Complete covalent structure of 60-kDa esterase isolated from 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced rabbit liver microsomes

The 60-kDa esterase was isolated from liver microsomes of 2,3,7,8-tetrachlorodibenzo-p-dioxin-induced rabbits and its complete amino acid sequence determined. Automated sequence analysis of intact protein, as well as characterization of the peptides obtained from enzymatic and chemical cleavages, led to the elucidation of the primary structure. The protein is a single polypeptide consisting of 539 residues and molecular weight 59,478. The active site serine is 195, and another diisopropylphospho binding site is at histidyl 441. Carbohydrate chains are attached at aspariginyl residues 61 and 363. Although 2,3,7,8-tetrachlorodibenzo-p-dioxin treatment induces this esterase severalfold, the amino acid sequence of the induced enzyme is identical to that of the enzyme isolated from liver microsomes of untreated rabbits. The sequence of the microsomal esterase is 30% identical with the sequences of human serum cholinesterase and the acetylcholinesterase from Torpedo californica. There is also a close homology between the 60-kDa esterase and the COOH-terminal domain of bovine thyroglobulin.     

The covalent structure of dog myoglobin.

The primary structure of the myoglobin of the domestic dog (German shepherd) was studied. Tryptic and thermolytic peptides were compared with the sequence of other known myoglobins; the stepwise automatic Edman's degradation of the whole globin and also the chymotryptic digestion of the median fragment obtained by CNBr cleavage completed this sequence. Comparison of the established dog myoglobin structure with those from other carnivora shows 16 differences versus badger, 20 versus harbour seal and 15 versus California sea lion.     

Crystal structure of the covalent intermediate of human cytosolic beta-glucosidase

Human cytosolic β-glucosidase, also known as klotho-related protein (KLrP, GBA3), is an enzyme that hydrolyzes various β-d-glucosides, including glucosylceramide. We recently reported the crystal structure of KLrP in complex with glucose [Y. Hayashi, N. Okino, Y. Kakuta, T. Shikanai, M. Tani, H. Narimatsu, M. Ito, Klotho-related protein is a novel cytosolic neutral beta-glycosylceramidase, J. Biol. Chem. 282 (2007) 30889-30900]. Here, we report the crystal structure of a covalent intermediate of the KLrP mutant E165Q, in which glucose was covalently bound to a nucleophile, Glu³⁷³. The structure confirms the double displacement mechanism of the retaining β-glucosidase. In addition, the structure suggests that a water molecule could be involved in the stabilization of transition states through a sugar, 2-hydroxyl.     

Complete covalent structure of a proline-rich phosphoprotein, PRP-2, an inhibitor of calcium phosphate crystal growth from human parotid saliva

Human salivary secretions contain many proteins in which proline forms an unusually large fraction of the amino-acid residues present, typically from 20% to over 40%. These proteins are also unusually rich in glycine and glutamine, generally account for over half the total protein in saliva, and include acidic, basic and glycosylated molecules. The functions of most of these are not clearly defined. One group, however, the acidic proline-rich phosphoproteins (PRP), have been shown to be potent inhibitors of secondary precipitation (crystal growth) of calcium phosphate salts. Acting together with a salivary protein inhibitor of primary precipitation of calcium phosphates, statherin, the PRP stabilize saliva which is supersaturated with respect to the calcium phosphate salts which form dental enamel. These inhibitory activities act to provide a protective, reparative, but stable environment for dental enamel, which is important for maintaining the health of the teeth. The PRP are a complex group of phosphoproteins which include four major and at least eight minor members. The primary structures of three of the major proteins have been determined. These are PRP-1, also designated Protein-C, PRP-3, also designated Protein-A (17), and PRP-4. The designations PRP-1,-2,-3 and -4 will be used here. The purpose of this paper is to report the complete primary structure of PRP-2 as a further step towards establishing the structural basis of the biological activity of the PRP, and clarifying the genetic and biosynthetic relationships of these closely related proteins.     

Solution structure of the covalent sterigmatocystin-DNA adduct.

