Purification,cofactor analysis,and site-directed mutagenesis of Synechococcus ferredoxin-nitrate reductase

The narB gene of the cyanobacterium Synechococcus sp. strain PCC 7942 encodes an assimilatory nitrate reductase that uses photosynthetically reduced ferredoxin as the physiological electron donor. This gene was expressed in Escherichia coli and electrophoretically pure preparations of the enzyme were obtained using affinity chromatography with either reduced-ferredoxin or NarB antibodies. The electronic absorption spectrum of the oxidized enzyme showed a shoulder at around 320 nm and a broad absorption band between 350 and 500 nm. These features are indicative of the presence of an iron-sulfur centre(s) and accordingly metal analysis showed ca. 3 atoms of Fe per molecule of protein that could represent a [3Fe-4S] cluster. Further analysis indicated the presence of 1 atom of Mo and 2 molecules of ribonucleotide-conjugated molybdopterin per molecule of protein. This, together with the requirement of a mobA gene for production of an active enzyme, strongly suggests the presence of Mo in the form of the bis-MGD (bis-molybdopterin guanine dinucleotide) cofactor in Synechococcusnitrate reductase. A model for the coordination of the Mo atom to the enzyme is proposed. Four conserved Cys residues were replaced by site-directed mutagenesis. The effects of these changes on the enzyme activity and electronic absorption spectra support the participation of those residues in iron-sulfur cluster coordination. This revised version was published online in June 2006 with corrections to the Cover Date.     

Probing electrostatic channeling in protozoal bifunctional thymidylate synthase-dihydrofolate reductase using site-directed mutagenesis

In this study we used site-directed mutagenesis to test the hypothesis that substrate channeling in the bifunctional thymidylate synthase-dihydrofolate reductase enzyme from Leishmania major occurs via electrostatic interactions between the negatively charged dihydrofolate produced at thymidylate synthase and a series of lysine and arginine residues on the surface of the protein. Accordingly, 12 charge reversal or charge neutralization mutants were made, with up to 6 putative channel residues changed at once. The mutants were assessed for impaired channeling using two criteria: a lag in product formation at dihydrofolate reductase and an increase in dihydrofolate accumulation. Surprisingly, none of the mutations produced changes consistent with impaired channeling, so our findings do not support the electrostatic channeling hypothesis. Burst experiments confirmed that the mutants also did not interfere with intermediate formation at thymidylate synthase. One mutant, K282E/R283E, was found to be thymidylate synthase-dead because of an impaired ability to form the covalent enzyme-methylene tetrahydrofolate-deoxyuridate complex prerequisite for chemical catalysis.     

In vivo site-directed mutagenesis using oligonucleotides

Functional characterization of the genes of higher eukaryotes has been aided by their expression in model organisms and by analyzing site-specific changes in homologous genes in model systems such as the yeast Saccharomyces cerevisiae. Modifying sequences in yeast or other organisms such that no heterologous material is retained requires in vitro mutagenesis together with subcloning. PCR-based procedures that do not involve cloning are inefficient or require multistep reactions that increase the risk of additional mutations. An alternative approach, demonstrated in yeast, relies on transformation with an oligonucleotide, but the method is restricted to the generation of mutants with a selectable phenotype. Oligonucleotides, when combined with gap repair, have also been used to modify plasmids in yeast; however, this approach is limited by restriction-site availability. We have developed a mutagenesis approach in yeast based on transformation by unpurified oligonucleotides that allows the rapid creation of site-specific DNA mutations in vivo. A two-step, cloning-free process, referred to as delitto perfetto, generates products having only the desired mutation, such as a single or multiple base change, an insertion, a small or a large deletion, or even random mutations. The system provides for multiple rounds of mutation in a window up to 200 base pairs. The process is RAD52 dependent, is not constrained by the distribution of naturally occurring restriction sites, and requires minimal DNA sequencing. Because yeast is commonly used for random and selective cloning of genomic DNA from higher eukaryotes such as yeast artificial chromosomes, the delitto perfetto strategy also provides an efficient way to create precise changes in mammalian or other DNA sequences.     

