Structural basis for the stereospecific inhibition of the dual proline/hydroxyproline catabolic enzyme ALDH4A1 by trans-4-hydroxy-L-proline

Alexandra N. Bogner, Kyle M. Stiers, Cole M. McKay, Donald F. Becker, John J. Tanner

Research output: Contribution to journalArticlepeer-review

3 Scopus citations


Aldehyde dehydrogenase 4A1 (ALDH4A1) catalyzes the final steps of both proline and hydroxyproline catabolism. It is a dual substrate enzyme that catalyzes the NAD+-dependent oxidations of L-glutamate-γ-semialdehyde to L-glutamate (proline metabolism), and 4-hydroxy-L-glutamate-γ-semialdehyde to 4-erythro-hydroxy-L-glutamate (hydroxyproline metabolism). Here we investigated the inhibition of mouse ALDH4A1 by the six stereoisomers of proline and 4-hydroxyproline using steady-state kinetics and X-ray crystallography. Trans-4-hydroxy-L-proline is the strongest of the inhibitors studied, characterized by a competitive inhibition constant of 0.7 mM, followed by L-proline (1.9 mM). The other compounds are very weak inhibitors (approximately 10 mM or greater). Insight into the selectivity for L-stereoisomers was obtained by solving crystal structures of ALDH4A1 complexed with trans-4-hydroxy-L-proline and trans-4-hydroxy-D-proline. The structures suggest that the 10-fold greater preference for the L-stereoisomer is due to a serine residue that hydrogen bonds to the amine group of trans-4-hydroxy-L-proline. In contrast, the amine group of the D-stereoisomer lacks a direct interaction with the enzyme due to a different orientation of the pyrrolidine ring. These results suggest that hydroxyproline catabolism is subject to substrate inhibition by trans-4-hydroxy-L-proline, analogous to the known inhibition of proline catabolism by L-proline. Also, drugs targeting the first enzyme of hydroxyproline catabolism, by elevating the level of trans-4-hydroxy-L-proline, may inadvertently impair proline catabolism by the inhibition of ALDH4A1.

Original languageEnglish (US)
Pages (from-to)1714-1722
Number of pages9
JournalProtein Science
Issue number8
StatePublished - Aug 2021


  • ALDH4A1
  • L-glutamate-γ-semialdehyde dehydrogenase
  • X-ray crystallography
  • aldehyde dehydrogenase
  • enzyme inhibition
  • hydroxyproline catabolism
  • proline metabolism

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology


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