Molecular diversity of glucose-6-phosphate dehydrogenase: rat enzyme structure identifies NH2-terminal segment, shows initiation from sites nonequivalent in different organisms, and establishes otherwise extensive sequence conservation.

The NH2-terminal region of rat liver glucose-6-phosphate dehydrogenase (EC 1.1.1.49) is shown to differ radically from a reported amino acid sequence for the fruit fly enzyme and from one for the human enzyme. The results indicate considerable differences in the translational start point. However, a close relationship with another reported sequence for the human enzyme is established, now showing agreement between an indirectly deduced and a directly analyzed NH2-terminal structure of this enzyme type. The results provide evidence of one structural motif common to mammalian species but also suggest that genetic inconstancy 5' to, or at the start of, the region coding for the enzyme protein could be a source of intra- and interspecies diversity. This is of interest in relation to the large number of genetic variants of human glucose-6-phosphate dehydrogenase.     

Bacillus anthracis comparative genome analysis in support of the Amerithrax investigation

Before the anthrax letter attacks of 2001, the developing field of microbial forensics relied on microbial genotyping schemes based on a small portion of a genome sequence. Amerithrax, the investigation into the anthrax letter attacks, applied high-resolution whole-genome sequencing and comparative genomics to identify key genetic features of the letters' Bacillus anthracis Ames strain. During systematic microbiological analysis of the spore material from the letters, we identified a number of morphological variants based on phenotypic characteristics and the ability to sporulate. The genomes of these morphological variants were sequenced and compared with that of the B. anthracis Ames ancestor, the progenitor of all B. anthracis Ames strains. Through comparative genomics, we identified four distinct loci with verifiable genetic mutations. Three of the four mutations could be directly linked to sporulation pathways in B. anthracis and more specifically to the regulation of the phosphorylation state of Spo0F, a key regulatory protein in the initiation of the sporulation cascade, thus linking phenotype to genotype. None of these variant genotypes were identified in single-colony environmental B. anthracis Ames isolates associated with the investigation. These genotypes were identified only in B. anthracis morphotypes isolated from the letters, indicating that the variants were not prevalent in the environment, not even the environments associated with the investigation. This study demonstrates the forensic value of systematic microbiological analysis combined with whole-genome sequencing and comparative genomics.     

Evolution of highly active enzymes by homology-independent recombination

The theta-class GST enzymes hGSTT1-1 (human GSTTheta-1-1) and rGSTT2-2 (rat GSTTheta-2-2) share 54.3% amino acid identity and exhibit different substrate specificities. Homology-independent techniques [incremental truncation for the creation of hybrid enzymes (ITCHY) and SCRATCHY] and low-homology techniques (recombination-dependent exponential amplification PCR) were used to create libraries of chimeric enzymes containing crossovers (C/Os) at positions not accessible by DNA family shuffling. High-throughput flow cytometric screening using the fluorogenic rGSTT2-2-specific substrate 7-amino-4-chloromethyl coumarin led to the isolation of active variants with either one or two C/Os. One of these enzymes, SCR23 (83% identity to hGSTT1-1), was encoded by a gene that exchanged helices 4 and 5 of hGSTT1-1 with the corresponding sequence from rGSTT2-2. Compared with either parent, this variant was found to have an improved k(cat) with the selection substrate and also exhibited activity for the conjugation of glutathione to ethacrynic acid, a compound that is not recognized by either parental enzyme. These results highlight the power of combinatorial homology-independent and low-homology recombination methods for the generation of unique, highly active enzymes and also suggest a possible means of enzyme "humanization."     

Directed evolution of mammalian paraoxonases PON1 and PON3 for bacterial expression and catalytic specialization

Serum paraoxonases (PONs) are a group of enzymes that play a key role in organophosphate (OP) detoxification and in prevention of atherosclerosis. However, their structure and mechanism of action are poorly understood. PONs seem like jacks-of-all-trades, acting on a very wide range of substrates, most of which are of no physiological relevance. Family shuffling and screening lead to the first PON variants that express in a soluble and active form in Escherichia coli. We describe variants with kinetic parameters similar to those reported for PONs purified from sera and others that show dramatically increased activities. In particular, we have evolved PON1 variants with OP-hydrolyzing activities 40-fold higher than wild type and a specificity switch of >2,000-fold, producing PONs specialized for OP rather than ester hydrolysis. Analysis of the newly evolved variants provides insights into the evolutionary relationships between different family members.     

