Recombinant human serine racemase: enzymologic characterization and comparison with its mouse ortholog.
D-serine plays a key role in glutamatergic neurotransmission in mammalian brain as a co-agonist of N-methyl-D-aspartate receptors. The enzyme responsible for D-serine biosynthesis, serine racemase (SR), is therefore a promising target for treatment of neuropathologies related to glutamate receptor excitotoxicity, such as stroke or Alzheimer's disease. Much of the experimental work to date has been performed on mouse serine racemase, which shares a high level of sequence identity with its human ortholog. In this work, we report the synthesis of a human SR gene variant optimized for heterologous expression in Escherichia coli and describe the expression and purification of active recombinant human SR. This strategy may be of general interest to researchers wishing to express mammalian proteins in a bacterial system. Furthermore, we conduct a thorough analysis of the kinetics and inhibitor-sensitivity of the recombinant enzyme, and we provide the first direct comparison of human and mouse SR based on our kinetic data. The orthologs behave similarly overall and exhibit identical inhibition profiles, validating the use of mouse models in SR research.
Hoffman
Hillary E
HE
Gilead Sciences and IOCB Research Center, Institute of Organic Chemistry and Biochemistry of the Academy of Sciences of the Czech Republic, v.v.i., Flemingovo n. 2, 166 10 Prague 6, Czech Republic.
Jirásková
Jana
J
Ingr
Marek
M
Zvelebil
Marketa
M
Konvalinka
Jan
J
D000595:Amino Acid Sequence; D000645:Ammonium Sulfate; D000818:Animals; D002846:Chromatography, Affinity; D002942:Circular Dichroism; D003001:Cloning, Molecular; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D019998:Racemases and Epimerases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D000099300:Serine Racemase
D000645:Ammonium Sulfate; D019998:Racemases and Epimerases; D011994:Recombinant Proteins; D000099300:Serine Racemase