Lack of catalytic activity of a murine mRNA cytoplasmic serine hydroxymethyltransferase splice variant: Evidence against alternative splicing as a regulatory mechanism

Mammalian serine hydroxymethyltransferase (SHMT) is a tetrameric, pyridoxal phosphate-dependent enzyme that catalyzes the reversible interconversion o f serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. Thi s reaction generates single-carbon units for purine, thymidine, and methion ine biosynthesis. Cytoplasmic SHMT (cSHMT) has been postulated to channel o ne-carbon substituted folates to various folate-dependent enzymes, and alte rnative splicing of the cSHMT transcript may be a mechanism that enables sp ecific protein-protein interactions. The cytoplasmic isozyme is expressed f rom species-specific and tissue-specific alternatively spliced transcripts that encode proteins with modified carboxy-terminal domains, while the mito chondrial isozyme is expressed from a single transcript. While the full-len gth mouse and human cSHMT proteins are 91% identical, their alternatively s pliced transcripts differ. The murine cSHMT gene is expressed as two transc ripts. One transcript encodes a full-length 55 kDa active enzyme (cSHMT), w hile the other transcript encodes a 35 kDa protein (McSHMTtr). The McSHMTtr protein present in mouse liver and kidney does not bind 5-formyltetrahy-dr ofolate, nor does it oligomerize with the full-length cSHMT enzyme. While r ecombinant cSHMT-glutathione S-transferase fusion proteins form tetramers a nd are catalytically active, McSHMTtr-glutathione S-transferase fusion prot eins are catalytically inactive, do not form heterotetramers, and do not bi nd pyridoxal phosphate. Analysis of the murine cSHMT crystal structure indi cates that the active site lysine that normally binds pyridoxal phosphate i n the cSHMT protein is exposed to solvent in the McSHMTtr protein, preventi ng stable formation of a Schiff base with pyridoxal phosphate. Modeling stu dies suggest that the human cSHMT proteins expressed from alternatively spl iced transcripts are inactive as well. Therefore, channeling mechanisms ena bling specific protein-protein interactions of active enzymes are not based on cSHMT alternative splicing.