Function of a conserved sequence motif in biotin holoenzyme synthetases

The biotin holoenzyme synthetases (BHS) are essential enzymes in all organisms that catalyze post-translational linkage of biotin to biotin-dependent carboxylases. The primary sequences of a large number of these enzymes are now available and homologies are found among all. The glycine-rich sequence, GRGRXG, constitutes one of the homologous regions in these enzymes and, based on its similarity to sequences found in a number of mononucleotide binding enzymes, has been proposed to function in ATP binding in the BHSs. In the Escherichia coli enzyme, the only member of the family for which a three-dimensional structure has been determined, the conserved sequence is found in a partially disordered surface loop. Mutations in the sequence have previously been isolated and characterized in vivo. In this work these single-site mutants, G115S, R118G, and R119W, of the E. coli BHS have been purified and biochemically characterized with respect to binding of small molecule substrates and the intermediate in the biotinylation reaction. Results of this characterization indicate that, rather than functioning in ATP binding, this glycine-rich sequence is required for binding the substrate biotin and the intermediate in the biotinylation reaction, biotinyl-5'-AMP. These results are of general significance for understanding structure-function relationships in biotin holoenzyme synthetases.     

Enzymatic biosynthesis of ergotamine and investigation on some aminoacyl-tRNA synthetases in Claviceps

An enzyme system from Claviceps purpurea (Fr.) Tul. catalyzing the incorporation of l-phenylalanine into ergotamine - ergotamine synthetase - was purified 172-fold. This was done by a combination of ammonium sulfate precipitation, gel filtration, ion-exchange chromatography on DEAE-Sepharose CL-6B, and hydroxyapatite chromatography. The activation of ergotamine specific amino acids as well as d-lysergic acid and dihydrolysergic acid via adenylates, as determined by the ATP-³²PP_i exchange, was investigated. Phenylalanyl-tRNA synthetase, catalyzing the same type of activation reaction, could not be separated from ergotamine synthetase by the purification procedure applied. Therefore, at the present stage of enzyme purification, phenylalanine-dependent ATP-³²PP_i exchange cannot be used to measure ergotamine synthetase activity specifically.Phenylalanyl-tRNA synthetase and leucyl-tRNA synthetase were separated into mitochondrial and cytoplasmic isoenzymes by hydroxyapatite chromatography. Their charging activities of procaryotic versus eucaryotic tRNA and their molecular masses were determined.     

Crystallization and preliminary X-ray diffraction analysis of arginyl-tRNA synthetase from Escherichia coli.

Arginyl-tRNA Synthetase, a class I aminoacyl tRNA synthetase playing a crucial role in protein biosynthesis, has been crystallized for the first time. Polyethylene glycol (PEG) was used as a precipitant, and the crystallization proceeded at pH 6.5. These single crystals diffracted to 2.8 A with a rotating anode X-ray source and R-axis IIc image plate detector. They have an orthorhombic space group P2(1)2(1)2 with unit cell parameters of a = 251.51 A, b = 53.12 A, and c = 52.35 A. A complete native data set has been collected at 3.1 A resolution for these crystals.     

The 50th anniversary of the publication of the operon theory in the Journal of Molecular Biology: past, present and future

A series of eight review articles that appear in the present issue of the Journal of Molecular Biology celebrates the 50th anniversary for the landmark publication of François Jacob and Jacques Monod entitled “Genetic Regulatory Mechanisms in the Synthesis of Proteins”. In this publication, the authors presented a model for the regulation of gene expression deduced from genetic and biochemical studies. They proposed that a new class of genes, regulatory genes, would code for repressors that bind to operator sequences upstream of operons consisting of a group of catabolic or biosynthetic genes with related functions. Binding is controlled by small metabolites, substrates or end products. The repressors control the transmission of information from genes to mRNA that is translated into proteins. The present review articles demonstrate how this publication influenced our thinking and how it stimulated the studies on the regulation of gene expression all the way to present day epigenetics and systems biology.     

Analyses of the stability and function of three surface mutants (R82C, K69H, and L32R) of the gene V protein from Ff phage by X-ray crystallography.

