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1.
Golinelli-Pimpaneau B Lüthi C Christen P 《The Journal of biological chemistry》2006,281(33):23969-23977
Antibody 15A9, raised with 5'-phosphopyridoxyl (PPL)-N(epsilon)-acetyl-L-lysine as hapten, catalyzes the reversible transamination of hydrophobic D-amino acids with pyridoxal 5'-phosphate (PLP). The crystal structures of the complexes of Fab 15A9 with PPL-L-alanine, PPL-D-alanine, and the hapten were determined at 2.3, 2.3, and 2.5A resolution, respectively, and served for modeling the complexes with the corresponding planar imine adducts. The conformation of the PLP-amino acid adduct and its interactions with 15A9 are similar to those occurring in PLP-dependent enzymes, except that the amino acid substrate is only weakly bound, and, due to the immunization and selection strategy, the lysine residue that covalently binds PLP in these enzymes is missing. However, the N-acetyl-L-lysine moiety of the hapten appears to have selected for aromatic residues in hypervariable loop H3 (Trp-H100e and Tyr-H100b), which, together with Lys-H96, create an anion-binding environment in the active site. The structural situation and mutagenesis experiments indicate that two catalytic residues facilitate the transamination reaction of the PLP-D-alanine aldimine. The space vacated by the absent L-lysine side chain of the hapten can be filled, in both PLP-alanine aldimine complexes, by mobile Tyr-H100b. This group can stabilize a hydroxide ion, which, however, abstracts the C alpha proton only from D-alanine. Together with the absence of any residue capable of deprotonating C alpha of L-alanine, Tyr-H100b thus underlies the enantiomeric selectivity of 15A9. The reprotonation of C4' of PLP, the rate-limiting step of 15A9-catalyzed transamination, is most likely performed by a water molecule that, assisted by Lys-H96, produces a hydroxide ion stabilized by the anion-binding environment. 相似文献
2.
Salzmann D Christen P Mehta PK Sandmeier E 《Biochemical and biophysical research communications》2000,270(2):576-580
The pyridoxal-5'-phosphate-dependent enzymes (B(6) enzymes), that operate in the metabolism of amino acids, are of multiple evolutionary origin. To estimate their rates of evolution, a total of 180 sequences of 21 B(6) enzymes from distantly related eukaryotic species were compared. The enzymes belong to all four evolutionarily independent families of B(6) enzymes with different folds, i.e., the large alpha family, the beta family, the d-alanine aminotransferase family, and the alanine racemase family. Their unit evolutionary periods, i.e., the time for a 1% sequence difference to accumulate between branches, ranged from 4.6 to 45.1 million years. Both, fastest changing serine pyruvate aminotransferase and most slowly changing glutamate decarboxylase are members of the alpha family. The evolutionary rates of the few enzymes belonging to the other three families were interspersed among those of the alpha family members. Enzymes that catalyze the same reaction, e.g., transamination or amino acid decarboxylation, with different substrates show widely varying rates. The absence of correlations of the rate of evolution with either protein fold or type of catalyzed reaction suggests that individual functional constraints have determined the differential rates of evolution of B(6) enzymes. 相似文献
3.
Sun L Bartlam M Liu Y Pang H Rao Z 《Protein science : a publication of the Protein Society》2005,14(3):791-798
L-serine dehydratase (SDH), a member of the beta-family of pyridoxal phosphate-dependent (PLP) enzymes, catalyzes the deamination of L-serine and L-threonine to yield pyruvate or 2-oxobutyrate. The crystal structure of L-serine dehydratase from human liver (hSDH) has been solved at 2.5 A-resolution by molecular replacement. The structure is a homodimer and reveals a fold typical for beta-family PLP-dependent enzymes. Each monomer serves as an active unit and is subdivided into two distinct domains: a small domain and a PLP-binding domain that covalently anchors the cofactor. Both domains show the typical open alpha/beta architecture of PLP enzymes. Comparison with the rSDH-(PLP-OMS) holo-enzyme reveals a large structural difference in active sites caused by the artifical O-methylserine. Furthermore, the activity of hSDH-PLP was assayed and it proved to show catalytic activity. That suggests that the structure of hSDH-PLP is the first structure of the active natural holo-SDH. 相似文献
4.
