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 of serine and tetrahydrofolate to glycine and methylenetetrahydrofolate. This reaction generates single-carbon units for purine, thymidine, and methionine biosynthesis. Cytoplasmic SHMT (cSHMT) has been postulated to channel one-carbon substituted folates to various folate-dependent enzymes, and alternative splicing of the cSHMT transcript may be a mechanism that enables specific protein-protein interactions. The cytoplasmic isozyme is expressed from species-specific and tissue-specific alternatively spliced transcripts that encode proteins with modified carboxy-terminal domains, while the mitochondrial isozyme is expressed from a single transcript. While the full-length mouse and human cSHMT proteins are 91% identical, their alternatively spliced transcripts differ. The murine cSHMT gene is expressed as two transcripts. One transcript encodes a full-length 55 kDa active enzyme (cSHMT), while the other transcript encodes a 35 kDa protein (McSHMTtr). The McSHMTtr protein present in mouse liver and kidney does not bind 5-formyltetrahydrofolate, nor does it oligomerize with the full-length cSHMT enzyme. While recombinant cSHMT-glutathione S-transferase fusion proteins form tetramers and are catalytically active, McSHMTtr-glutathione S-transferase fusion proteins are catalytically inactive, do not form heterotetramers, and do not bind pyridoxal phosphate. Analysis of the murine cSHMT crystal structure indicates that the active site lysine that normally binds pyridoxal phosphate in the cSHMT protein is exposed to solvent in the McSHMTtr protein, preventing stable formation of a Schiff base with pyridoxal phosphate. Modeling studies suggest that the human cSHMT proteins expressed from alternatively spliced transcripts are inactive as well. Therefore, channeling mechanisms enabling specific protein-protein interactions of active enzymes are not based on cSHMT alternative splicing.
Liu
X
X
Division of Nutritional Sciences and Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York 14853, USA.
Szebenyi
D M
DM
Anguera
M C
MC
Thiel
D J
DJ
Stover
P J
PJ
D017398:Alternative Splicing; D000818:Animals; D002384:Catalysis; D018360:Crystallography, X-Ray; D003593:Cytoplasm / Q000201:enzymology; D004789:Enzyme Activation / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D012696:Glycine Hydroxymethyltransferase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007527:Isoenzymes / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008958:Models, Molecular; D009928:Organ Specificity / Q000235:genetics; D012333:RNA, Messenger / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D017384:Sequence Deletion; D013329:Structure-Activity Relationship
D007527:Isoenzymes; D012333:RNA, Messenger; D011994:Recombinant Proteins; D012696:Glycine Hydroxymethyltransferase