The high-resolution crystal structure of the gene V protein (GVP) from the Ff filamentous phages (M13, fl, fd) has been solved recently for the wild-type and two surface mutant (Y41F and Y41H) proteins, leading to a plausible model for the polymeric GVP-ssDNA complex (Guan Y, Zhang H, Wang AHJ, 1995, Protein Sci 4:187-197). The model of the complex shows extensive contacts between neighboring dimer GVPs involving electrostatic interactions between the K69 from one and the D79 and R82 from the next dimer. In addition, hydrophobic interactions between the amino acids L32 and L44 from one and G23 from the next dimer also contribute to the dimer-dimer interactions. Mutations at the L32, K69, and R82 amino acid sites generally destabilize the protein and many of these affect the function of the phage. We have studied the structural effects of three mutant proteins involving those sites, i.e., L32R, K69H, and R82C, by X-ray crystallographic analysis at 2.0 A resolution. In L32R GVP, the structural perturbation is localized, whereas in K69H and R82C GVPs, some long-range effects are also detected in addition to the local perturbation. We have interpreted the protein stability and the functional properties associated with those mutations in terms of the observed structural perturbations.     

Electrostatic potential distribution of the gene V protein from Ff phage facilitates cooperative DNA binding: a model of the GVP-ssDNA complex.

The crystal structure of the gene V protein (GVP) from the Ff filamentous phages (M13, fl, fd) has been solved for the wild-type and two mutant (Y41F and Y41H) proteins at high resolution. The Y41H mutant crystal structure revealed crystal packing interactions, which suggested a plausible scheme for constructing the polymeric protein shell of the GVP-single-stranded DNA (ssDNA) complex (Guan Y, et al., 1994, Biochemistry 33:7768-7778). The electrostatic potentials of the isolated and the cooperatively formed protein shell have been calculated using the program GRASP and they revealed a highly asymmetric pattern of the electrostatic charge distribution. The inner surface of the putative DNA-binding channel is positively charged, whereas the opposite outer surface is nearly neutral. The electrostatic calculation further demonstrated that the formation of the helical protein shell enhanced the asymmetry of the electrostatic distribution. A model of the GVP-ssDNA complex with the n = 4 DNA-binding mode could be built with only minor conformational perturbation to the GVP protein shell. The model is consistent with existing biochemical and biophysical data and provides clues to the properties of GVP, including the high cooperatively of the protein binding to ssDNA. The two antiparallel ssDNA strands form a helical ribbon with the sugar-phosphate backbones at the middle and the bases pointing away from each other. The bases are stacked and the Phe 73 residue is intercalated between two bases. The optimum binding to a tetranucleotide unit requires the participation of four GVP dimers, which may explain the cooperativity of the GVP binding to DNA.     

Structural analysis of the multienzyme aminoacyl-tRNA synthetase complex: a three-domain model based on reversible chemical crosslinking.

A subset of eukaryotic aminoacyl-tRNA synthetases (a-RS) are contained in a multienzyme complex for which little structural detail is known. Three reversible chemical crosslinking reagents have been used to investigate the arrangement of polypeptides within this particle as isolated from rabbit reticulocytes. Identification of the crosslinked protein pairs was accomplished by two-dimensional SDS diagonal gel electrophoresis. Seventeen neighboring protein pairs have been identified. Eight are seen with at least two reagents: K-RS:p38, D-RS:K-RS, R-RS dimer, K-RS dimer, K-RS:Q-RS, E/P-RS:K-RS, E/P-RS:I-RS, and Q-RS with one of the nonsynthetase proteins. Nine more are observed with one reagent: D-RS dimer, R-RS:p43, D-RS:Q-RS, D-RS:M-RS, K-RS:L-RS, I-RS:R-RS, D-RS:E/P-RS, I-RS:Q-RS, I-RS:L-RS. One trimeric association is seen: E/P-RS:I-RS:L-RS. The observed neighboring protein pairs suggest that the polypeptides within the aminoacyl-tRNA synthetase complex are distributed in three structural domains of similar mass. These can be arranged in a U-shaped particle in which each "arm" is considered a domain and the third forms the "base" of the structure. The arms have been termed domain I (D-RS, M-RS, Q-RS) and domain II (K-RS, R-RS), with domain III (E/P-RS, I-RS, L-RS) assigned to the base. The smaller proteins (p38, p43) may bridge the domains. This proposed spatial relationship of these domains, as well as their compositions, are consistent with earlier studies. Thus, this study provides an initial three-dimensional working model of the arrangement of polypeptides within the multienzyme aminoacyl-tRNA synthetase complex.     