Characterization of ferredoxin:thioredoxin reductase modified by site-directed mutagenesis

Ferredoxin:thioredoxin reductase (FTR) is a key regulatory enzyme of oxygenic photosynthetic cells involved in the reductive regulation of important target enzymes. It catalyzes the two-electron reduction of the disulfide of thioredoxins with electrons from ferredoxin involving a 4Fe-4S cluster and an adjacent active-site disulfide. We replaced Cys-57, Cys-87, and His-86 in the active site of Synechocystis FTR by site-directed mutagenesis and studied the properties of the mutated proteins. Mutation of either of the active-site cysteines yields inactive enzymes, which have different spectral properties, indicating a reduced Fe-S cluster when the inaccessible Cys-87 is replaced and an oxidized cluster when the accessible Cys-57 is replaced. The oxidized cluster in the latter mutant can be reversibly reduced with dithionite showing that it is functional. The C57S mutant is a very stable protein, whereas the C87A mutant is more labile because of the missing interaction with the cluster. The replacement of His-86 greatly reduces its catalytic activity supporting the proposal that His-86 increases the nucleophilicity of the neighboring cysteine. Ferredoxin forms non-covalent complexes with wild type (WT) and mutant FTRs, which are stable except with the C87A mutant. WT and mutant FTRs form stable covalent heteroduplexes with active-site modified thioredoxins. In particular, heteroduplexes formed with WT FTR represent interesting one-electron-reduced reaction intermediates, which can be split by reduction of the Fe-S cluster. Heteroduplexes form non-covalent complexes with ferredoxin demonstrating the ability of FTR to simultaneously dock thioredoxin and ferredoxin, which is in accord with the proposed reaction mechanism and the structural analyses.     

Intact protein analysis for site-directed mutagenesis overexpression products: plasmid-encoded R67 dihydrofolate reductase

Mass spectrometry is currently the method of choice for the analysis of recombinant protein expression products. By combining proteolytic digestion with peptide mapping and tandem mass spectrometry techniques, verification of site-directed mutagenesis products can be obtained. The proteolytic digestion step converts a purified recombinant protein into a mixture that must be reseparated, thus greatly increasing the analysis time associated with the confirmation of site-directed mutagenesis products. Ion/ion reaction chemistry combined with quadrupole ion trap mass spectrometry provides a fast and efficient way to analyze intact proteins for the correct site-directed mutagenesis products, without heavy reliance on the proteolytic digestion step. Analysis of a series of protein variants (I68M, I68Q, Y69F, and Q67Y) from plasmid-encoded R67 dihydrofolate reductase using ion/ion reaction chemistry confirmed the presence of the correct site-directed mutagenesis products. For the I68M mutant, ion/ion separations detected the presence of extensive degradation from the N-terminal end of the protein. In the case of the Q67Y mutant, a mixture of Q67Y and Q67C species was detected by employing tandem mass spectrometry combined with ion/ion reactions. The ion/ion reaction technique was also performed on a partially purified lysate of the Q67Y/C mixture and successfully screened for the presence of both components in a complex mixture. The ion/ion reaction approach achieved the same results as the proteolytic-digestion-based methodology in a much shorter analysis time.     

利用定点突变法研究精氨酸脱亚胺酶活性的影响机制

精氨酸脱亚胺酶(ADI)是一种针对精氨酸缺陷型癌症(如:肝癌、黑素瘤)的新药,目前处于临床三期试验。文中通过定点突变技术分析了精氨酸脱亚胺酶的特定氨基酸位点对酶活力的影响机制。针对已报道的关键氨基酸残基A128、H404、I410,采用QuikChange法进行定点突变,获得ADI突变株M1(A128T)、M2(H404R)、M3(I410L)和M4(A128T/H404R)。将突变株在大肠杆菌BL21(DE3)中进行重组表达,并对纯化获得的突变蛋白进行酶学性质研究。结果表明,突变位点A128T和H404R对ADI最适pH的提高,生理中性(pH 7.4)条件下的酶活力和稳定性的提高,以及Km值的降低均具有显著的作用。研究结果为阐明ADI的酶活力影响机制和蛋白质的理性改造提供了一定的依据。     

A putative glutathione-binding site in T4 glutaredoxin investigated by site-directed mutagenesis

A glutathione monomer has been docked into the active site cleft of T4 glutaredoxin (previously called T4 thioredoxin) using molecular graphics. The central part of the cleft is formed by the side chain of Tyr-16 on one side and the residues Thr-64, Met-65, and Pro-66 on the other. The entire glutathione molecule fits well into the cleft. A cis-peptide bond between the residues Met-65 and Pro-66 allows glutathione to bind in an anti-parallel fashion to residues 64-66. Hydrogen bonds can be formed between Met-65 and the glutathione cysteine. This binding positions the glutathione sulfur atom ideally for reaction with the glutaredoxin disulfide. In the model, glutathione can form a hydrogen bond to the hydroxyl group of Tyr-16. Charged interactions at opposite ends of the binding cleft are provided by His-12 and Asp-80. The negatively charged alpha-carboxyl group of glutathione may interact with a positive helix dipole of the protein. Fifteen mutant T4 glutaredoxins have been produced and assayed for glutathione binding by determining thioltransferase activity. Mutant proteins with substitutions in the sides of the cleft (Tyr-16, Pro-66) exhibited the most marked decreases in thioltransferase activity. Mutation of His-12 to a serine decreases the catalytic efficiency whereas substitution of Asp-80 by serine increases the catalytic efficiency. A double mutant, D80S;H12S, has much less affinity for glutathione than either single mutant. Substitution of Cys-14 produces an inactive protein, whereas C17S retains some thioltransferase activity.     