Malthouse JP 《Biochimica et biophysica acta》2003,1647(1-2):138-142
A NMR method for quantifying the catalytic efficiency and stereospecificity of the exchange of the alpha-protons of glycine is described. It is used to determine how the binding of the alpha-carboxylate group of amino acids contributes to the stereospecificity of exchange reactions catalysed by tryptophan synthase, serine hydroxymethyltransferase and a catalytic antibody utilising pyridoxal-5'-phosphate (PLP) as a cofactor. Using larger substrates, it is shown how the size of the amino acid side chain contributes to the stereospecificity of exchange. Mutants of aspartate aminotransferase are used to determine how substrate binding controls the catalytic efficiency and stereospecificity of the exchange of the alpha-protons of aspartate and glutamate. Evidence is presented which shows that with serine hydroxymethyltransferase, L-norleucine is not bound at the same catalytic site as glycine. Finally the catalytic efficiency and stereospecificity of the alpha-proton exchange reactions catalysed by all the PLP-dependent catalysts examined are compared. 相似文献
5.
The Arabidopsis thaliana S-adenosylmethionine decarboxylase (AdoMetDC) cDNA (GenBank(TM) U63633) was cloned, and the AdoMetDC protein was expressed, purified, and characterized. The K(m) value for S-adenosylmethionine (AdoMet) is 23.1 microM and the K(i) value for methylglyoxal bis-(guanylhydrazone) (MGBG) is 0.15 microM. Site-specific mutagenesis was performed on the AdoMetDC to introduce mutations at conserved cysteine (Cys(50), Cys(83), and Cys(230)) and lysine(81) residues, chosen by examination of the conserved sequence and proved to be involved in enzymatic activity by chemical modification. The AdoMetDC mutants K81A and C83A retained up to 60 and 10% of wild type activity, respectively, demonstrating that lysyl and sulfhydryl groups are required for full catalytic activity. However, changing Cys(50) and Cys(230) to alanine had minimal effects on the catalytic activity. Changing Lys(81) to alanine produced an altered substrate specificity. When lysine was used as a substrate instead of AdoMet, the substrate specificity for lysine increased 6-fold. The K(m) value for AdoMet is 11-fold higher than that of the wild type, but the V(max) value is more than 60%. Taken together, the results suggest that the lysine(81) residue is critical for substrate binding. 相似文献
6.
Transducin (T), a GTP-binding protein involved in phototransduction of rod photoreceptor cells, is a heterotrimer arranged as two units, the alpha-subunit (T alpha) and the beta gamma-complex (T beta gamma). The role of the carboxyl groups in T was evaluated by labeling with N,N'-dicyclohexylcarbodiimide (DCCD) and 1-ethyl 3-(3-dimethylaminopropyl) carbodiimide (EDC). Only a minor effect on the binding of beta, gamma-imido guanosine 5'-triphosphate (GMPpNp) to T was observed in the presence of the hydrophobic carbodiimide, DCCD. Similarly, the GMPpNp binding activity of the reconstituted holoenzyme was not significantly affected when T alpha was combined with DCCD-treated T beta gamma. However, the binding of guanine nucleotides to the reconstituted T was approximately 50% inhibited when DCCD-labeled T alpha was incubated with T beta gamma. In contrast, treatment of T with the hydrophilic carbodiimide, EDC, completely impaired its GMPpNp-binding ability. EDC-modified T was incapable of interacting with illuminated rhodopsin, as determined by sedimentation experiments. However, rhodopsin only partially protected against the inactivation of T. Additionally, analyses of trypsin digestion patterns showed that fluoroaluminate was not capable of activating the EDC-labeled T sample. The function of the reconstituted holoenzyme was also disrupted when EDC-modified T alpha was combined with T beta gamma, and when EDC-treated T beta gamma was incubated with T alpha. 相似文献
7.
