DELIGIO, J.T.*; ELLINGTON, W.R.; Florida State Univ., Tallahassee; Florida State Univ., Tallahassee: The Key Enzyme for Creatine Biosynthesis is Expressed in the Gonad of the Tunicate Ciona intestinalis
Creatine phosphate (CP) and its precursor creatine (Cr), as mediated by the creatine kinase reaction (CK), play a central role in energy homeostasis for cells that display high and variable rates of energy turnover. The key enzyme of de novo Cr biosynthesis is guanidinoacetate methyltransferase (GAMT), which catalyzes the methylation of guanidinoacetate (glycocyamine) using S-adenosylmethionine yielding Cr and S-adenosylhomocysteine. GAMT activities are present in lampreys and other vertebrates but appear to be lacking in hagfish (non-vertebrate craniates), protochordates and invertebrates in spite of the presence of high levels of CP/Cr in certain species. The primitive-type spermatozoa of tunicates contain high CK activities to mitigate energy transport and diffusion constraints in these cells. We have cloned and sequenced three unique GAMT transcripts, most likely alleles, from the gonad of the tunicate Ciona intestinalis. The deduced amino acid sequences correspond with striking similarity to GAMT sequences from vertebrates. The full length cDNAs for two of these GAMTs were ligated into the pETBlue1 expression vector which was then used to transform DE3 E. coli expression host cells. Expression of both constructs yielded considerable soluble protein. The recombinant GAMTs were purified to homogeneity and had native Mr values, as determined using MALDI mass spectrometry, virtually identical to predicted Mr values based on the deduced amino acid sequences. Using an HPLC-based assay protocol, we have shown that recombinant protein catalyzes the formation of Cr with a specific activity comparable to that of the mammalian enzyme. GAMT is expressed in the gonad of the tunicate C. intestinalis indicating that this species has the capability for de novo biosynthesis of Cr to package into primitive-type spermatozoa. Supported by NSF grant IBN-0130024 to WRE.