Active site analysis of fructosyl amine oxidase using homology modeling and site-directed mutagenesis

A three-dimensional structural model of fructosyl amine oxidase from the marine yeast Pichia N1-1 was generated using the crystal structure of monomeric sarcosine oxidase from Bacillus sp. B-0618 as template. The putative active site region was investigated by site-directed mutagenesis, identifying several amino acid residues likely playing important roles in the enzyme reaction. Asn354 was identified as a residue that plays an important role in substrate recognition and that can be substituted in order to change substrate specificity while maintaining high catalytic activity. While the Asn354Ala substitution had no effect on the V _max K _m⁻¹ value for fructosyl valine, the V _max K _m⁻¹ value for fructosyl-^ε N-lysine was decreased 3-fold, thus resulting in a 3-fold improvement in specificity for fructosyl valine over fructosyl-^ε N-lysine.     

A site-directed mutagenesis analysis of tNOX functional domains

Constitutive NADH oxidase proteins of the mammalian cell surface exhibit two different activities, oxidation of hydroquinones (or NADH) and protein disulfide-thiol interchange which alternate to yield oscillatory patterns with period lengths of 24 min. A drug-responsive tNOX (tumor-associated NADH oxidase) has a period length of about 22 min. The tNOX cDNA has been cloned and expressed. These two proteins are representative of cycling oxidase proteins of the plant and animal cell surface. In this report, we describe a series of eight amino acid replacements in tNOX which, when expressed in Escherichia coli, were analyzed for enzymatic activity, drug response and period length. Replacement sites selected include six cysteines that lie within the processed plasma membrane (34 kDa) form of the protein, and amino acids located in putative drug and adenine nucleotide (NADH) binding domains. The latter, plus two of the cysteine replacements, resulted in a loss of enzymatic activity. The recombinant tNOX with the modified drug binding site retained activity but the activity was no longer drug-responsive. The four remaining cysteine replacements were of interest in that both activity and drug response were retained but the period length for both NADH oxidation and protein disulfide-thiol interchange was increased from 22 min to 36 or 42 min. The findings confirm the correctness of the drug and adenine nucleotide binding motifs within the tNOX protein and imply a potential critical role of cysteine residues in determining the period length.     

Sulfide-induced sulfide-quinone reductase activity in thylakoids of Oscillatoria limnetica

Sulfide-dependent partial electron-transport reactions were studied in thylakoids isolated from cells of the cyanobacterium Oscillatoria limnetica, which had been induced to perform sulfide-driven anoxygenic photosynthesis. It was found that these thylakoids have the capacity to catalyze electron transfer, from sulfide to externally added quinones, in the dark. Assay conditions were developed to measure the reaction either as quinone-dependent sulfide oxidation (colorimetrically) or as sulfide-dependent quinone reduction (by UV dual-wavelength spectrophotometry). The main features of this reaction are as follows. (i) It is exclusively catalyzed by thylakoids of sulfide-induced cells. Noninduced thylakoids lack this reaction. (ii) Plastoquinone-1 or -2 are equally good substrates. Ubiquinone-1 and duroquinone yield somewhat slower rates. (iii) The apparent Km for plastoquinone-1 was 32 microM and for sulfide about 4 microM. Maximal rates (at 25 degrees C) were about 75 mumol of quinone reduced per mg of chlorophyll.h. (iv) The reaction was not affected by extensive washes of the membranes. (v) Unlike sulfide-dependent NADP photoreduction activity of these thylakoids, which is sensitive to all the specific inhibitors of the cytochrome b6f complex, the new dark reaction exhibited differential sensitivity to these inhibitors. 2-n-Nonyl-4-hydroxyquinoline-N-oxide was the most potent inhibitor of both light and dark reactions, working at submicromolar concentrations. 5-n-Undecyl-6-hydroxy-4,7-dioxobenzothiazole also inhibited the two reactions to a similar extent, but at 10 times higher concentrations than 2-n-nonyl-4-hydroxyquinoline-N-oxide. 2,5-Dibromo-3-methyl-6-isopropyl-p-benzoquinone, 2-iodo-6-isopropyl-3-methyl-2',4,4'-trinitrodiphenyl ether, and stigmatellin had no effect on the dark reaction at concentrations sufficient to fully inhibit the light reaction from sulfide. We propose that the sulfide-induced factor which enables the use of sulfide as the electron donor for anoxygenic photosynthesis in Oscillatria limnetica is a membrane-bound sulfide-quinone reductase. Its site of interaction is suggested to be either the cytochrome b6 (at the Qc quinone binding site or the bH site) or the plastoquinone pool. The analogy to other anoxygenic photosynthetic systems is discussed.     

