Evidence for a conserved binding motif of the dinuclear metal site in mammalian and plant purple acid phosphatases: 1H NMR studies of the di-iron derivative of the Fe(III)Zn(II) enzyme from kidney bean

Abstract

The di-iron core of mammalian purple acid phosphatases has been reproduced in the plant enzyme from kidney bean (Mr 111000) upon insertion of an Fe(II) ion in place of the native zinc(II) in the dinuclear Fe(III)Zn(II) core. The shortening of the electronic relaxation time of the metal centre allows detection of hyperfine-shifted 1H NMR resonances, although severe broadening due to Curie relaxation prevents independent signal assignment. Nevertheless, comparison of the spectral features of the structurally characterized plant enzyme with those of the mammalian species, which were previously extensively assigned, is consistent with a close similarity of the metal-binding sites, also suggested by previous sequence-alignment studies. Some differences appear to be mainly localized at the M(II) site. Spectral comparison was also carried out on the Fe(III)Co(II) derivatives.

Structural Studies on a Mitochondrial Glyoxalase II

“…There were at least eight paramagnetically shifted resonances in-between 110 and Ϫ30 ppm. Previous studies have shown that the T 1e of Fe(III) when part of a Fe(III)Zn(II) center is too slow to observe paramagnetically shifted 1 H resonances (48,49). Therefore, the peaks observed in Fig.…”

“…It has been suggested that this peak arises from a backbone NH in close proximity to the metal center (53). Although unambiguous assignments of the remaining peaks are not possible, it is likely that peaks b and c are due to either meta protons (ϪCH) on Fe(II)-bound histidines or to ␤-CH 2 protons on metal-bound histidines (49,50). Ortho protons on metal-bound histidines are usually too broad to detect (50).…”

“…Ortho protons on metal-bound histidines are usually too broad to detect (50). Peaks f and g are probably due to ␤-CH 2 protons on bound Asp/Glu ligands (49,50). Therefore, the NMR spectrum of GLX2-5 confirmed the presence of an Fe(III)Fe(II) center in the enzyme.…”

The Catalytic Mechanisms of Binuclear Metallohydrolases

“…29,75 Also, 1 H NMR studies of the di-iron derivative of the red kidney bean enzyme and native mammalian enzymes provided strong evidence that the coordination environment of the chromophoric Fe(III) centers is very similar in both systems, an observation also supported by comparable charge-transfer transitions in the visible region. 76 The Fe(III) site in PAPs is coordinated to the oxygen of a deprotonated tyrosine, the nitrogen atom of a histidine, and the oxygen atoms of two aspartate residues, one of which bridges the two metal sites (Figure 4). Although no binding constants have been reported, metal ion replacement studies indicate that Fe(III) is tightly bound.…”

The UDP-diacylglucosamine Pyrophosphohydrolase LpxH in Lipid A Biosynthesis Utilizes Mn2+ Cluster for Catalysis

“…It has been postulated that the metal ions serve to bridge an oxide that performs a nucleophilic attack, to stabilize the formation of a phosphorane intermedi-ate, and to coordinate a nucleophilic hydroxyl group to allow for deprotonation (12,13). Besides calcineurin, enzymes of the CLP superfamily also include protein serine/threonine phosphatases (14), purple acid phosphatases (15), nucleotidases, sphingomyelin phosphodiesterases (16), exonucleases, and cyclic nucleotide phosphodiesterases (11). These enzymes have broad functions but are thought to have evolved from the same ancestor (17).…”