Improvement of Cd2+ uptake ability of SmtA protein by Lys/Cys mutation; experimental and theoretical studies

The improved Cd²⁺ surface affinity characteristics of a mutated cyanobacterial metallothionein SmtA (K45C) were investigated via experimental and theoretical methods. Molecular dynamics simulations were carried out using a model of Cd²⁺ and other ions enclosed in a fully hydrated simulation box with the wild-type or mutated SmtA protein. The theoretical results suggested that mutated SmtA was more powerful in absorption of Cd²⁺ than the wild-type protein. Then, the mutated smtA gene (from Synechococcus PCC 7942) was synthesized by simplified gene synthesis method and expressed on isopropyl-beta-d-thiogalactopyranoside induction. The protein expression was investigated by SDS-PAGE and verified by Western blotting. Finally, cadmium uptake ratio of mutant protein toward wild type was analyzed by atomic absorption. This study is the first example of cytoplasmic expression of a mutant protein. Experimental results also verified that the mutation intensifies uptake of Cd²⁺ ions.     

Improvement of the enzymatic activity of the hyperthermophilic cellulase from <Emphasis Type="Italic">Pyrococcus horikoshii</Emphasis>

A hyperthermophilic β-1,4 endoglucanase (EGPh) from the hyperthermophilic archaeon Pyrococcus horikoshii exhibits a strong hydrolyzing activity toward crystalline cellulose. The characteristic features of EGPh are: (1) it appears to have disulfide bonds, which is rare among anaerobic hyperthermophilic archaeon proteins, and (2) it lacks a carbohydrate-binding domain, which is necessary for effective hydrolysis of cellulose. We first examined the relationship between the disulfide bonds and the catalytic activity by analyzing various cysteine mutations. The activities of the mutated enzymes toward carboxy methyl cellulose (CMC) increased without any loss in thermostability. Second, we prepared a fusion enzyme so that the thermostable chitin-binding domain of chitinase from P. furiosus was joined to the C-terminus of EGPh and its variants. These fusion enzymes showed stronger activities than did the wild-type EGPh toward both CMC and crystalline cellulose (Avicel).     

Transport of haemin across the cytoplasmic membrane through a haemin-specific periplasmic binding-protein-dependent transport system in Yersinia enterocolitica

The Yersinia enterocolitica O:8 periplasmic binding-protein-dependent transport (PBT) system for haemin was cloned and characterized. It consisted of four proteins: the periplasmic haemin-binding protein HemT, the haemin permease protein HemU, the ATP-binding hydrophilic protein HemV and the putative haemin-degrading protein HemS. Y. enterocolitica strains mutated in hemU or hemV genes were unable to use haemin as an iron source whereas those mutated in the hemT gene were able to use haemin as an iron source. As Escherichia coli strains expressing only the haemin outer membrane receptor protein HemR from Y. enterocolitica were capable of using haemin as an iron source the existence of an E. coli K-12 haemin-specific PBT system is postulated. The first gene in the Y. enterocolitica haemin-specific PBT system encoded a protein, HemS, which is probably involved in the degradation of haemin in the cytoplasm. The presence of the hemS gene was necessary to prevent haemin toxicity in E. coli strains that accumulate large amounts of haemin in the cytoplasm. We propose a model of haemin utilization in Y. enterocolitica in which HemT, HemU and HemV proteins transport haemin into the cytoplasm where it is degraded by HemS thereby liberating the iron.     

Cysteine mutants of herpes simplex virus type 1 glycoprotein D exhibit temperature-sensitive properties in structure and function.

