Abstract

L-Serine deaminase #1 (L-SD#1) from E. coli, synthesized in an inactive form that requires activation in vitro and in vivo, was purified in 2 part fusion (2PF) and 3 part fusion (3PF) $\beta$-galactosidase fusion protein forms, which demonstrate possible conformational differences between one another. The 3PF protein contained a collagen sequence linking the L-SD#1 and $\beta$-galactosidase protein molecules, whose digestion using collagenase to yield separate $\beta$-galactosidase and L-SD#1 protein molecules was demonstrated. Purified protein was used to raise antibodies to 2PF protein, which were used for identifying L-SD#1 in bacterial extracts. This work proved that L-SD#1 (48.8 kDa) is indeed coded for by sdaA. It was also revealed that the translational start site of sdaA was 18 bp or 6 amino acids upstream of what was previously thought. An analysis of L-SD#1 activation and activity in vitro revealed the Km for activated enzyme as being 25.5 mM for L-serine and found glycine to be an inhibitor of L-SD#1 activity. Two forms of inactive L-SD#1 may exist, IN#1 and IN#2, which can be activated with low and high amounts of DTT respectively. The activation mechanism of L-SD#1 had been theorized to involve a proteolytic cleavage, for which no evidence was found in this investigation, but evidence suggesting a conformational change was revealed

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