Sterigmatocystin and aflatoxin are potent mutagens that contaminate foodstuffs stored under conditions that permit fungal growth. These food mycotoxins can be metabolically activated to their epoxides, which subsequently form covalent adducts with DNA and can eventually induce tumor development. We have generated the sterigmatocystin-d(A1-A2-T3-G4-C5-A6-T7-T8) covalent adduct (two sterigmatocystins per duplex) by reacting sterigmatocystin-1,2-epoxide with the self-complementary d(A-A-T-G-C-A-T-T) duplex and determined its solution structure by the combined application of two-dimensional NMR experiments and molecular dynamics calculations. The self-complementary duplex retains its 2-fold symmetry following covalent adduct formation of sterigmatocystin at the N7 position of G4 residues on each strand of the duplex. The H8 proton of [ST]G4 exchanges rapidly with water and resonates at 9.58 ppm due to the presence of the positive charge on the guanine ring following adduct formation. We have assigned the exchangeable and nonexchangeable proton resonances of sterigmatocystin and the duplex in the covalent adduct and identified the intermolecular proton-proton NOEs that define the orientation and mode of binding of the mutagen to duplex DNA. The analysis was aided by intermolecular NOEs between the sterigmatocystin protons with both the major groove and minor groove protons of the DNA. The molecular dynamics calculations were aided by 180 intramolecular nucleic acid constraints, 16 intramolecular sterigmatocystin constraints, and 56 intermolecular distance constraints between sterigmatocystin and the nucleic acid protons in the adduct. The sterigmatocystin chromophore intercalates between the [ST]G4.C5 and T3.A6 base pairs and stacks predominantly over the modified guanine ring in the adduct duplex. The overall conformation of the DNA remains right-handed on adduct formation with unwinding of the helix, as well as widening of the minor groove. Parallel NMR studies on the sterigmatocystin-d(A1-A2-A3-G4-C5-T6-T7-T8) covalent adduct (two sterigmatocystins per duplex) provide supportive evidence that the mutagen covalently adducts the N7 position of G4 and its chromophore intercalates to the 5' side of the guanine and stacks over it. The present NMR-molecular dynamics studies that define a detailed structure for the sterigmatocystin-DNA adduct support key structural conclusions proposed previously on the basis of a qualitative analysis of NMR parameters for the adduct formed by the related food mutagen aflatoxin B1 and DNA [Gopalakrishnan, S., Harris, T. M., & Stone, M. P. (1990) Biochemistry 29, 10438-10448].     

Crystal structure of a covalent intermediate of endogenous human arylsulfatase A

The structures of human arylsulfatase A crystals soaked in solutions containing 4-methylumbelliferyl phosphate and O-phospho-DL-tyrosine have been determined at 2.7- and 3.2-A resolution, respectively. The formylglycine in position 69, a residue crucial for catalytic activity, was unambiguously identified in both structures as forming a covalent bond to the phosphate moiety. A hydroxyl group is present at the Cbeta of residue 69 and the formation of one out of two possible stereomeric forms is strongly favoured. The structures confirm the importance of the gem-diol intermediate in the arylsulfatase's catalytic mechanism. The presence of an apparently stable covalent bond is consistent with the weak phosphatase activity observed for human arylsulfatase A. The structures of the complexes suggest that phosphate ions and phosphate esters inhibit arylsulfatase in non-covalent and covalent modes, respectively. The metal ion present in the active site of arylsulfatase A isolated from human placenta is Ca(2+) and not Mg(2+) as was found in the structure of the recombinant enzyme.     

Partial covalent structure of two basic chromosomal proteins from human spermatozoa

The partial covalent structure of the basic chromosomal proteins I and II isolated from human spermatozoa was determined by automatic Edman degradation and digestion with carboxypeptidases A and B. The partial covalent structures obtained are compared with complete and partial known sequences of the basic chromosomal proteins from other animals.A. H. J. K. was supported pursuant to Contract NIH-NICHD-73-2700 with the National Institute of Child Health and Human Development, U.S. Department of Health, Education, and Welfare.     

Complete covalent structure of nisin Q, new natural nisin variant, containing post-translationally modified amino acids

The third member of the nisin variant, nisin Q, produced by Lactococcus lactis 61-14, is a ribosomally-synthesized antimicrobial peptide, the so-called lantibiotic containing post-translationally modified amino acids such as lanthionine and dehydroalanine. Here, we determined the complete covalent structure of nisin Q, consisting of 34 amino acids, by two-dimensional (1)H nuclear magnetic resonance (NMR) spectroscopy. Sequential assignment of nisin Q containing the unusual amino acids was performed by total correlation spectroscopy (TOCSY) and nuclear Overhauser enhancement spectroscopy (NOESY). The observed long range nuclear Overhauser effect (NOE) in nisin Q indicated assignment of all five sets of lanthionines that intramolecularly bridge residues 3-7, 8-11, 13-19, 23-26, and 25-28. Consequently, the covalent structure of nisin Q was determined to hold the same thioether linkage formation as the other two nisins, but to harbor the four amino acid substitutions, in contrast with nisin A.     