Compensatory mutations in receptor function: a reevaluation of the role of methylation in bacterial chemotaxis.

During bacterial chemotaxis membrane receptor proteins are methylated and demethylated at glutamate residues. The generally accepted view is that these reactions play an essential role in the chemosensing mechanism. Strains may be isolated, however, that exhibit chemotaxis in the complete absence of methylation. These are readily obtained by selecting for chemotactic variants of a mutant that completely lacks the methylating enzyme. Methyltransferase activity is not restored; instead, the sensory-motor apparatus is genetically restructured to compensate for the methylation defect. Genetic and biochemical analyses show that the compensatory mutational locus is the structural gene for the demethylating enzyme. Thus, although mutants lacking either the methylating or demethylating enzymes are nonchemotactic, strains defective in both activities exhibit almost-wild-type chemotactic ability.     

Novel sequence variants in the human xylosyltransferase I gene and their role in diabetic nephropathy.

Aims Decreased content of heparan sulphate proteoglycans (HSPGs) is a characteristic of the glomerular basement membrane (GBM) in diabetes and contributes to the development of diabetic nephropathy (DN). Xylosyltransferase I (XT‐I) is the chain‐initiating enzyme involved in the biosynthesis of HSPGs. This study investigated a possible association between XYLT‐I sequence variants and susceptibility to DN. Methods Screening of all XYLT‐I exons was performed in 74 caucasians with Type 1 diabetes (48 with and 26 without DN) and in 13 non‐diabetic control subjects using denaturing high‐performance liquid chromatography. Results Fifteen XYLT‐I sequence variants were identified. Of these, six were previously unknown. There were significant differences in the allele frequencies of the three polymorphisms (c.343G→T (p.A115S), IVS3+10C→T, IVS3+30G→C) in Type 1 diabetic patients and healthy controls. Conclusions The occurrence of DN is independent of the XYLT‐I variants detected in our study. However, three XYLT‐I polymorphisms may be linked to Type 1 diabetes. Since we have previously proposed that one of these polymorphisms was not associated with Type 1 diabetes (Schön S et al. Kidney Int 2005; 68: 1483–1490), larger‐scale analysis is clearly necessary to pinpoint the significance of this mutation.     

Molecular heterogeneity and cellular localization of carboxypeptidase H in the islets of Langerhans

The intracellular distribution and molecular heterogeneity of carboxypeptidase H was studied in rat insulinoma tissue and isolated islets of Langerhans by a combination of immunohistochemical, ultrastructural, subcellular fractionation, and immunoblotting analyses. Immunofluorescence microscopy of islets demonstrated the presence of carboxypeptidase H in both insulin-containing B cells and glucagon-containing A cells. Quantitative ultrastructural analyses of islet B cells indicated that the enzyme was concentrated in mature insulin secretory granules, clathrin-coated condensing granules, and to a lesser extent the Golgi apparatus. Carboxypeptidase H activity was localized principally to secretory granule subfractions of insulinoma tissue, where it was present for the major part (70%) as a form which is readily solubilizable at pH values prevailing in the granule interior (5.5). This species migrated as a diffuse band of 53-57 kilodaltons (kDa) on immunoblot analysis using antisera raised against the purified native enzyme. In contrast, the insoluble form which was associated with the granule membrane at pH 5.5, migrated as a relatively compact band of 55-57 kDa. Carboxypeptidase H activity was also present in subcellular fractions which contained Golgi membranes together with elements of the endoplasmic reticulum, and in a low density secretory granule fraction which may represent immature granules. The enzyme in these compartments, like the granule membrane species, migrated as a compact 55-57 kDa band on immunoblots. Two-dimensional electrophoretic immunoblot analysis of secretory granules suggested that both membrane and soluble forms of the enzyme were glycoproteins and that the terminal glycosylation was similar in both instances. Antiserum raised against the deduced C-terminal 11 amino acids of the cloned carboxypeptidase H sequence recognized the 55-57 kDa membrane component in granules but did not react with the 53-57 kDa soluble species. A major difference between the soluble and membrane forms therefore appears to be a structural modification or proteolytic removal of the C-terminal domain in the trans-Golgi or early secretory granule compartment. The concept that proteolysis is involved is further supported by the observation that the relative proportion of the high and low mol wt forms of the enzyme in different subcellular fractions correlated with that of proinsulin and insulin, respectively. The membrane association of the 55-57 kDa form of carboxypeptidase H is disrupted at pH values of 9 and is dependent on ionic strength. This further suggests that the C-terminus of the protein may have an important role in the sorting or concentration of the enzyme in vesicular elements of the regulated pathway of secretion.     

Structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma brucei determined from Laue data.

The three-dimensional structure of glycosomal glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating), EC 1.12.1.12] from the sleeping-sickness parasite Trypanosoma brucei was solved by molecular replacement at 3.2-A resolution with an x-ray data set collected by the Laue method. For data collection, three crystals were exposed to the polychromatic synchrotron x-ray beam for a total of 20.5 sec. The structure was solved by using the Bacillus stearothermophilus enzyme model [Skarzyński, T., Moody, P. C. E. & Wonacott, A. J. (1987) J. Mol. Biol. 193, 171-187] with a partial data set which was 37% complete. The crystals contain six subunits per asymmetric unit, which allowed us to overcome the absence of > 60% of the reflections by 6-fold density averaging. After molecular dynamics refinement, the current molecular model has an R factor of 17.6%. Comparing the structure of the trypanosome enzyme with that of the homologous human muscle enzyme, which was determined at 2.4-A resolution, reveals important structural differences in the NAD binding region. These are of great interest for the design of specific inhibitors of the parasite enzyme.     

White lines in L-edge x-ray absorption spectra and their implications for anomalous diffraction studies of biological materials.

We have measured high-resolution x-ray absorption spectra of lanthanide (Ln) and heavy transition metal complexes that display prominent narrow absorption peaks near the L2 and L3 absorption edges. The anomalous scattering factors (f' and f"), which are mathematically related to the absorption cross section, have correspondingly sharp changes in their magnitude within 5-10 eV of the absorption edge. Calculations of the magnitude of the change in f' and f" demonstrate that significant changes (on the order of 20 electrons in f') can be expected for these materials. These substantial changes in the anomalous scattering factors have applications to deriving structural information for macromolecules from x-ray diffraction studies. The magnitude of the changes indicate that the anomalous scattering technique is a powerful means of obtaining structural characteristics for macromolecules in single crystals, in solution, and in biological membranes.     

Pseudomonas stutzeri N2O reductase contains CuA-type sites.

N2O reductase (N2O----N2) is the terminal enzyme in the energy-conserving denitrification pathway of soil and marine denitrifying bacteria. The protein is composed of two identical subunits and contains eight copper ions per enzyme molecule. The magnetic circular dichroism spectrum of resting (oxidized) N2O reductase is strikingly similar to the magnetic circular dichroism spectrum of the CuA site in mammalian cytochrome c oxidase [Greenwood, C., Hull, B. C., Barber, D., Eglinton, D. G. & Thomson, A. J. (1983) Biochem. J. 215, 303-316] and is unlike the magnetic circular dichroism spectra of all other biological copper chromophores obtained to date. Sulfur (or chlorine) scatterers are required to fit the copper extended x-ray absorption fine structure data of both the oxidized and reduced forms of N2O reductase. Satisfactory fits require a Cu-N or Cu-O [denoted Cu-(N, O)] interaction at 2.0 A, a Cu-(S, Cl) interaction at 2.3 A and an additional Cu(S, Cl) interaction at approximately 2.6 A (oxidized) or approximately 2.7 A (reduced). Approximately eight sulfur ions (per eight copper ions) at approximately 2.3 A are required to fit the extended x-ray absorption fine structure data for both the oxidized and reduced N2O reductase. The 2.3-A Cu-(S, Cl) distance is nearly identical to that previously determined for the CuA site in cytochrome c oxidase. A 2.6-2.7 A Cu-(S, Cl) interaction is also present in resting and fully reduced cytochrome c oxidase. Comparison of the N2O reductase sequence, determined by translating the structural NosZ gene, with cytochrome c oxidase subunit II sequences from several sources indicates that a Gly-Xaa-Xaa-Xaa-Xaa-Xaa-Cys-Ser-Xaa-Xaa-Cys-Xaa-Xaa-Xaa-His stretch is highly conserved. This sequence contains three of the probable ligands (two cysteines and one histidine) in a CuA-type site. Collectively these data establish that Pseudomonas stutzeri N2O reductase contains CuA-type sites.     

