AbstractThe early studies on chicken embryos revealed that exposition to 4-oxo-L-proline resulted in the explicit increase in 4-hydroxy-L-proline content in their tissues. In 1962, 4-oxo-L-proline reductase, an enzyme responsible for the reduction of 4-oxo-L-proline, was partially purified from rabbit kidneys and characterized biochemically, but only recently the molecular identity of the enzyme has been unveiled in our laboratory. The present investigation reports the purification, identification as well as biochemical characterization of 4-oxo-L-proline reductase. The enzyme was purified from rat kidneys about 280-fold. Following mass spectrometry analysis of the purified protein preparation, the mammalian cytosolic type 2 (R)-β-hydroxybutyrate dehydrogenase (BDH2) emerged as the only meaningful candidate for the reductase. Rat and human BDH2 were expressed in E. coli, purified, and shown to catalyze the reversible reduction of 4-oxo-L-proline to cis-4-hydroxy-L-proline, as confirmed by chromatographic and mass spectrometry analysis. Specificity studies carried out on both enzymes showed that 4-oxo-L-proline was the best substrate, particularly the human enzyme acted with 9400-fold higher catalytic efficiencies on 4-oxo-L-proline than on (R)-β-hydroxybutyrate. Finally, HEK293T cells efficiently metabolized 4-oxo-L-proline to cis-4-hydroxy-L-proline and simultaneously accumulated trans-4-hydroxy-L-proline in the culture medium, suggesting that 4-oxo-L-proline is most likely an inhibitor of trans-4-hydroxy-L-proline metabolism in human cells. We conclude that BDH2 is mammalian 4-oxo-L-proline reductase that converts 4-oxo-L-proline to cis-4-hydroxy-L-proline, and not to trans-4-hydroxy-L-proline as currently thought, and hypothesize that the enzyme may be considered as a potential source of cis-4-hydroxy-L-proline in mammalian tissues.
Modifications of proteins by 4-hydroxy-2-nonenal in the ventilatory muscles of rats