We previously constructed seven mutations in the gene for glycoprotein D (gD) of herpes simplex virus type 1 in which the codon for one of the cysteine residues was replaced by a serine codon. Each of the mutant genes was cloned into a eucaryotic expression vector, and the proteins were transiently expressed in mammalian cells. We found that alteration of any of the first six cysteine residues had profound effects on protein conformation and oligosaccharide processing. In this report, we show that five of the mutant proteins exhibit temperature-sensitive differences in such properties as aggregation, antigenic conformation, oligosaccharide processing, and transport to the cell surface. Using a complementation assay, we have now assessed the ability of the mutant proteins to function in virus infection. This assay tests the ability of the mutant proteins expressed from transfected plasmids to rescue production of infectious virions of a gD-minus virus, F-gD beta, in Vero cells. Two mutant proteins, Cys-2 (Cys-106 to Ser) and Cys-4 (Cys-127 to Ser), were able to complement F-gD beta at 31.5 degrees C but not at 37 degrees C. The rescued viruses, designated F-gD beta(Cys-2) and F-gD beta(Cys-4), were neutralized as efficiently as wild-type virus by anti-gD monoclonal antibodies, indicating that gD was present in the virion envelope in a functional form. Both F-gD beta(Cys-2) and F-gD beta(Cys-4) functioned normally in a penetration assay. However, the infectivity of these viruses was markedly reduced compared with that of the wild type when they were preincubated at temperatures above 37 degrees C. The results suggest that mutations involving Cys-106 or Cys-127 in gD-1 confer a temperature-sensitive phenotype on herpes simplex virus. These and other properties of the cysteine-to-serine mutants allowed us to predict a disulfide bonding pattern for gD.     

Identification of a key functional region in harpins from <Emphasis Type="Italic">Xanthomonas</Emphasis> that suppresses protein aggregation and mediates harpin expression in <Emphasis Type="Italic">E. coli</Emphasis>

In the current study, we identified a key functional region in harpins from Xanthomonas that suppressed protein aggregation and mediated its expression in E. coli. Our data suggested that the presence of two common features in harpins [Wei et al. (1992) Science 257:85-88], namely, high glycine content and lack of cysteine residues, were not sufficient for Xanthomonas to elicit hypersensitive response (HR) activity or heat stability. Additionally, bioinformatic analyses revealed that the secondary structure of a conserved N-terminal region consisting of 12 highly hydrophilic amino acids (QGISEKQLDQLL) was α-helical. Following site-directed mutagenesis deletion of this region, the three mutated harpin proteins, in cultures induced at 37°C, failed to elicit a HR in tobacco leaves. However, at 24°C, two mutated harpins retained the ability to elicit HR, albeit with lower expression levels than that noted with the wild-type. SDS-PAGE and Western blot data suggested the HpaG mutant protein was found almost entirely in the inclusion body. These data demonstrated that these conserved amino acid residues played a critical role in protein aggregation and inclusion body formation in harpins from Xanthomonas.     

Site-Directed Mutagenesis of <Emphasis Type="Italic">Aeromonas hydrophila</Emphasis> Enoyl Coenzyme A Hydratase Enhancing 3-Hydroxyhexanoate Fractions of Poly(3-hydroxybutyrate-<Emphasis Type="Italic">co</Emphasis>-3-hydroxyhexanoate)

The aim of this study is to enhance 3-hydroxyhexanoate (3HHx) fractions of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate), abbreviated as PHBHHx, through site-directed mutagenesis of Aeromonas hydrophila enoyl Coenzyme A hydratase (PhaJ_Ah). Two amino acids (Leu-65 and Val-130) were selected as a substitutional site based on the structural information of PhaJ_Ah. The purified proteins from the wild-type enzyme and mutants were used to determine hydratase activities. Hydratase activities of four single-mutation enzymes were similar to those of the wild type PhaJ_Ah, while hydratase activities of two double-mutation enzymes were much lower. In addition, the mutated phaJ _Ah was individually co-transformed into E. coli BL21 (DE3) together with pFH21, which carried the PHA synthase (PhaC_Ah) gene from A. hydrophila. The recombinant E. coli harboring plasmid pETJ1 (L65A), pETJ2 (L65V) or plasmid pETJ3 (V130A) synthesized the enhanced 3HHx fractions of PHBHHx from dodecanoate, indicating that Leu-65 and Val-130 of PhaJ_Ah play an important role in determining the acyl chain length substrate specificity. The mutated PhaJ_Ah (L65A, L65V, or V130A) provided higher 3HHx precursors for PHA synthase, resulting in the enhanced 3HHx fractions of PHBHHx. It is possible to change the acyl chain length substrate specificity of PhaJ through site-directed mutagenesis and produce PHBHHx with a wider range of alterable monomer composition.     

