Elucidation of consensus amino acid sequence and potential alternate substrates of Rubisco large subunit methyltransferase (LSMT)

Abstract

Rubisco LSMT (EC 2.1.1.127), a SET domain protein, specifically catalyzes the trimethylation of Lys-14 of the enzyme Rubisco, in an N-terminus which contains three other lysines (Lys8, 18, and 21). Such specificity is analogous to the SET domain protein methyltransferases that modify the N-terminal tail of histones. Given that histone methyltransferases have recently been shown to methylate other substrates, a determination of Rubisco LSMT's substrate specificity may lead to alternative substrates. Fortuitously, the fusion of the N-terminally processed sequence of Rubisco (Pro3 - Tyr25) to the N-terminus of human carbonic anhydrase (HCAII) was a substrate for Rubisco LSMT. The facile purification of soluble carbonic anhydrase made this an ideal system to systematically study the side chain functional groups of amino acids in positions which are part of the protein substrate cleft according to the crystal structure. The consensus sequence that these mutational studies identify will be used to screen databases for putative alternative substrates in the chloroplast. Such potential protein sequences will be cloned as HCAII fusions and, through direct structural determination after in vitro assay, verified as Rubisco LSMT substrates. The source fundings are DOE, NSF and KTRDC. The first and second authors equally contributed to this study