Fig 1.
Hydrolysis of L,L-SDAP and analogs by HiDapE.
L,L-SDAP (1a) and analogs N6-methyl SDAP (1b) and N6-acetyl-SDAP (1c) with formation of hydrolysis products succinate (2) and L,L-diaminopimelic acid derivatives (3a-c). Enzyme-mediated hydrolysis was not observed for N-acetyl analog 1c which would afford 3c.
Fig 2.
Minimized substrate analogs docked and modeled in the HiDapE active site.
The diaminopimelate moiety is depicted in yellow and the succinate in turquoise. A) Native substrate L,L-SDAP, B) N6-methyl-L,L-SDAP, and C) N6-acetyl-SDAP. The catalytic domain of Chain A is depicted in green, whereas the dimerization domain of Chain B is shown in orange.
Fig 3.
Asymmetric synthesis of N6-methyl-L,L-SDAP 1b.
Synthetic route for preparation of monomethyl substrate analog as the hydrochloride salt (1b.HCl) via the methyl ester or the trifluoroacetate salt (1b.TFA) via the benzyl ester.
Fig 4.
Circular dichroism thermal denaturation study of HiDapE.
The α-helical structures are represented in red and β-sheets are represented in blue with (A) percent secondary structure observed over the course of heating from 20–80 °C, and (B) percent secondary structure remaining with continued heating at 80 °C.
Fig 5.
Glutamic acid control standard curve for the development of ninhydrin and primary amine in 50 mM HEPES buffer at pH 7.5.
Fig 6.
Enzyme saturation curve of HiDapE using 2 mM of N6-methyl-L,L-SDAP as the substrate (50 mM HEPES buffer at pH 7.5).
Optimal enzyme concentration selected for absorbance of primary amine product at or around 1 AU (absorbance unit).