Characterization of Active-Site Residues of the NIa Protease from Tobacco Vein Mottling Virus

Nuclear inclusion a (NIa) protease of tobacco vein mottling virus is responsible for the processing of the viral polyprotein into functional proteins. In order to identify the active-site residues of the TVMV NIa protease, the putative active-site residues, His-46, Asp-81 and Cys-151, were mutated individually to generate H46R, H46A, D81E, D81N, C151S, and C151A, and their mutational effects on the proteolytic activities were examined. Proteolytic activity was completely abolished by the mutations of H46R, H46A, D81N, and C151A, suggesting that the three residues are crucial for catalysis. The mutation of D81E decreased k_cat marginally by about 4.7-fold and increased K_m by about 8-fold, suggesting that the aspartic acid at position 81 is important for substrate binding but can be substituted by glutamate without any significant decrease in catalysis. The replacement of Cys-151 by Ser to mimic the catalytic triad of chymotrypsin-like serine protease resulted in the drastic decrease in k_cat by about 1,260-fold. This result might be due to the difference of the active-site geometry between the NIa protease and chymotrypsin. The protease exhibited a bell-shaped pH-dependent profile with a maximum activity approximately at pH 8.3 and with the abrupt changes at the respective pK_m values of approximately 6.6 and 9.2, implying the involvement of a histidine residue in catalysis. Taken together, these results demonstrate that the three residues, His-46, Asp-81, and Cys-151, play a crucial role in catalysis of the TVMV NIa protease.     

Structural requirements for dimerization,glycosylation, secretion,and biological function of VPF/VEGF

Vascular permeability factor (VPF) also known as vascular endothelial growth factor (VEGF), is a dimeric protein that affects endothelial cell (EC) and vascular functions including enhancement of microvascular permeability and stimulation of EC growth. To investigate the structural features of VPF/VEGF necessary for efficient dimerization, secretion, and biological activities, we employed site-directed mutagenesis with a Cos-1 cell expression system. Several cysteine residues essential for VPF dimerization were identified by mutation analysis of the Cys-25, Cys-56, and Cys-67 residues. Mutant VPF isoforms lacking either of these cysteines were secreted as monomers and were completely inactive in both vascular permeability and endothelial cell mitotic assays. VPF Cys-145 mutant protein was efficiently secreted as a glycosyaated, dimeric polypeptide, but had a reduction in biological activities. The site of N-linked glycosylation was directly identified as Asn-74, which, when mutated produced an inefficiently secreted dimeric protein without post-translational glycosylation, yet maintained full vascular permeability activity. Finally, we found that one VPF mutant isoform Cys-101 was not secreted and this mutant functioned as a dominant-negative suppressor of wild-type VPF secretion as demonstrated by co-expression assays in Cos-1 cells.     

Cloning,production and expression of the bacteriocin enterocin A produced by <Emphasis Type="Italic">Enterococcus faecium</Emphasis> PLBC21 in <Emphasis Type="Italic">Lactococcus lactis</Emphasis>

Two genes, ctc and ctc2, responsible for surface layer (S-layer) protein synthesis in Bacillus thuringiensis CTC, were mutated and resulted in B. thuringiensis Tr5. To synthesize and express the N-acyl-homoserine lactonase (AHL-lactonase) in the extracellular space of B. thuringiensis, the aiiA _4Q7 gene (an AHL-lactonase gene from B. thuringiensis 4Q7), which confers the ability to inhibit plant soft rot disease in B. thuringiensis 4Q7, was fused with the upstream sequence of the ctc gene, which in turn is essential for S-layer protein secretion and anchoring on the cell surface. The resulting fusion gene, slh-aiiA, was expressed in B. thuringiensis Tr5 to avoid competition for the extracellular space with the native S-layer protein. Our results indicate that B. thuringiensis Tr5 containing the fusion gene slh-aiiA displayed high extracellular AHL-degrading activity. When compared with wild-type B. thuringiensis strains, the ability of the constructed strain to inhibit soft rot disease caused by Erwinia carotovora SCG1 was markedly increased. These findings provide evidence for a significant advance in our ability to inhibit soft rot disease caused by E. carotovora.     