Efficient site-directed saturation mutagenesis using degenerate oligonucleotides.

We describe a reliable protocol for constructing single-site saturation mutagenesis libraries consisting of all 20 naturally occurring amino acids at a specific site within a protein. Such libraries are useful for structure-function studies and directed evolution. This protocol extends the utility of Stratagene's QuikChange Site-Directed Mutagenesis Kit, which is primarily recommended for single amino acid substitutions. Two complementary primers are synthesized, containing a degenerate mixture of the four bases at the three positions of the selected codon. These primers are added to starting plasmid template and thermal cycled to produce mutant DNA molecules, which are subsequently transformed into competent bacteria. The protocol does not require purification of mutagenic oligonucleotides or PCR products. This reduces both the cost and turnaround time in high-throughput directed evolution applications. We have utilized this protocol to generate over 200 site-saturation libraries in a DNA polymerase, with a success rate of greater than 95%.     

A novel strictly NADPH-dependent Pichia stipitis xylose reductase constructed by site-directed mutagenesis

Xylose reductase (XR) and xylitol dehydrogenase (XDH) are the key enzymes for xylose fermentation and have been widely used for construction of a recombinant xylose fermenting yeast. The effective recycling of cofactors between XR and XDH has been thought to be important to achieve effective xylose fermentation. Efforts to alter the coenzyme specificity of XR and HDX by site-directed mutagenesis have been widely made for improvement of efficiency of xylose fermentation. We previously succeeded by protein engineering to improve ethanol production by reversing XDH dependency from NAD⁺ to NADP⁺. In this study, we applied protein engineering to construct a novel strictly NADPH-dependent XR from Pichia stipitis by site-directed mutagenesis, in order to recycle NADPH between XR and XDH effectively. One double mutant, E223A/S271A showing strict NADPH dependency with 106% activity of wild-type was generated. A second double mutant, E223D/S271A, showed a 1.27-fold increased activity compared to the wild-type XR with NADPH and almost negligible activity with NADH.     

Functional analysis of ISC1 by site-directed mutagenesis

We previously reported that the yeast Saccharomyces cerevisiae ISC1 gene (Yer019w), which has homology to the bacterial sphingomyelinase gene, encodes inositol phosphosphingolipids-phospholipase C, Isc1p [Sawai, H., Okamoto, Y., Luberto, C., Mao, C., Bielawska, A., Domae, M., and Hannun, Y. A. (2000) J. Biol. Chem. 275, 39793-39798]. The present study was conducted to determine specific domains in Isc1p required for catalysis. Several amino acid residues are conserved from bacterial sphingomyelinase to mammalian sphingomyelinase and are also found in ISC1. Individual mutation of the conserved E100, N233, and H334 resulted in complete loss of Isc1p activity, suggesting an essential role in catalysis for these amino acid residues. Isc1p also contains a domain (from G162 to S169) with homology to P-loop domains, found in nucleotide-binding proteins. In addition, two amino acid residues from this domain, D163 and K168, are conserved from bacterial to mammalian sphingomyelinases in this "P-loop-like domain". G162, D163, G167, K168, and S169 were replaced individually with alanine using site-directed mutagenesis. D163A and K168A lost activity completely. Mutations in the other three positions rendered enzyme versions with much reduced but detectable activity. The V(max) values for G162A, G167A, and S169A were reduced, compared with wild type, but the K(m) values for G162A, G167A, and S169A were similar to that of wild type, indicating that the substrate binding efficiency was not greatly altered in these mutants and that the P-loop-like domain of ISC1 might be essential in catalysis of Isc1p. Furthermore, the Mg(2+) K(a) constants for G162A, G167, and S169A were higher than that for wild type, suggesting that this P-loop-like domain may be involved in Mg(2+) binding. Although cell lysates from yeast cells overexpressing all mutants similarly bound to phosphatidylserine (PS), an anionic lipid activator of Isc1p, G162A and G167A required 13.3 mol % PS to achieve maximum activity compared to 6.7 mol % for the wild-type enzyme, suggesting that PS might play a role in optimal catalytic efficiency of Isc1p via this P-loop-like domain. This study provides novel insight into a new domain found in Isc1p and related enzymes.     

