In the pathway from aspartate semialdehyde. the first two enzymes, dihydrodipicolinate synthase and dihydrodipicolinate reductase as well as the last, meso diaminopimelate decarboxylase, have been reported in plant extracts [l].It has, however. remained unclear how plants convert the central intermediate, tetrahydrodipicolinate (THDP) through to mesodiaminopimelate (DAP), the immediate precursor of lysine. The supposition that the route is similar to that in most bacteria (i.e. v i a hydration of THDP. Nacetyl or N-succinyl transfer from CoA. transamination, N-deacylation and, epimerisation) was strengthened by the report [2] of DAP epimerase in maize leaf. However the reported [3] isolation of a functional DAP dehydrogenase gene from a soybean nuclear gene library suggested that, similar to some bacteria (41. the transformation in plants could also occur V f 8 reductive amination. We used a sensitive HPLC method [5] to reinvestigate [cf Fig 11.Pea extract was a broad 0-25% polyethylene glycol precipitate of pea seedlings [6]. Stromal extracts were made by bursting spinach or pea chloroplasts [7] in hypotonic buffer (pH 7.0) and centrifuging. Extracts were exchanged (625) into buffers and, if not used fresh, kept as frozen beads over liquid Nz. DD/LL and meso DAP (15% DDILL). their mono N-succinyl and acetyl derivatives and THDP were made [8.9].High levels of DAP epimerase and decarboxylase were found in all extracts (e.g. from 18 mM DD/LL DAP > 8 nmol lysine formed min-' m g ' ' in pea stroma). However, none of our extracts deacylated either N-acetyl or N-succinyl DD/LL DAP under a wide range of conditions (e.g. in fresh stroma at pH's 6 , 7 or 8 both with and without ZnZ* or Co2*). These experiments were partly obscured by lysine forming from the trace of unacylated material in the N-acyl DAP; within error, unacylated LL accounted for all the lysine detected. Thus. deacylase activity was less than 0.02 -0.20 nmol min-' mg-' (detection limits varying with [N-acyl DAP] between 0.4 and 10 mM). We considered the possibility LVDD-DAP 1 I Fig. 1. Conversion of LL to meso diaminoDimelate and lysine. 0 . 6 mg of pea extract in 0.1 M Kt Pi (pH 6 . 8 ) contained 10 mM EDTA, 10 mM p-mercaptoethanol, 1 mM pyridoxal phosphate and 0.4 mM LL/DD DAP. Aliquots were automatically pre-derivatized with 0phthaldialdehyde for HPLC [ 5 ] . Y-axis. fluoresence. Top chromatogram. zero time; lower. after 2 h at 3OOC.that, just as plants make ornithine [lo], there might be no deacylase but rather a direct transfer of the Nacyl group from DAP back to THDP. However, there was also no reaction in the presence of 4 mM THDP. Similarly, we found no evidence for the enzyme activities (THDP N-acyl transferase and N-acyl Z-oxopimelate/ glutamate transaminase) which bacteria use to form Nacyl DAP. The HPLC experiment provided sensitive (> 10 fold better than lysine) detection of all species of N-acyl DAP. However, none (< 0.3 nmol) was formed in any experiment (e.g. after 2 h in 0.4 mg of spinach stroma in HEPES buffer, pH 7.0, containing 0.2 m...
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.