Novel mutations in <Emphasis Type="Italic">katG</Emphasis> gene of a clinical isolate of isoniazid-resistant <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis>

Most of isoniazid-resistant Mycobacterium tuberculosis evolved due to mutation in the katG gene encoding catalase-peroxidase. A set of new mutations, namely T1310C, G1388T, G1481A, T1553C, and A1660G, which correspond to amino acid substitutions of L437P, R463L, G494D, I518T, and K554E, in the katG gene of the L10 clinical isolate M. tuberculosis was identified. The wild-type and mutant KatG proteins were expressed in Escherichia coli BL21(DE3) as a protein of 80 kDa based on sodium dodecyl sulphate-polyacrylamide gel electrophoresis analysis. The mutant KatG protein exhibited catalase and peroxidase activities of 4.6% and 24.8% toward its wild type, respectively, and retained 19.4% isoniazid oxidation activity. The structure modelling study revealed that these C-terminal mutations might have induced formation of a new turn, perturbing the active site environment and also generated new intramolecular interactions, which could be unfavourable for the enzyme activities.     

Aspartate aminotransferase is involved in cold adaptation in psychrophilic Pseudomonas syringae

CSM2, a cold-sensitive mutant of psychrophilic Pseudomonas syringae, grows like wild-type cells when cultured at 22 and 28°C; but at 4°C, the growth is retarded. In CSM2, AAT (coding for aspartate aminotransferase) is identified as the mutated gene. The expression of AAT in Pseudomonas syringae was transiently enhanced when cells were shifted from 22 to 4°C indicating that AAT is cold-inducible. Complementation of the mutated AAT transformed CSM2 from a cold-sensitive phenotype to a cold-resistant phenotype like the wild-type cells, thus providing evidence for the first time that AAT is required for low-temperature growth.     

Ribosomal Proteins S27E, P2, and L37A from Marine Invertebrates

Single complementary DNAs encoding sequences for 40S ribosomal proteins related to S27E from the American lobster Homarus americanus and mussel Mytilus galloprovincialis were characterized. Single genes for ribosomal proteins L37A and P2 from the gumboot chiton Cryptochiton stellerii are similarly described. The lobster S27E protein contains the highly conserved cysteine residues, suggesting its likely designation in the C₄ protein family containing zinc finger motifs. The lobster S27E protein also appears to have an intermediate gene copy number between lower and higher euckaryotes. Expression of the S27E protein in lobster hepatopancreas was slightly elevated during several postmolt and premolt stages. Chlorinated pesticide treatment significantly reduced S27E expression in hepatopancreas, indicating that this gene is responsive to endogenous and exogenous cues. Received March 6, 1998; accepted October 2, 1998.     

Regulation of Escherichia coli RelA requires oligomerization of the C-terminal domain

The E. coli RelA protein is a ribosome-dependent (p)ppGpp synthetase that is activated in response to amino acid starvation. RelA can be dissected both functionally and physically into two domains: The N-terminal domain (NTD) (amino acids [aa] 1 to 455) contains the catalytic domain of RelA, and the C-terminal domain (CTD) (aa 455 to 744) is involved in regulating RelA activity. We used mutational analysis to localize sites important for RelA activity and control in these two domains. We inserted two separate mutations into the NTD, which resulted in mutated RelA proteins that were impaired in their ability to synthesize (p)ppGpp. When we caused the CTD in relA⁺ cells to be overexpressed, (p)ppGpp accumulation during amino acid starvation was negatively affected. Mutational analysis showed that Cys-612, Asp-637, and Cys-638, found in a conserved amino acid sequence (aa 612 to 638), are essential for this negative effect of the CTD. When mutations corresponding to these residues were inserted into the full-length relA gene, the mutated RelA proteins were impaired in their regulation. In attempting to clarify the mechanism through which the CTD regulates RelA activity, we found no evidence for competition for ribosomal binding between the normal RelA and the overexpressed CTD. Results from CyaA complementation experiments of the bacterial two-hybrid system fusion plasmids (G. Karimova, J. Pidoux, A. Ullmann, and D. Ladant, Proc. Natl. Acad. Sci. USA 95:5752–5756, 1998) indicated that the CTD (aa 564 to 744) is involved in RelA-RelA interactions. Our findings support a model in which RelA activation is regulated by its oligomerization state.     

