I. An Improved Platform for Human Serine Racemase Expression and Characterization. II. Identification of a Possible Mannose 6-Phosphate Receptor in Dictyostelium discoideum

Human serine racemase (hSR) is the vitamin B6 dependent enzyme responsible for the biosynthesis of the NMDA (N-methyl D-aspartate) receptor co-agonist, D-serine. There is evidence that abnormal D-serine levels may be associated with certain degenerative diseases including amyotrophic lateral sclerosis (ALS) and schizophrenia. Additionally, elevated D-serine levels may lead to neuronal infarction pursuant to ischemic stroke. The previously described constructs for the heterologous expression of hSR have been plagued by a tendency toward aggregation. This chapter describes a new N-terminal fusion protein, MBP-hSR, with maltose binding tag. This tag greatly improves solubility without changing the observable kinetic characteristics of serine racemase. This construct also serves as an excellent platform for the study of structure/activity relationships in the racemase, with a focus in this work on serine-84, the accepted re-face base. Four mutants have been expressed and characterized, S84A, S84T, S84N, and S84D. Computational methods have been employed to provide a rational model for the inverted substrate specificity of the S84D mutant. The results of these studies suggest that R135 functions to position the substrates with negatively charged side chains such as L-THA (L-threo-beta-hydroxyaspartate) and L-SOS (L-serine-O-sulfate). The same interaction in both L-THA and L-EHA (L-erythro-beta-hydroxyaspartate) provides an explanation as to why L-THA is a substrate, and L-EHA is an inhibitor. Furthermore, sequence data of fold type II serine eliminases suggest that R135 may have originated from an L-THA eliminase predecessor enzyme. The cation independent mannose 6-phosphate receptor(CI-MPR)/insulin-like growth factor II(IGF-II) receptor has a well-established role of binding and transporting phosphomannosolated glycoproteins to the lysosome. It is a type I glycoprotein found only in higher eukaryotes, and recent evidence has shown it to be involved in growth regulation and motility. Preliminary gene mining efforts suggest the existence of an evolutionarily distant homologue of this receptor in the slime mold, Dictyostelium discoideum ( D.d.). Furthermore, D. d. is known to have unusual glycosylations to include combinations of mannose-6-sulfates and phosphomannodiesters. This discovery potentially unveils the sequence of the most distant evolutionary M6P receptor reported to date; however, the function of this receptor in D.d. has yet to be fully understood