Crystal structure of the schiff base intermediate prior to decarboxylation in the catalytic cycle of aspartate alpha-decarboxylase

l-Aspartate alpha-decarboxylase (ADC), encoded by the panD gene, catalyzes the conversion of l-aspartate into beta-alanine. In the microorganisms, beta-alanine is required for the synthesis of pantothenate (vitamin B(5)), which is the precursor of 4'-phosphopantetheine and coenzyme A. We have determined the crystal structure of Helicobacter pylori ADC, a tetrameric enzyme, in two forms: the apo structure at 2.0 A resolution and the isoasparagine complex structure at 1.55 A resolution. All subunits of the tetramer are self-processed at the Gly24-Ser25 linkage, producing the smaller beta chain (residues 1-24) and the larger alpha chain (residues 25-117). Each subunit contains nine beta-strands and three alpha-helices; it is folded into the double-psi beta-barrel structure. In the apo structure, the new amino terminus of the alpha chain, Ser25, is converted into a pyruvoyl group. In the isoasparagine complex structure, the substrate analog is covalently attached to the pyruvoyl group. This structure represents the enzyme-substrate Schiff base intermediate that was proposed to form prior to the decarboxylation step in the catalytic cycle of ADC. Thus our study provides direct structural evidence for the reaction mechanism of ADC.     

Effect of continuous-wave and amplitude-modulated 2.45 GHz microwave radiation on the liver and brain aminoacyl-transfer RNA synthetases of in utero exposed mice

Investigations have been carried out concerning the effects of microwave (MW) exposure on the aminoacyl-transfer ribonucleic acid (tRNA) synthetase of the progeny of females that were exposed during their entire period of gestation (19 days). The changes caused by continuous-wave (CW) and amplitude-modulated (AM) MW radiation have been compared. CFLP mice were exposed to MW radiation for 100 min each day in an anechoic room. The MW frequency was 2.45 GHz, and the amplitude modulation had a 50 Hz rectangular waveform (on/off ratio, 50/50%). The average power density exposure was 3 mW/cm², and the whole body specific absorption rate (SAR) was 4.23 ± 0.63 W/kg. The weight and mortality of the progeny were followed until postnatal day 24. Aminoacyl-tRNA synthetase enzymes and tRNA from the brains and livers of the offspring (461 exposed, 487 control) were isolated. The aminoacyl-tRNA synthetase activities were determined. The postnatal increase of body weight and organ weight was not influenced by the prenatal MW radiation. The activity of enzyme isolated from the brain showed a significant decrease after CW MW exposure, but the changes were not significant after 50 Hz AM MW exposure. The activity of the enzyme isolated from liver increased under CW and 50 Hz modulated MW. © 1996 Wiley-Liss, Inc.     

Functional Compartmentalization of the Translation Apparatus and Channeling of tRNA/Aminoacyl-tRNA in Cells of Higher Eukaryotes

Literature data and authors' results on the structural and functional organization of the translation apparatus in higher eukaryotes are considered. Proofs are presented of the channeling of tRNA/aminoacyl-tRNA in the course of eukaryotic protein synthesis. The concept of the shuttle role of eEF1A is grounded; the factor, being in a GTP-bound form, delivers aminoacyl-tRNA to the ribosome and then, in the having undergone to a GDP -form after hydrolysis of GTP on the ribosome, forms a complex with the deacylated tRNA and delivers it to the aminoacyl-tRNA synthetase. The notion of a translational compartment is defined.     

Monophyly of class I aminoacyl tRNA synthetase,USPA, ETFP,photolyase, and PP-ATPase nucleotide-binding domains: implications for protein evolution in the RNA

Protein sequence and structure comparisons show that the catalytic domains of Class I aminoacyl-tRNA synthetases, a related family of nucleotidyltransferases involved primarily in coenzyme biosynthesis, nucleotide-binding domains related to the UspA protein (USPA domains), photolyases, electron transport flavoproteins, and PP-loop-containing ATPases together comprise a distinct class of alpha/beta domains designated the HUP domain after HIGH-signature proteins, UspA, and PP-ATPase. Several lines of evidence are presented to support the monophyly of the HUP domains, to the exclusion of other three-layered alpha/beta folds with the generic "Rossmann-like" topology. Cladistic analysis, with patterns of structural and sequence similarity used as discrete characters, identified three major evolutionary lineages within the HUP domain class: the PP-ATPases; the HIGH superfamily, which includes class I aaRS and related nucleotidyltransferases containing the HIGH signature in their nucleotide-binding loop; and a previously unrecognized USPA-like group, which includes USPA domains, electron transport flavoproteins, and photolyases. Examination of the patterns of phyletic distribution of distinct families within these three major lineages suggests that the Last Universal Common Ancestor of all modern life forms encoded 15-18 distinct alpha/beta ATPases and nucleotide-binding proteins of the HUP class. This points to an extensive radiation of HUP domains before the last universal common ancestor (LUCA), during which the multiple class I aminoacyl-tRNA synthetases emerged only at a late stage. Thus, substantial evolutionary diversification of protein domains occurred well before the modern version of the protein-dependent translation machinery was established, i.e., still in the RNA world.     