Yanase H Maeda M Hagiwara E Yagi H Taniguchi K Okamoto K 《Journal of biochemistry》2002,132(4):565-572
The catalytic residues of levansucrase (sucrose:2,6-beta-D-fructan 6-beta-D-fructosyltransferase, EC 2.4.1.10) from Zymomonas mobilis were analyzed by random mutation and site-directed mutagenesis. We found that substitution of Glu278 with Asp and His reduced the k(cat) for sucrose hydrolysis 30- and 210-fold, respectively, strongly suggesting Glu278 plays a key role in catalyzing this reaction. Given the likelihood that another acidic amino residue was also involved, we constructed variants in which acidic amino acids located within homologous regions among bacterial levansucrases and fructosyltransferases were substituted, and found that substitution of Asp194, located in homologous region III, abolished sucrose hydrolysis. In addition, Glu278 was determined to be situated within the DXXER motif in homologous region IV conserved among bacterial levansucrases and fructosyltransferases, while Asp194 was within the triplet RDP motif conserved among bacterial levansucrases, fructosyltransferases and fructofuranosidases. Finally, comparison of our findings with published data on other site-directed mutated enzymes indicated His296, also located in homologous region IV, is crucial for catalysis of the transfructosylation reaction. 相似文献
8.
Identification of functionally important residues in the DNA binding region of the mnt repressor 总被引:7,自引:0,他引:7
Single amino acid substitutions have been introduced throughout the N-terminal DNA binding region of the Mnt repressor, and the operator binding properties of the resulting mutant repressors have been assayed. These studies show that the side chains of Arg2, His6, Asn8, and Arg10 are critical for high affinity binding to operator DNA. Other side chains in the N-terminal region do not appear to play major roles in DNA recognition and binding. Specific alterations in the pattern of methylation protection afforded by the Arg2----Lys mutant protein suggest that Arg2 contacts the N7 groups of guanines 10 and 12 in the operator. In conjunction with previous results, these findings suggest that part of the N-terminal region of Mnt binds as an extended polypeptide strand within the major groove of the mnt operator. 相似文献
9.
A A Antson B V Strokopytov G N Murshudov M N Isupov E H Harutyunyan T V Demidkina D G Vassylyev Z Dauter H Terry K S Wilson 《FEBS letters》1992,302(3):256-260
The tyrosine phenol lyase (EC 4.1.99.2) from Citrobacter intermedius has been crystallised in the apo form by vapour diffusion. The space group is P2(1)2(1)2. The unit cell has dimensions a = 76.0 A, b = 138.3 A, c = 93.5 A and it contains two subunits of the tetrameric molecule in the asymmetric unit. Diffraction data for the native enzyme and two heavy atom derivatives have been collected with synchrotron radiation and an image plate scanner. The structure has been solved at 2.7 A resolution by isomorphous replacement with subsequent modification of the phases by averaging the density around the non-crystallographic symmetry axis. The electron density maps clearly show the relative orientation of the subunits and most of the trace of the polypeptide chain. Each subunit consists of two domains. The topology of the large domain appears to be similar to that of the aminotransferases. 相似文献
10.
H K?ck J Sandmark K Gibson G Schneider Y Lindqvist 《Journal of molecular biology》1999,291(4):857-876
The three-dimensional structure of diaminopelargonic acid synthase, a vitamin B6-dependent enzyme in the pathway of the biosynthesis of biotin, has been determined to 1.8 A resolution by X-ray crystallography. The structure was solved by multi-wavelength anomalous diffraction techniques using a crystal derivatized with mercury ions. The protein model has been refined to a crystallographic R -value of 17.5% (R -free 22.6%). Each enzyme subunit consists of two domains, a large domain (residues 50-329) containing a seven-stranded predominantly parallel beta-sheet, surrounded by alpha-helices, and a small domain comprising residues 1-49 and 330-429. Two subunits, related by a non-crystallographic dyad in the crystals, form the homodimeric molecule, which contains two equal active sites. Pyridoxal-5'-phosphate is bound in a cleft formed by both domains of one subunit and the large domain of the second subunit. The cofactor is anchored to the enzyme by a covalent linkage to the side-chain of the invariant residue Lys274. The phosphate group interacts with main-chain nitrogen atoms and the side-chain of Ser113, located at the N terminus of an alpha-helix. The pyridine nitrogen forms a hydrogen bond to the side-chain of the invariant residue Asp245. Electron density corresponding to a metal ion, most likely Na(+), was found in a tight turn at the surface of the enzyme. Structure analysis reveals that diaminopelargonic acid synthase belongs to the family of vitamin B6-dependent aminotransferases with the same fold as originally observed in aspartate aminotransferase. A multiple structure alignment of enzymes in this family indicated that they form at least six different subclasses. Striking differences in the fold of the N-terminal part of the polypeptide chain are one of the hallmarks of these subclasses. Diaminopelargonic acid synthase is a member of the aminotransferase subclass III. From the structure of the non-productive complex of the holoenzyme with the substrate 7-keto-8-aminopelargonic acid the location of the active site and residues involved in substrate binding have been identified. 相似文献
11.