Complete structure of the human gene encoding neuron-specific enolase

At least three genes encode the different isoforms of the glycolytic enzyme enolase. We have isolated the gene for the human gamma- or neuron-specific enolase and determined the nucleotide sequence from upstream to the 5' end to beyond the polyadenylation site. The gene contains 12 exons distributed over 9213 nucleotides. Introns occur at positions identical to those reported for the homologous rat gene, as well as for the human alpha- or nonneuronal enolase gene, supporting the existence of a single ancestor for the members of this gene family. Primer extension analysis indicates that the gene has multiple start sites. The putative promoter region lacks canonical TATA and CAAT boxes, is very G + C-rich, and contains several potential regulatory sequences. Furthermore, an inverted Alu sequence is present approximately 572 nucleotides upstream of the major start site. A comparison of the 5'-flanking region of the human gamma-enolase gene with the same region of the rat gene revealed a high degree of sequence conservation.     

Complete primary structure of human C4a anaphylatoxin

C4a anaphylatoxin is derived from the fourth component (C4) of the blood complement system. The C4 alpha-chain is selectively cleaved between positions 77 and 78 by the protease C1s, a subcomponent of C1, generating the fragments C4a and C4b. Human C4a was isolated directly from fresh serum after C1 of the classical pathway of complement was activated by heat-aggregated gamma-globulin. The C4a anaphylatoxin is a cationic polypeptide of Mr = 9000 composed of 77 residues and devoid of histidine, tryptophan, and carbohydrate. The primary structure of human C4a was deduced from sequence analysis of two cyanogen bromide fragments and of peptides obtained after chymotryptic digestion of the COOH-terminal cyanogen bromide fragment. The proposed sequence is: (formula, see text) Manual alignment of the linear structures of human C3a, C4a, and C5a, based primarily on the location of two Cys-Cys sequences in each indicate a 30% homology between C3a and C4a and a 36% homology between C5a and C4a. It was concluded from the sequence comparison that C3a, C4a, and C5a are a family of bioactive factors derived from precursor molecules that share a common genetic origin. Although the human anaphylatoxins share a partial structural identity and express similar biological activities, these factors ae immunologically distinct molecules having no antigenic determinants in common as judged by radioimmunoassay.     

Partial covalent structure of the human alpha 2 type V collagen chain

Human cDNA libraries were screened with a cDNA fragment presumably encoding the 3' terminus of a procollagen carboxyl propeptide not identifiable as types I, II, III, or IV by protein sequence or Northern blot hybridization. One clone contained a 1350-base pair insert coding in part for 55 uninterrupted Gly-X-Y triplets. Comparison with the amino acid composition of the COOH-terminal cyanogen bromide (CB) peptides of the alpha 1 and alpha 2 type V collagen chains showed similarity only to the alpha 2(V)CB fragment. To identify the NH2 terminus of the peptide designated by methionine, an additional isolate was sequenced and found to contain a Gly-Met-Pro triplet. Thirty-one amino acids from the NH2 terminus of the alpha 2(V)CB9 fragment were then determined by Edman degradation and found to be identical to those derived from the cDNA clone. The DNA sequence encoding part of the triple helical region establishes for the first time the partial structure of a type V collagen chain. Although comparison of residues 796-1020 of the alpha 2(V) collagenous region with alpha 1 (III), alpha 1(I), and alpha 2(I) shows strong conservation of charged positions, the latter three chains appear considerably more similar to each other than to alpha 2(V). A striking feature of the alpha 2(V) sequence between 918-944 is the absence of proline residues. In the analogous region of alpha 1(I) where this amino acid is also lacking, a flexible site in the rigid triple helical structure of type I collagen has been observed (Hofmann, H., Voss, T., Kuhn, K. and Engel, J. (1984) J. Mol. Biol. 172, 325-343).