Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics
Structure of bovine lactoperoxidase with a partially linked heme moiety at 1.98 Å resolution
Graphical abstract
Two positions of side chain of Glu258 indicated by yellow and green.
Introduction
Heme peroxidases catalyze the H2O2-dependent oxidation of a number of inorganic [1], [2], [3] and small organic substrates [4], [5], [6], [7], [8]. These enzymes can be broadly divided into two super families consisting of (i) bacterial, fungal and plant (non-animal) heme peroxidases and (ii) mammalian heme peroxidases which are represented by myeloperoxoidase (MPO), lactoperoxidase (LPO), eosinophil peroxidase (EPO) and thyroid peroxidase (TPO). Evolutionarily, the two families emerged independently [9] and differ greatly in their amino acid sequences with sequence identities between the members of two superfamilies ranging from 10% to 20% only. They also differ in their three-dimensional structures although carry out similar functional roles. However, the most striking difference between the members of the two superfamilies is attributed to the way their prosthetic heme groups are associated with the protein. There are also further differences in the fine architectures of their substrate binding sites as well as between the hydrogen bonding patterns involving the important His351 (LPO numbering scheme) on the proximal side. The functionally critical ferroprotophorphyrin IX moiety is held non-covalently in the enzymes of non-animal heme peroxidases while in the mammalian heme peroxidases, as a result of post-translational modification, the heme moiety is bound firmly with the help of two or three covalent bonds. In LPO, EPO and TPO, the heme moiety is covalently linked to the protein via two ester bonds through conserved aspartate and glutamate residues [10] while in MPO, it has an additional sulfonium ion linkage between the 2-vinyl group of the heme moiety and a methionine residue of the protein [11]. These linkages contribute to the rigidification of the prostheic heme group and the formation of a well defined substrate binding site on the distal heme side which may, in turn, be responsible for the peculiar actions of mammalian heme peroxidases. The studies have also shown that the heme moiety of lactoperoxidase can be cleaved using various procedures [12].
The fine differences and similarities in the functional properties of mammalian heme peroxidases have been summarized in a recent publication [13]. The amino acid sequence identities among the four members of the mammalian heme peroxidase superfamily range from 46% to 57%. Several crystal structures of LPO [14], [15], [16], [17], [18], [19], [20], [21], [22], [23], [24] and MPO [25], [26], [27], [28], [29], [30], [31] in both bound and unbound forms have been determined. In spite of ongoing significant efforts, the crystal structures of EPO and TPO have not yet been determined. As revealed by structures of LPO and MPO, the overall-folds of protein chains in these two members of the mammalian heme peroxidase superfamily are identical. The stereochemical features of their substrate binding sites on the distal heme side are also very similar. Because of the highly conserved amino acid sequences of the substrate binding sites and similar functional properties, the substrate binding sites of all the four proteins of the mammalian heme peroxidase superfamily are expected to have similar heme environments.
The biological activities of LPO, MPO and EPO are concerned primarily with their roles in the host defence [32], [33], [34] while TPO is involved in the biosynthesis of thyroid hormones, thyroxine and tri-iodothyronine [35]. The substrates for these four enzymes include small anions such as chloride, bromide, iodide, thiocynate and nitrite [3]. As shown by crystal structure of LPO with four inorganic substrates [21], there is a subtle difference in the preference for the binding of these substrates. The role of two covalent linkages between protein and heme moiety involving conserved glutamate and aspartate residues is considered to be essential for fulfilling the structural requirements of the substrate binding site. It was presumed that these two ester linkages are firmly formed for stabilizing the structure. The roles of glutamate and aspartate residues in linking to heme moiety through two ester linkages via 1-methyl and 5-methyl groups respectively were investigated biochemically and spectroscopically [36], [37], [38], [39]. Here, we report a new structure of LPO in which the covalent bond between Glu258 and 1-methyl group of heme moiety is present only in 50% of protein molecules. In the rest of the 50% molecules, the side chain of Glu258 occupies a position in the middle of the substrate binding site on the distal heme side. At this position, the side chain of Glu258 forms hydrogen bonded and van der Waals contacts with several other residues thus making it a stable arrangement for Glu258. At this position, the side chain of Glu258 occupies a significant space of the distal heme cavity thus blocking the substrate binding site. This may prevent the binding of ligands in the substrate binding site. This is the first structural observation that demonstrates the absence of an ester covalent bond involving Glu258 and 1-methyl group of pyrrole ring C of the heme moiety in LPO. Because of structural and functional similarities, the similar conditions may be present in other members of the mammalian heme peroxidase superfamily.
