Stereochemistry of lysine formation by meso-diaminopimelate decarboxylase from wheat germ: use of proton-carbon-13 NMR shift correlation to detect stereospecific deuterium labeling

Abstract

The stereochemical course of the wheat germ meso-diaminopimelate (DAP) decarboxylase reaction is compared to that of the decarboxylase isolated from Bacillus sphaericus, which has been reported to proceed with an unusual inversion of configuration [Asada, Y., Tanizawa, K., Sawada, S., Suzuki, T., Misono, H., & Soda, K. (1981) Biochemistry 20, 6881-6886]. Reaction of each enzyme with either unlabeled diaminopimelic acid in D2O or [2,6-2H2]diaminopimelic acid in H2O gave stereospecifically deuterium-labeled lysine samples that were derivatized with (-)-camphanoyl chloride and diazomethane. Analysis by two-dimensional 1H-13C heteronuclear NMR shift correlation spectroscopy with 2H decoupling confirmed the stereochemistry of the B. sphaericus enzyme reaction and showed that the eukaryotic wheat germ meso-DAP decarboxylase also operates with inversion of configuration. This suggests similar mechanisms for the prokaryotic and eukaryotic enzymes and contrasts the retention mode observed with other pyridoxal phosphate dependent alpha-decarboxylases.

Effect of analogues of diaminopimelic acid on the meso‐diaminopimelate‐adding enzyme from Escherichia coli

“…However, these in vitro effects were not translated into conspicuous antibacterial effects: none of the isomers of 3 or 4 caused any inhibition of growth when tested against a series of organisms on defined media on plates; only 4c inhibited slightly B. subtilis growth at high doses (->350 p,g per test disc) [11]. On the other hand, it was shown that A2pm auxotrophs could grow, in the presence of lysine, with exogenous lanthionine ( [15], and references therein), the meso isomer of the sulfur amino acid being incorporated into peptidoglycan [15].…”

“…In this regard, the synthesis of stereochemically defined N-hydroxy analogues would be of great interest. Previously, we showed that the mixture of isomers 10 was an alternative substrate for A2pm dehydrogenase from B. sphaericus (22% velocity relative to meso-A2pm), a moderate inhibitor of LysA from B. sphaericus (Ki = 0,91 mM) and a very potent inhibitor of DapF from E. coli (Ki =5.6 ~tM); it inhibited by 75% the growth of B. megaterium at 20 ~g m1-1, and totally that of B. subtilis at 500 p.g m1-1 [5,11].…”

“…10 (mixture of i somers) Moreover, for lanthionine derivatives 2-4 and phosphonic analogue 5, all the possible stereoisomers were available (except for meso-lanthionine sulfoxide 3a, which was presumably an optically inactive mixture of two isomers at the sulfur atom [11]). We first determined whether these compounds were substrates, at 10 mM, for the meso-A2pm-adding enzyme from E. coli (Table 1).…”

“…There has been growing interest in the design and synthesis of A2pm analogues, some of which have been assayed either for antibacterial activity (see [3] for a recent example) or for a specific effect on the epimerase [4][5][6][7][8][9], the decarboxylase [9][10][11], the dehydrogenase [5,8,9] or less frequently the A2pm-adding enzyme [12][13][14][15]. Herein we take *Corresponding author.…”

“…[17] and meso-A2pm [18] were obtained according to published procedures, meso-[laC]A2pm (11.6 TBq mo1-1) was purchased from CEA (Saclay, France). The synthesis of A2pm analogues 2-8 and 10 has already been described [5,6,8,9,11]; analogue 9 was synthesized according to the procedure of Girodeau et al [10]. The MurE activity was partially purified from E. coli JM83(pHE5) as described previously [13].…”