Attenuation of GTPase activity of recombinant G(o) alpha by peptides representing sequence permutations of mastoparan.

There is convincing evidence that the cytoplasmic domains of multispanning receptors interact with guanine nucleotide-binding proteins (G proteins). What are the rules governing these interactions? In an attempt to answer this question, we focused our attention on mastoparan, an amphiphilic tetradecapeptide from wasp venom, and on nine of its variants, produced by sequence permutation, which have altered amphiphilicity or no amphiphilicity at all. Mastoparan enhances the GTPase activity of recombinant G(o) alpha 5-fold in phospholipid vesicles. Like mastoparan, four of the synthetic variants can form amphiphilic alpha-helices and two of them indeed stimulate the GTPase activity of the G protein, whereas the other two have no effect. This confirms that the activation of certain G proteins by a number of peptides is mainly due to their cationic amphiphilicity. However, this structural feature is clearly not sufficient. The relative orientation of the positively charged residues as well as that of the hydrophobic side chains appear to be of fundamental importance. The other five peptides are not amphiphilic and do not enhance the rate of GTP hydrolysis. Surprisingly, three of them almost completely inhibit the G protein''s intrinsic GTPase activity. This finding is of interest because of the possible role differential regulation of G protein activity can play in cellular functions.     

Amino acid substitutions in genetic variants of human serum albumin and in sequences inferred from molecular cloning.

The structural changes in four genetic variants of human serum albumin were analyzed by tandem high-pressure liquid chromatography (HPLC) of the tryptic peptides, HPLC mapping and isoelectric focusing of the CNBr fragments, and amino acid sequence analysis of the purified peptides. Lysine-372 of normal (common) albumin A was changed to glutamic acid both in albumin Naskapi, a widespread polymorphic variant of North American Indians, and in albumin Mersin found in Eti Turks. The two variants also exhibited anomalous migration in NaDodSO4/PAGE, which is attributed to a conformational change. The identity of albumins Naskapi and Mersin may have originated through descent from a common mid-Asiatic founder of the two migrating ethnic groups, or it may represent identical but independent mutations of the albumin gene. In albumin Adana, from Eti Turks, the substitution site was not identified but was localized to the region from positions 447 through 548. The substitution of aspartic acid-550 by glycine was found in albumin Mexico-2 from four individuals of the Pima tribe. Although only single-point substitutions have been found in these and in certain other genetic variants of human albumin, five differences exist in the amino acid sequences inferred from cDNA sequences by workers in three other laboratories. However, our results on albumin A and on 14 different genetic variants accord with the amino acid sequence of albumin deduced from the genomic sequence. The apparent amino acid substitutions inferred from comparison of individual cDNA sequences probably reflect artifacts in cloning or in cDNA sequence analysis rather than polymorphism of the coding sections of the albumin gene.     

Structure-based design of Taq DNA polymerases with improved properties of dideoxynucleotide incorporation.