Peroxisome-targeted and tandem repeat multimer expressions of human antimicrobial peptide LL37 in Pichia pastoris

Although the human antimicrobial peptide LL37 has a broad spectrum of antimicrobial activities, it easily damages host cells following heterologous expressions. This study attempted two strategies to alleviate its damage to host cells when expressed in Pichia pastoris using the AOX1 promoter. Tandem repeat multimers of LL37 were first designed, and secretion expression strains GS115-9K-(DPLL37DP)_n (n?=?2, 4, 6 and 8) containing different copies of the LL37 gene were constructed. However, LL37 tandems still killed the cells after 96?hr of induction. Subsequently, peroxisome-targeted expression was performed by adding a peroxisomal targeting signal 1 (SKL) at the C-terminus of LL37. The LL37 expression strain GS115-3.5K-LL37-SKL showed no significant inhibition in the cells after induction. Antibacterial activity assays showed that the recombinant LL37 expressed in peroxisomes had good antimicrobial activities. Then, a strain GS115-3.5K-LL37-GFP-SKL producing LL37, green fluorescent protein, and SKL fusion proteins was constructed, and the fusion protein was confirmed to be targeting the peroxisomes. However, protein extraction analysis indicated that most of the fusion proteins were still located in the cell debris after cell disruption, and further studies are required to extract more proteins from the peroxisome membrane.     

Bacillus anthracis pXO1 virulence plasmid encodes a type 1 DNA topoisomerase

The virulence plasmid pXO1 is responsible for toxin production in Bacillus anthracis. A DNA fragment from pXO1 was isolated and was shown, by sequence analysis, to contain part of a type 1 DNA topoisomerase gene. Attempts to clone the entire wild-type gene, designated topX, in Escherichia coli, were unsuccessful. In order to obtain the complete gene, it was first insertionally inactivated and then cloned in the mutated form. The deduced amino acid sequence of Topo X1 shows similarities to that of the two E. coli type 1 DNA topoisomerases. The N-terminal two-thirds of the putative B. anthracis protein exhibits strongest sequence similarity to topoisomerase III, whereas the C-terminal portion contains cysteine residues that could form three zinc-binding domains, as they do in topoisomerase I. The suggested active-site tyrosine is conserved in all three proteins. The regulation of expression from the topX promoter is modified by addition of a gyrase inhibiting antibiotic. The Topo X1 protein is likely to be involved in the stability of pXO1.     

The FG loop of a heme-based gas sensor enzyme, Ec DOS, functions in heme binding, autoxidation and catalysis

Ec DOS is a heme-based gas sensor enzyme that catalyzes conversion from cyclic-di-GMP to linear-di-GMP in response to gas molecules, such as oxygen, CO and NO. Ec DOS contains an N-terminal heme-binding PAS domain and C-terminal phosphodiesterase domain. Based on crystal structures of the isolated heme-binding domain, it is suggested that the FG loop is involved in intra-molecular signal transduction to the catalytic domain. We generated nine full-length proteins mutated at ionic and non-ionic polar residues between positions 83 and 96 corresponding to the F-helix and FG loop, and examined the heme binding properties, autoxidation rates, and catalytic activities of mutant proteins. N84A and R85A mutant proteins displayed lower heme binding affinities, consistent with the finding that Asn84 interacts with propionate of protoporphyrin IX, and Arg85 with Asp40 on the heme proximal side. Autoxidation rates (0.058-0.54 min⁻¹) of R91A, S96A and K89A/R91A/E93A mutant proteins were significantly higher than that (0.0053 min⁻¹) of wild-type protein, suggesting that these residues in the FG loop form heme distal architecture conferring stability to the Fe(II)-O₂ complex. Catalytic activities of N84A and R85A mutant proteins with low heme affinity were significantly higher than those of wild-type protein in the absence of gas molecules. Accordingly, we propose that loss of heme binding enhances basal catalysis without the gas molecule, consistent with previous reports on heme inhibition of Ec DOS catalysis.     