Section snippets
Purification of LPO
LPO was isolated from the samples of milk collected during the late lactation from an indigenous variety of Sahiwal cows maintained at a private farm in Delhi. The protein was purified using a procedure reported earlier [15] which was modified slightly as described here. The buffer containing 50 mM Tris-HCl, pH 8.0 and 2 mM CaCl2 was added to the skimmed colostrum. The cation exchanger CM-Sephadex C-50 (7 g l− 1) (GE Healthcare, Uppsala, Sweden) was dissolved in 50 mM Tris-HCl, pH 8.0. The unbound
Results and discussion
The present structure of LPO (Fig. 2) has been determined at 1.98 Å resolution and refined to values of 0.188 and 0.235 for Rcryst and Rfree factors respectively. The quality of this structure is reasonably good as 95.8% amino acid residues belong to the most favored regions of the Ramachandran plot [46]. As reported in the previously determined structures of LPO, the heme moiety was expected to be covalently linked to the protein through two ester bonds involving 1-methyl and 5-methyl groups of
Conclusions
In mammalian heme peroxidases, the heme moiety is linked to the protein through two or three covalent bonds. In LPO, the prosthetic heme moiety is linked through two covalent ester bonds involving Glu258 and Asp108 of protein molecule and 1-methyl and 5-methyl groups of the heme moiety respectively. The present structure of LPO shows that the covalent bond involving Glu258 is not formed in 50% molecules (PDB ID, 5B72) while another structure of LPO (PDB ID, 5GLS) shows that Glu258 is not
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Author contributions
P.K.S.-structure determination and preparation of manuscript; H.V.S.-collection of samples, isolation, purification and crystallization; N.I. structure analysis and preparation of manuscript; P.T.-structure determination using another dataset on a different crystal that supported this finding; P.K.-discussion on refinement and structure analysis; S.S.-purification, crystallization and preparation of manuscript; T.P.S.-sample preparation, structure determination, interpretation of results and
Acknowledgments
This work was supported by the FIST grant from the Department of Science and Technology, New Delhi and an investigator grant from the Department of Biotechnology, New Delhi, Government of India (D-298). A grant from the Indian National Science Academy, New Delhi (N-1527) under INSA Senior Scientist programme to TPS is gratefully acknowledged.
References (49)
- et al.
Oxidation of the substituted catechols dihydroxyphenylalanine methyl ester and trihydroxyphenylalanine by lactoperoxidase and its compounds
Arch. Biochem. Biophys.
(1989) - et al.
Evidence for a peroxidatic oxidation of norepinephrine, a catecholamine, by lactoperoxidase
Biochem. Biophys. Res. Commun.
(1989) - et al.
Oxidation of catechols and catecholamines by horseradish-peroxidase and lactoperoxidase: ESR spin stabilization approach combined with optical methods
Spectrochim. Acta
(1993) - et al.
Active site structure and catalytic mechanisms of human peroxidases
Arch. Biochem. Biophys.
(2006) - et al.
Lactoperoxidase: I. The prosthetic group of lactoperoxidase
J. Biol. Chem.
(1963) - et al.
Crystal structure of lactoperoxidase at 2.4 Å resolution
J. Mol. Biol.
(2008) - et al.
Inhibition of lactoperoxidase by its own catalytic product: crystal structure of the hypothiocyanate-inhibited bovine lactoperoxidase at 2.3 Å resolution
Biophys. J.
(2009) - et al.
Structural evidence of substrate specificity in mammalian peroxidases: structure of the thiocyanate complex with lactoperoxidase and its interactions at 2.4 Å resolution
J. Biol. Chem.
(2009) - et al.
Binding modes of aromatic ligands to mammalian heme peroxidases with associated functional implications: crystal structures of lactoperoxidase complexes with acetylsalicylic acid, salicylhydroxamic acid, and benzylhydroxamic acid
J. Biol. Chem.
(2009) - et al.
Mode of binding of the tuberculosis prodrug isoniazid to heme peroxidases: binding studies and crystal structure of bovine lactoperoxidase with isoniazid at 2.7 Å resolution
J. Biol. Chem.
(2010)
X-ray crystal structure of canine myeloperoxidase at 3 Å resolution
J. Mol. Biol.
Structure of the green heme in myeloperoxidase
Arch. Biochem. Biophys.
X-ray crystal structure and characterization of halide-binding sites of human myeloperoxidase at 1.8 Å resolution
J. Biol. Chem.
Essential role of proximal histidine-asparagine interaction in mammalian peroxidases
J. Biol. Chem.
2-Thioxanthines are mechanism-based inactivators of myeloperoxidase that block oxidative stress during inflammation
J. Biol. Chem.
Potent reversible inhibition of myeloperoxidase by aromatic hydroxamates
J. Biol. Chem.
Oxidation of chloride and thiocyanate by isolated leukocytes
J. Biol. Chem.
Thiocyanate is the major substrate for eosinophil peroxidase in physiologic fluids. Implications for cytotoxicity
J. Biol. Chem.
Autocatalytic processing of heme by lactoperoxidase produces the native protein bound prosthetic group
J. Biol. Chem.
Biochemical evidence for heme linkage through esters with Asp-93 and Glu-241 in human eosinophil peroxidase. The ester with Asp-93 is only partially formed in vivo
J. Biol. Chem.
Asp-225 and glu-375 in autocatalytic attachment of the prosthetic heme group of lactoperoxidase
J. Biol. Chem.
Processing of X-ray diffraction data collected in oscillation mode
Methods Enzymol.
Glu375Gln and Asp225Val mutants: about the nature of the covalent linkages between heme group and apo-protein in bovine lactoperoxidase
Bioorg. Med. Chem. Lett.
Role of heme in intracellular trafficking of thyroperoxidase and involvement of H2O2 generated at the apical surface of thyroid cells in autocatalytic covalent heme binding
J. Biol. Chem.
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