The determination of one-carbon metabolic intermediates in plants and the development of a protocol for the identification of pyridoxal-l-phosphate modified residues in proteins.

摘要

Plants produce specialized molecules that are designed to protect them from specific environmental threats. These specialized molecules fall under the category of secondary metabolites. In addition to the physiological importance of secondary metabolites to plants, many of these molecules possess pharmaceutical and industrial value. This has sparked an interest in controlling the output of specific secondary metabolites in plants. The synthetic pathways for secondary metabolites require methyl groups from one-carbon metabolism in plants. However, relatively little is known about plant one-carbon metabolism that would allow scientists to engineer plants for greater yields of secondary metabolites.; This dissertation discusses some efforts that have been made to study some of these intermediates and enzymes, particularly those that are involved in a poorly understood pathway in plant one-carbon metabolism, the S-methylmethionine (SMM) cycle. Presented here is the development of some strategies used to quantify key intermediates that are involved, or related, to the SMM cycle. These protocols were applied to wild type and mutant Arabidopsis thaliana plants. The mutant plant lacked S-methionine methyltransferase (MMT) activity. The established function of MMT is methylating methionine which effectively starts the SMM cycle. Measurements reported here of the intermediates involved in the SMM cycle in both the wild type and mutant plants imply a function of the SMM cycle in regulating the methylation ratio in plants. The methylation ratio is the relative concentration of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH), (SAM/SAH). SAM is the one-carbon metabolic intermediate responsible for the donation of methyl groups to primary and secondary metabolite synthesis, and SAH is the demethylated byproduct.; A protocol was also developed to study the key enzyme initiating the SMM cycle, MMT. Sequence analysis of MMT suggests a possible association with pyridoxal-l-phosphate (PLP). Before this could be assessed, a protocol had to be established that could be applied to MMT in future studies. Tandem mass spectrometric studies were performed with PLP modified peptides and the MS/MS spectra reveal that these peptides fragment to yield two distinct products corresponding to the neutral losses of phosphoric acid, H3PO 4, and PLP. Although no sequence information was available, a fragmentation pattern was established that could be used to identify MS/MS spectra that correspond to PLP modified peptides (by neutral loss analysis). This strategy was applied to two known PLP binding enzymes that were digested with enzymatic proteases. The peptide products were fractionated with liquid chromatography and analyzed with tandem mass spectrometry. Neutral loss analysis of the resulting MS/MS spectra highlighted those that displayed the characteristic neutral loss patterns of PLP modified peptides. The masses of these peptides matched the theoretical peptides from the digests that contained the previously determined PLP modified residue.