12.
Lack of crystal structure data of folate binding proteins has left so many questions unanswered (for example, important residues in active site, binding domain, important amino acid residues involved in interactions between ligand and receptor). With sequence alignment and PROSITE motif identification, we attempted to answer evolutionarily significant residues that are of functional importance for ligand binding and that form catalytic sites. We have analyzed 46 different FRs and FBP sequences of various organisms obtained from Genbank. Multiple sequence alignment identified 44 highly conserved identical amino acid residues with 10 cysteine residues and 12 motifs including ECSPNLGPW (which might help in the structural stability of FR). 相似文献
13.
S Gasparini P Vincendon G Eriani J Gangloff Y Boulanger J Reinbolt D Kern 《Biochemistry》1991,30(17):4284-4289
Cytoplasmic aspartyl-tRNA synthetase from Saccharomyces cerevisiae is an alpha 2 dimer (alpha, Mr 63,000), each alpha containing 12 histidines. The covalent incorporation of 6-7 mol of diethyl pyrocarbonate per monomer corresponded to complete enzyme inactivation. This inactivation was reversed by hydroxylamine hydrolysis which regenerates free histidine (and tyrosine) while leaving the carbethoxy group still attached to the epsilon-amino group of lysine. Three histidines, one tyrosine, and four lysines were the main targets of the reagent. Site-directed mutagenesis was also tried to replace each of these modified residues. Given the unstability of the carbethoxy-imidazole bond, the nine histidines that were not modified by diethyl pyrocarbonate were mutated too. For these experiments, the enzyme was expressed in Escherichia coli by using a vector bearing the structural gene in which the first 13 codons were replaced by the first 14 of the CII lambda gene. This substitution had no effect on the kinetic parameters. The combined results of chemical modification and site-directed mutagenesis show that one histidine seems to be part of the active site while two others play an important structural role. On the other hand, labeled lysines and tyrosine are nonessential residues. These results are discussed in light of two recent articles establishing the existence of a second family of aminoacyl-tRNA synthetases devoid of the HIGH and KMSKS consensus sequences and containing no Rossmann's domain in their three-dimensional structures. 相似文献
14.
Takahashi S Takahashi K Kaneko T Ogasawara H Shindo S Saito K Kawamura Y 《Journal of biochemistry》2001,129(4):529-535
Renin-binding protein (RnBP) is an endogenous renin inhibitor originally isolated from porcine kidney. It was recently identified as the enzyme N-acetyl-D-glucosamine (GlcNAc) 2-epimerase [Takahashi, S. et al. (1999) J. Biochem. 125, 348-353] and its active site residue was determined to be cysteine 380 by site-directed mutagenesis [Takahashi, S. et al. (1999) J. Biochem. 126, 639-642]. To further investigate the relationship between structure and function of recombinant human (rh) RnBP as a GlcNAc 2-epimerase, we have constructed several C-terminal deletion and multi-cysteine/serine mutants of rhGlcNAc 2-epimerase and expressed them in Escherichia coli cells. The expression was detected by Western blotting using anti-rhRnBP antiserum. The C-terminal deletion mutant, Delta400-417, had approximately 50% activity relative to the wild-type enzyme, but other C-terminal deletion mutants, Delta380-417, Delta386-417, and Delta390-417, had no enzymatic activity. Mutational analysis of multi-cysteine/serine mutants revealed that cysteines 41 and 390 were critical for the activity or stabilization of the enzyme, while cysteine residues in the middle of the enzyme, cysteines 125, 210, 239, and 302, had no essential function in relation to the activity. 相似文献
15.
