Type of Document Thesis Author Conejo, Maria Soledad URN etd-07112008-115116 Title Functional Properties and Evolution of Arginine Kinases From Organisms at the Protozoan-Metazoan Interface Degree Master of Science Department Biological Science, Department of Advisory Committee
Advisor Name Title Brian G. Miller Committee Member Gavin Naylor Committee Member William R. Ellington Committee Member Keywords
- Arginine Kinase
- Hexactinellid Sponges
Date of Defense 2008-06-11 Availability unrestricted AbstractArginine kinase (AK; arginine phosphate + MgADP + H+ ²³ arginine + MgATP) and creatine kinase (CK; creatine phosphate + MgADP + H+ ²³ creatine + MgATP) play a key role in energy homeostasis in cells that display high and variable rates of ATP turnover. AK and CK are members of a highly conserved family of phosphotransferases called phosphagen kinases (PK). CK is widely distributed in lower and higher invertebrates and vertebrates while AK is lacking in vertebrates but is present in most invertebrate groups as well as in certain protozoans. Conventional wisdom suggests that CK evolved from an AK-like ancestor. Sponges, the oldest extant group of metazoans, have two CK genes, one targeted to the cytoplasm while the other is targeted to the mitochondrial intermembrane space. This thesis probes the early origin of CK by looking at choanoflagellate protozoans which are thought to be a direct sister group of metazoans.
Assays of lysates of four choanoflagellate species displayed AK activity but appeared to be lacking CK. PK cDNAs from three choanoflagellates (Monosiga brevicollis, Monosiga ovata and Codonosiga gracilis) were amplified by reverse transcription PCR. All three had deduced amino acid sequences with the requisite residues for arginine/arginine phosphate specificity indicating that they are likely AKs. The deduced amino acid sequences were used as queries in tBLASTn searches of the newly released genome sequences of M. brevicollis. The M. brevicollis, M. ovata, and C. gracilis sequences each matched unique M. brevicollis gene scaffolds implying that there are at least three unique AK genes in choanoflagellates. Phylogenetic analyses indicated that the M. ovata and C. gracilis AKs form a unique clade outside of a super clade consisting of M. brevicollis AK and protozoan and invertebrate AKs. Pair wise amino acid sequence comparisons showed that the M. ovata and C. gracilis AKs, although true AKs, are more similar to sponge CKs than to AKs. The multiple AK genes in choanoflagellates may represent the earliest stage of divergence of AKs into the CK lineage.
Based on comparisons with the residues required for formation of dimers in CKs, it was determined that M. brevicollis AK is likely a functional monomer. To validate this hypothesis and to further characterize this enzyme, the cDNA for M. brevicollis AK was ligated into an expression vector. This AK was subsequently expressed at high yield in E. coli and purified to homogeneity by low pressure column chromatography. Size exclusion chromatography of the recombinant M. brevicollis AK showed that the native Mr was close to the predicted subunit Mr indicating that this protein is a functional monomer. Assays of this AK in both the forward (ATP³ arginine phosphate) and reverse (arginine phosphate ³ ATP) directions yielded somewhat higher activities than found in typical AKs. M. brevicollis AK showed high specificity for L-arginine and limited activity with L-arginine analogs. Minimal activity or no activity was observed with other PK substrates. The AK from M. brevicollis appears to have more sequence similarity to typical invertebrate AKs. Studies of the more CK-like choanoflagellate AKs with respect to quaternary structure and substrate specificity should be most revealing.
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