The Taq DNA polymerase is the most commonly used enzyme in DNA sequencing. However, all versions of Taq polymerase are deficient in two respects: (i) these enzymes incorporate each of the four dideoxynucleoside 5' triphosphates (ddNTPs) at widely different rates during sequencing (ddGTP, for example, is incorporated 10 times faster than the other three ddNTPs), and (ii) these enzymes show uneven band-intensity or peak-height patterns in radio-labeled or dye-labeled DNA sequence profiles, respectively. We have determined the crystal structures of all four ddNTP-trapped closed ternary complexes of the large fragment of the Taq DNA polymerase (Klentaq1). The ddGTP-trapped complex structure differs from the other three ternary complex structures by a large shift in the position of the side chain of residue 660 in the O helix, resulting in additional hydrogen bonds being formed between the guanidinium group of this residue and the base of ddGTP. When Arg-660 is mutated to Asp, Ser, Phe, Tyr, or Leu, the enzyme has a marked and selective reduction in ddGTP incorporation rate. As a result, the G track generated during DNA sequencing by these Taq polymerase variants does not terminate prematurely, and higher molecular-mass G bands are detected. Another property of these Taq polymerase variants is that the sequencing patterns produced by these enzymes are remarkably even in band-intensity and peak-height distribution, thus resulting in a significant improvement in the accuracy of DNA sequencing.     

Computational and experimental analysis of DNA shuffling

We describe a computational model of DNA shuffling based on the thermodynamics and kinetics of this process. The model independently tracks a representative ensemble of DNA molecules and records their states at every stage of a shuffling reaction. These data can subsequently be analyzed to yield information on any relevant metric, including reassembly efficiency, crossover number, type and distribution, and DNA sequence length distributions. The predictive ability of the model was validated by comparison to three independent sets of experimental data, and analysis of the simulation results led to several unique insights into the DNA shuffling process. We examine a tradeoff between crossover frequency and reassembly efficiency and illustrate the effects of experimental parameters on this relationship. Furthermore, we discuss conditions that promote the formation of useless "junk" DNA sequences or multimeric sequences containing multiple copies of the reassembled product. This model will therefore aid in the design of optimal shuffling reaction conditions.     

Coexpression of nuclear receptor partners increases their solubility and biological activities

The biological activities of the retinoids are mediated by two nuclear hormone receptors: the retinoic acid receptor (RAR) and the retinoid-X receptor (RXR). RXR (and its insect homologue ultraspiracle) is a common heterodimeric partner for many other nuclear receptors, including the insect ecdysone receptor. As part of a continuing analysis of nuclear receptor function, we noticed that, whereas RXR can be readily expressed in Escherichia coli to produce soluble protein, many of its heterodimeric partners cannot. For example, overexpression of RAR results mostly in inclusion bodies with the residual soluble component unable to interact with RXR or ligand efficiently. Similar results are seen with other RXR/ultraspiracle partners. To overcome these problems, we designed a novel double cistronic vector to coexpress RXR and its partner ligand-binding domains in the same bacterial cell. This resulted in a dramatic increase in production of soluble and apparently stable heterodimer. Hormone-binding studies using the purified RXR–RAR heterodimer reveal increased ligand-binding capacity of both components of 5- to 10-fold, resulting in virtually complete functionality. Based on these studies we find that bacterially expressed receptors can exist in one of three distinct states: insoluble, soluble but unable to bind ligand, or soluble with full ligand-binding capacity. These results suggest that coexpression may represent a general strategy for biophysical and structural analysis of receptor complexes.     

Human recombinant soluble guanylyl cyclase: expression, purification, and regulation

The alpha1- and beta1-subunits of human soluble guanylate cyclase (sGC) were coexpressed in the Sf9 cells/baculovirus system. In addition to the native enzyme, constructs with hexahistidine tag at the amino and carboxyl termini of each subunit were coexpressed. This permitted the rapid and efficient purification of active recombinant enzyme on a nickel-affinity column. The enzyme has one heme per heterodimer and was readily activated with the NO donor sodium nitroprusside or 3-(5'-hydroxymethyl-2'furyl)-1-benzyl-indazole (YC-1). Sodium nitroprusside and YC-1 treatment potentiated each other in combination and demonstrated a remarkable 2,200-fold stimulation of the human recombinant sGC. The effects were inhibited with 1H-(1,2, 4)oxadiazole(4,3-a)quinoxalin-1one (ODQ). The kinetics of the recombinant enzyme with respect to GTP was examined. The products of the reaction, cGMP and pyrophosphate, inhibited the enzyme. The extent of inhibition by cGMP depended on the activation state of the enzyme, whereas inhibition by pyrophosphate was not affected by the enzyme state. Both reaction products displayed independent binding and cooperativity with respect to enzyme inhibition. The expression of large quantities of active enzyme will facilitate structural characterization of the protein.     