16.
Each of four conserved glutamate residues of Bacillus stearothermophilus leucine aminopeptidase II (BsLAPII) was replaced with aspartate, lysine, and leucine respectively by site-directed mutagenesis. The over-expressed wild-type and mutant enzymes were purified to homogeneity by nickel-chelate chromatography and the molecular mass of the subunit was determined to be 44.5 kDa by SDS-PAGE. The specific activity for the Glu-316 and Glu-340 mutants was completely abolished, while Glu-249 mutants showed comparable activity to that of the wild-type BsLAPII. Compared with the wild-type enzyme, the E250D and E250L mutant enzymes retained less than 18% of the enzyme activity and exhibited a dramatic decrease in the value of k
cat/K
m. These observations indicate that Glu-250, Glu-316, and Glu-340 residues are critical for the catalytic activity of BsLAPII. 相似文献
17.
Relevance of mode coupling to energy/information transfer during protein function, particularly in the context of allosteric interactions is widely accepted. However, existing evidence in favor of this hypothesis comes essentially from model systems. We here report a novel formal analysis of the near‐native dynamics of myosin II, which allows us to explore the impact of the interaction between possibly non‐Gaussian vibrational modes on fluctutational dynamics. We show that an information‐theoretic measure based on mode coupling alone yields a ranking of residues with a statistically significant bias favoring the functionally critical locations identified by experiments on myosin II. Proteins 2014; 82:1777–1786. © 2014 Wiley Periodicals, Inc. 相似文献
18.
Evolutionarily conserved and functionally important residues in the I-CeuI homing endonuclease. 总被引:2,自引:1,他引:1
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Two approaches were used to discern critical amino acid residues for the function of the I- Ceu I homing endonuclease: sequence comparison of subfamilies of homologous proteins and genetic selection. The first approach revealed residues potentially involved in catalysis and DNA recognition. Because I- Ceu I is lethal in Escherichia coli , enzyme variants not perturbing cell viability were readily selected from an expression library. A collection of 49 variants with single amino acid substitutions at 37 positions was assembled. Most of these positions are clustered within or around the LAGLI-DADG dodecapeptide and the TQH sequence, two motifs found in all protein subfamilies examined. The Km and kcat values of the wild-type and nine variant enzymes synthesized in vitro were determined. Three variants, including one showing a substitution of the glutamine residue in the TQH motif, revealed no detectable endonuclease activity; five others showed reduced activity compared to the wild-type enzyme; whereas the remaining variant cleaved the top strand about three times more efficiently than the wild-type. Our results not only confirm recent reports indicating that amino acids in the LAGLI-DADG dodecapeptide are functionally critical, but they also suggest that some residues outside this motif directly participate in catalysis. 相似文献
19.
20.
Powered by ATP hydrolysis, PIB‐ATPases drive the energetically uphill transport of transition metals. These high affinity pumps are essential for heavy metal detoxification and delivery of metal cofactors to specific cellular compartments. Amino acid sequence alignment of the trans‐membrane (TM) helices of PIB‐ATPases reveals a high degree of conservation, with ~60–70 fully conserved positions. Of these conserved positions, 6–7 were previously identified to be important for transport. However, the functional importance of the majority of the conserved TM residues remains unclear. To investigate the role of conserved TM residues of PIB‐ATPases we conducted an extensive mutagenesis study of a Zn2+/Cd2+ PIB‐ATPase from Rhizobium radiobacter (rrZntA) and seven other PIB‐ATPases. Of the 38 conserved positions tested, 24 had small effects on metal tolerance. Fourteen mutations compromised in vivo metal tolerance and in vitro metal‐stimulated ATPase activity. Based on structural modelling, the functionally important residues line a constricted ‘channel’, tightly surrounded by the residues that were found to be inconsequential for function. We tentatively propose that the distribution of the mutable and immutable residues marks a possible trans‐membrane metal translocation pathway. In addition, by substituting six trans‐membrane amino acids of rrZntA we changed the in vivo metal specificity of this pump from Zn2+/Cd2+ to Ag+. 相似文献