A plant‐specific in vitro ubiquitination analysis system

Protein ubiquitination requires the concerted action of three enzymes: ubiquitin‐activating enzyme (E1), ubiquitin‐conjugating enzyme (E2) and ubiquitin ligase (E3). These ubiquitination enzymes belong to an abundant protein family that is encoded in all eukaryotic genomes. Describing their biochemical characteristics is an important part of their functional analysis. It has been recognized that various E2/E3 specificities exist, and that detection of E3 ubiquitination activity in vitro may depend on the recruitment of E2s. Here, we describe the development of an in vitro ubiquitination system based on proteins encoded by genes from Arabidopsis. It includes most varieties of Arabidopsis E2 proteins, which are tested with several RING‐finger type E3 ligases. This system permits determination of E3 activity in combination with most of the E2 sub‐groups that have been identified in the Arabidopsis genome. At the same time, E2/E3 specificities have also been explored. The components used in this system are all from plants, particularly Arabidopsis, making it very suitable for ubiquitination assays of plant proteins. Some E2 proteins that are not easily expressed in Escherichia coli were transiently expressed and purified from plants before use in ubiquitination assays. This system is also adaptable to proteins of species other than plants. In this system, we also analyzed two mutated forms of ubiquitin, K48R and K63R, to detect various types of ubiquitin conjugation.     

Heterologous expression of a <Emphasis Type="Italic">Ammopiptanthus mongolicus</Emphasis> late embryogenesis abundant protein gene (<Emphasis Type="Italic">AmLEA</Emphasis>) enhances <Emphasis Type="Italic">Escherichia coli</Emphasis> viability under cold and heat stress

A late embryogenesis abundant protein gene, AmLEA from Ammopiptanthus mongolicus, was introduced into Escherichia coli using the IMPACT™-TWIN system to analyze the possible function of AmLEA under heat and cold stresses. A fusion protein about 38 kD was expressed in E.coli cells harboring pTWIN-LEA after the induction of IPTG by SDS–PAGE analysis and the accumulation of the fusion protein peaked 3 h after IPTG addition when cultured at 37°C. Compared with control cells, the E. coli cells expressing AmLEA fusion protein showed improved chilling and heat resistence, illuminating the protein may play a protective role in cells under stress conditions. These results suggested the natively unstructured protein, similar to other members of LEA proteins, has high capacity for binding water and potential protective function against dehydration or action similar to the cold shock chaperones.     

MSMEG_4626 ribonuclease from <Emphasis Type="Italic">Mycobacterium smegmatis</Emphasis>

The MSMEG_4626 gene was cloned from Mycobacterium smegmatis MC2 155. It codes for a protein of 1,037 amino acids, identified as ribonuclease E by matching to the protein family HMM TIGR00757. The protein was expressed and purified. Although its calculated molecular weight is 112.7 kDa, it has an aberrant mobility in SDS-polyacrylamide gels, like other ribonuclease E enzymes (it migrates as a 180 kDa protein). The central part of the protein displays high similarity to the catalytic domains of other RNase E enzymes. Mass spectrometric analysis revealed the presence of the chaperonin GroEL, ribosomal proteins, a negative regulator of genetic competence and GTP pyrophosphokinase in the affinity-purified preparation. It is a very unstable protein; despite the use of protease inhibitors in addition to the full-length RNase E its proteolytic fragments were detected.