Residues in three conserved regions of the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase are required for quaternary structure.

To explore the role of individual residues in the small subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase (EC 4.1.1.39), small subunits with single amino acid substitutions in three regions of relative sequence conservation were produced by directed mutagenesis of the rbcS gene from Anabaena 7120. These altered small subunits were cosynthesized with large subunits (from an expressed Anabaena rbcL gene) in Escherichia coli. Mutants were analyzed for effects on quaternary structure and catalytic activity. Changing Glu-13S (numbering used is that of the spinach enzyme) to Val, Trp-67S to Arg, Pro-73S to His, or Tyr-98S to Asn prevented accumulation of stable holoenzyme. Interpretation of these results using a model for the three-dimensional structure of the spinach enzyme based on x-ray crystallographic data suggests that our small subunit mutants containing substitutions at positions 13S and 67S probably do not assemble because of mispairing or nonpairing of charged residues on the interfacing surfaces of the large and small subunits. The failure of small subunits substituted at positions 73S or 98S to assemble correctly may result from disruption of intersubunit or intrasubunit hydrophobic pockets, respectively.     

Meet Me Halfway: When Genomics Meets Structural Bioinformatics

The DNA sequencing technology developed by Frederick Sanger in the 1970s established genomics as the basis of comparative genetics. The recent invention of next-generation sequencing (NGS) platform has added a new dimension to genome research by generating ultra-fast and high-throughput sequencing data in an unprecedented manner. The advent of NGS technology also provides the opportunity to study genetic diseases where sequence variants or mutations are sought to establish a causal relationship with disease phenotypes. However, it is not a trivial task to seek genetic variants responsible for genetic diseases and even harder for complex diseases such as diabetes and cancers. In such polygenic diseases, multiple genes and alleles, which can exist in healthy individuals, come together to contribute to common disease phenotypes in a complex manner. Hence, it is desirable to have an approach that integrates omics data with both knowledge of protein structure and function and an understanding of networks/pathways, i.e. functional genomics and systems biology; in this way, genotype-phenotype relationships can be better understood. In this review, we bring this 'bottom-up' approach alongside the current NGS-driven genetic study of genetic variations and disease aetiology. We describe experimental and computational techniques for assessing genetic variants and their deleterious effects on protein structure and function.     

Specificity variants in monoclonal antibodies reactive with peptide epitopes of the ring-infected erythrocyte surface antigen (RESA) of Plasmodium falciparum

It has been suggested that repeat sequence antigens of Plasmodium falciparum may serve the parasite in immune evasion by modifying the host antibody response and impairing the development of protective immunity. According to this proposal networks of cross-reactive, repeat sequence malarial antigens have the ability to stimulate a high proportion of all somatically mutated B cells with altered antibody specificity, and thus to hinder the normal process of antibody affinity maturation. To determine the rate at which immunoglobulin mutations produce new reactivities with repeat sequence antigens, hybridoma cell lines specific for the ring-infected erythrocyte surface antigen (RESA) were examined for the incidence of specificity variants that arose naturally or as a result of treatment with the chemical mutagen ethylmethane sulphonate (EMS). From one of the cell lines variants were readily isolated having reactivity towards a very closely related repeat sequence epitope within the same RESA antigen. However, the other hybridoma/antigen combinations revealed no variants. In general, mutations giving rise to antibodies with altered specificity for related repetitive antigens were not readily induced and only limited support of the hypothesis was obtained.