Switching the function of PLP-dependent human serine racemase to serine dehydratase and vice versa by point mutation
Date Issued
2010
Date
2010
Author(s)
Wang, Cyong-Yi
Abstract
Serine racemase catalyzes the production of D-serine, a co-agonist of the
NMDARs in the brain. Mammalian serine racemase is involved in the reversible conversion of L- to D-serine, as well as the dehydration activity toward L- and Dserine. We observed human serine racemase gene shows 23% identity with that of the human serine dehydratase (SD), which catalyzes the dehydration of L-serine to yield ammonia and pyruvate. Sequence alignment shows that the corresponding residue Ala65 in the human serine dehydratase is aligned with the catalytic Ser84 in the
human serine racemase. One such mutant protein is a serine to alanine substitution at residue 84, located at the active site of human serine racemase. The S84A mutation caused the loss of isomerization activity and D-serine dehydratase of serine racemase. Whereas it retained the capability to act as an L-serine dehydratase activity. The single mutant of human serine dehydratase A65S protein increased 5-fold D-serine
dehydratase activity at pH=9. To improve our understanding of the relationship between human serine racemase and human serine dehydratase mechanism, we have determined the X-ray crystal structure of the human serine dehydratase A65S mutant protein at 1.54Å resolution. Our results show that the S84 residue in human serine
racemase, proximity to the substrate in an ideal orientation, plays an important role in shuttling the proton required for isomerization. The biological activity analysis of target mutagenesis and useful structural information have paved the way for mechanistic studies and have provided a framework for interpretation of those results.
Subjects
serine racemase
serine dehydratase
pyridoxal 5’-phosphate
NMDAR
X- ray diffraction
Type
thesis
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