additionally, these studies also revealed a negative effect on cofactor binding which is not fully explained by the interaction with the nicotinamide ring. However, it is suggestive that Thr-244 immediately precedes helix αG, which forms one-half of the primary binding interface for the coenzyme. Hence, in order to more fully investigate the role of this highly conserved residue, we generated valine, alanine, glycine and serine substitutions for Thr-244 in human ALDH2. All four substituted enzymes exhibited reduced catalytic efficiency toward substrate and coenzyme. We also determined the crystal structure of the T244A enzyme in the absence and presence of coenzyme. In the apo-enzyme, the alpha G helix, which is key to NAD binding, exhibits increased temperature factors accompanied by a small displacement toward the active site cysteine. This structural perturbation was reversed in the coenzyme-bound complex. Our studies confirm a role for the Thr-244 beta methyl in the accurate positioning of the nicotinamide ring for efficient catalysis. We also identify a new role for Thr-244 in the stabilization of the N-terminal end of helix αG. This suggests that Thr-244, although less critical than Glu-487, is also an important contributor toward coenzyme binding.

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