Structural analysis of the zinc hydroxide–Thr-199–Glu-106 hydrogen-bond network in human carbonic anhydrase II

The singnificance of the zinc hydroxide–Thr-199–Glu-106 hydrogen-bond network in the active site of human carbonic anhydrase II has been examined by X-ray crystallographic analyses of site-specific mutants. Mutants with Ala-199 and Ala-106 or Gln-106 have low catalytic activities, while a mutant with Asp-106 has almost full CO₂ hydration activity. The structures of these four mutants, as well as that of the bicarbonate complex of the mutant with Ala-199, have been determined at 1.7 to 2.2 Å resolution. Removal of the γ atoms of residue 199 leads to distorted tetrahedral geometry at the zine ion, and a catalytically important zinc-bound water molecule has moved towards Glu-106. In the bicarbonate complex of the mutant with Ala-199 one oxygen atom from bicarbonate binds to zinc without displacing this water molecule. Tetrahedral coordination geometries are retained in the mutants at position 106. The mutants with Ala-106 and Gln-106 have a zinc-bound sulfate ion, whereas this sulfate site is only partially occupied in the mutant with Asp-106. The hydrogen-bond network seems to be “reversed” in the mutants with Ala-106 and Gln-106. The network is preserved as in native enzyme in the mutant with Asp-106 but the side chain of Asp-106 is more extended than that of Glu-106 in the native enzyme. These results illustrate the importance of Glu-106 and Thr-199 for controlling the precise coordination geometry of the zinc ion and its ligand preferences with results in an optimal orientation of a zine-bound hydroxide ion for an attack on the CO₂ substrate. © 1993 Wiley-Liss, Inc.     

Crystallographic analysis of a full-length streptavidin with its C-terminal polypeptide bound in the biotin binding site

The structure of a full-length streptavidin has been determined at 1.7 A resolution and shows that the 20 residue extension at the C terminus forms a well-ordered polypeptide loop on the surface of the tetramer. Residues 150-153 of the extension are bound to the ligand-binding site, possibly competing with exogenous ligands. The binding mode of these residues is compared with that of biotin and peptidic ligands. The observed structure helps to rationalize the observations that full-length mature streptavidin binds biotinylated macromolecules with reduced affinity.     

非核糖体肽合成酶(NRPSs)作用机理与应用的研究进展

许多微生物能利用非核糖体肽合成酶(NRPSs)合成结构复杂、种类繁多的的生物活性肽。非核糖体肽因其独特的理化特性和药理学特性已被广泛关注,极具商业开发潜力。NRPSs由多个模块组成,模块的不同空间排列顺序决定其多肽产物的氨基酸序列特异性。NRPSs以多载体巯基化模板机理进行多肽合成,其底物特异性由腺苷酰化结构域和缩合结构域共同实现。目前,人们已经利用天然的NRPSs、某些特定结构域、将已知NRPSs的模块或特定结构域进行组合甚至杂合组合而构建成的新的NRPSs来合成目的多肽。     

Structural comparison of the prokaryotic ribosomal proteins L7/L12 and L30

The structures of two prokaryotic ribosomal proteins, the carboxyterminal half of L7/L12 from Escherichia coli (L12CTF) and L30 from Bacilus stearothermophilus display a remarkably similar fold in which alpha-helices pack onto one side of an antiparallel, three-stranded, beta-pleated sheet. A detailed comparison of the structures by least-squares methods reveals that more than two-thirds of the alpha carbons can be superimposed with a root mean square distance of 2.33 A. The principal difference is an extra alpha-helix in L12CTF. The sequences of the proteins display a distinct conservation in regions which are crucial to the common fold, in particular the hydrophobic core. It is proposed that the similarity is a result of divergent evolution.