Cyanobacterial sulfide-quinone reductase: cloning and heterologous expression

The gene encoding sulfide-quinone reductase (SQR; E.C.1.8.5.'), the enzyme catalyzing the first step of anoxygenic photosynthesis in the filamentous cyanobacterium Oscillatoria limnetica, was cloned by use of amino acid sequences of tryptic peptides as well as sequences conserved in the Rhodobacter capsulatus SQR and in an open reading frame found in the genome of Aquifex aeolicus. SQR activity was also detected in the unicellular cyanobacterium Aphanothece halophytica following sulfide induction, with a V(max) of 180 micromol of plastoquinone-1 (PQ-1) reduced/mg of chlorophyll/h and apparent K(m) values of 20 and 40 microM for sulfide and quinone, respectively. Based on the conserved sequences, the gene encoding A. halophytica SQR was also cloned. The SQR polypeptides deduced from the two cyanobacterial genes consist of 436 amino acids for O. limnetica SQR and 437 amino acids for A. halophytica SQR and show 58% identity and 74% similarity. The calculated molecular mass is about 48 kDa for both proteins; the theoretical isoelectric points are 7.7 and 5.6 and the net charges at a neutral pH are 0 and -14 for O. limnetica SQR and A. halophytica SQR, respectively. A search of databases showed SQR homologs in the genomes of the cyanobacterium Anabaena PCC7120 as well as the chemolithotrophic bacteria Shewanella putrefaciens and Thiobacillus ferrooxidans. All SQR enzymes contain characteristic flavin adenine dinucleotide binding fingerprints. The cyanobacterial proteins were expressed in Escherichia coli under the control of the T7 promoter. Membranes isolated from E. coli cells expressing A. halophytica SQR performed sulfide-dependent PQ-1 reduction that was sensitive to the quinone analog inhibitor 2n-nonyl-4-hydroxyquinoline-N-oxide. The wide distribution of SQR genes emphasizes the important role of SQR in the sulfur cycle in nature.     

Efficient site-directed in vitro mutagenesis using phagemid vectors

Several methods have been developed that enhance the efficiency of in vitro, site-directed mutagenesis. Kunkel (8,9) has developed a method which uses a strong selection for the mutated strand and, hence, is highly efficient, but yet simple and rapid. This method originally used M13 phage as the vector. In this paper, we describe a refinement of this method using phagemid vectors, which combine the advantages of plasmids (such as high copy number and stability of cloned DNA) with the single-stranded DNA generating capability of M13 phage. We demonstrate that high efficiency of mutant production can be obtained with these vectors. We also analyzed by sequencing 11 mutated clones and found no second-site mutations, suggesting that alterations other than the site-directed mutation rarely occur in our system.     

IL-1beta epitope mapping using site-directed mutagenesis and hydrogen-deuterium exchange mass spectrometry analysis

Hu007, a humanized IgG1 monoclonal antibody, binds and neutralizes human, cynomolgus, and rabbit IL-1beta but only weakly binds to mouse and rat IL-1beta. Biacore experiments demonstrated that Hu007 and the type-I IL-1 receptor competed for binding to IL-1beta. Increasing salt concentrations decrease the association rate with only moderate effects on the dissociation rate, suggesting that long-range electrostatics are critical for formation of the initial complex. To understand the ligand-binding specificity of Hu007, we have mapped the critical residues involved in the recognition of IL-1beta. Selected residues in cynomolgus IL-1beta were mutated to the corresponding residues in mouse IL-1beta, and the effects of the changes on binding were evaluated by surface plasmon resonance measurements using Biacore. Specifically, substitution of F150S decreased binding affinity by 100-fold, suggesting the importance of hydrophobic interactions in stabilizing the antibody/antigen complex. Substitution of three amino acids near the N- and C-terminal regions of cIL-1beta with those found in mouse IL-1beta (V3I/S5Q/F150S) decreased the binding affinity of Hu007 to IL-1beta by about 1000-fold. Conversely, mutating the corresponding residues in mouse IL-1beta to the human sequence resulted in an increase in binding affinity of about 1000-fold. Hydrogen-deuterium exchange/mass spectrometry analysis confirmed that these regions of IL-1beta were protected from exchange because of antibody binding. The results from this study demonstrate that Hu007 binds to a region located in the open end of the beta-barrel structure of IL-1beta and blocks binding of IL-1beta to its receptor.