We isolated a gene for serine acetyltransferase (SAT), a key enzyme in sulfate assimilation, from the primitive red alga Cyanidioschyzon merolae, an inhabitant of sulfurous hot springs, and designated this gene cmSAT. The N-terminal region of the cmSAT protein has characteristics of a chloroplast targeting peptide. cmSAT protein fused with a 6x histidine tag complemented a SAT deficient Escherichia coli mutant. The protein was purified with its SAT activity, which was inhibited by cysteine, using the high affinity of the histidine tag in an Ni-NTA column. The Km values for acetyl-CoA and l-serine were 0.3 and 0.1 mM, respectively. Southern blotting indicated the existence of other SAT isoforms in C. merolae. A 2.4 kb transcript was always detected when growth was synchronized under a 12-h light/dark cycle. Under these conditions, a 31-kDa protein was always detected on immunoblots, indicating processing of the cmSAT protein and constitutive expression of cmSAT. A 45-kDa protein, thought to be the unprocessed cmSAT protein, was detected in the dark period, from M phase to early G1 phase. No significant change in the level of protein expression was detected under continuous darkness or in a sulfate-deficient medium. Using immunoelectron microscopy, the cmSAT protein was primarily detected in the stroma and a few were detected in the cytoplasm, which indicate that cmSAT protein is transported to and functions in a chloroplast.

NCOAT is a bifunctional nucleo-cytoplasmic protein with both O-GlcNAcase and histone acetyltransferase domains. The O-GlcNAcase domain catalyzes the removal of O-linked GlcNAc modifications from proteins and we have found that it resides in the N-terminal third of NCOAT. The recognition of the substrate GlcNAc suggests that the O-GlcNAcase is related in structure and catalytic mechanism to chitinases, hexosaminidases and hyaluronidases. These families of glycosidases all possess a catalytic doublet of carboxylate-containing residues, with one providing an acid-base function, and the second acting to orient and use the N-acetyl group of GlcNAc during catalysis. Indeed, we show that the O-GlcNAcase also possesses the catalytic doublet motif shared among these enzymes and that these two essential residues are aspartic acids at positions 175 and 177, respectively, in mouse NCOAT. In addition, a conserved cysteine at 166 and a conserved aspartic acid at 174 were also found to be necessary for fully efficient enzymatic activity. Given this information, we propose that the O-GlcNAcase active site resembles those of the above glycosidases which carry out the hydrolysis of GlcNAc linkages in a substrate-assisted acid-base manner.