The involvement of coordinative interactions in the binding of dihydrolipoamide dehydrogenase to titanium dioxide—Localization of a putative binding site

Titanium (Ti) and its alloys are widely used in orthodontic and orthopedic implants by virtue to their high biocompatibility, mechanical strength, and high resistance to corrosion. Biointegration of the implants with the tissue requires strong interactions, which involve biological molecules, proteins in particular, with metal oxide surfaces. An exocellular high‐affinity titanium dioxide (TiO₂)–binding protein (TiBP), purified from Rhodococcus ruber , has been previously studied in our lab. This protein was shown to be homologous with the orthologous cytoplasmic rhodococcal dihydrolipoamide dehydrogenase (rhDLDH). We have found that rhDLDH and its human homolog (hDLDH) share the TiO₂‐binding capabilities with TiBP. Intrigued by the unique TiO₂‐binding properties of hDLDH, we anticipated that it may serve as a molecular bridge between Ti‐based medical structures and human tissues. The objective of the current study was to locate the region and the amino acids of the protein that mediate the protein‐TiO₂ surface interaction. We demonstrated the role of acidic amino acids in the nonelectrostatic enzyme/dioxide interactions at neutral pH. The observation that the interaction of DLDH with various metal oxides is independent of their isoelectric values strengthens this notion. DLDH does not lose its enzymatic activity upon binding to TiO₂, indicating that neither the enzyme undergoes major conformational changes nor the TiO₂ binding site is blocked. Docking predictions suggest that both rhDLDH and hDLDH bind TiO₂ through similar regions located far from the active site and the dimerization sites. The putative TiO₂‐binding regions of both the bacterial and human enzymes were found to contain a CHED (Cys, His, Glu, Asp) motif, which has been shown to participate in metal‐binding sites in proteins.     

赤红球菌二鸟苷酸环化酶基因的克隆、信息学分析和转录变化研究

本研究通过克隆赤红球菌SD3的二鸟苷酸环化酶(DGC)基因,并对该酶进行生物信息学分析,研究其在甲苯和苯酚胁迫下的转录水平变化,为进一步研究该酶和赤红球菌SD3有机溶剂耐受性之间的关系奠定基础。综合使用煮沸法和冻融法提取赤红球菌SD3的总DNA,利用PCR扩增和T载体克隆获得DGC基因序列;利用生物信息学手段对DGC的理化性质和结构特征进行分析;利用邻位相连法构建进化树;采用Discovery studio 3.5将DGC与底物GTP进行分子对接;采用qPCR方法分析DGC基因在甲苯和苯酚胁迫下的转录变化情况。克隆获得了赤红球菌SD3的DGC基因序列,在GenBank中的序列登录号为MH482549。该基因的开放阅读框长为1 332 bp,编码443个氨基酸,预测蛋白分子量为46.95 kD,是疏水性蛋白。无信号肽和二硫键,含有6段跨膜区,第181~316位氨基酸之间存在一个GGDEF保守结构域和c-di-GMP结合抑制位点(Ⅰ位点)。亚细胞定位分析表明,DGC属于质膜蛋白。DGC中超过50%的氨基酸参与形成α螺旋结构。进化树分析表明Rhodococcus ruber SD3和Rhodococcus aetherivorans的DGC聚为一簇,推测赤红球菌SD3的DGC也属于ClassⅢnucleotidyl cyclases家族,具有类似的分子功能。分子对接表明DGC和GTP分子通过疏水作用和氢键产生相互作用。qPCR结果表明在甲苯和苯酚胁迫下,赤红球菌SD3中DGC基因的转录分别上调了1.57倍和8.71倍。研究表明,赤红球菌SD3中的DGC催化GTP产生c-di-GMP,并且DGC基因的转录在有机溶剂胁迫下发生显著上调,这暗示c-di-GMP与赤红球菌SD3的有机溶剂耐受性可能相关。     

Purification and characterization of a 14-kilodalton protein that is bound to the surface of polyhydroxyalkanoic acid granules in Rhodococcus ruber.

The N-terminal amino acid sequence of the polyhydroxyalkanoic acid (PHA) granule-associated M(r)-15,500 protein of Rhodococcus ruber (the GA14 protein) was analyzed. The sequence revealed that the corresponding structural gene is represented by open reading frame 3, encoding a protein with a calculated M(r) of 14,175 which was recently localized downstream of the PHA synthase gene (U. Pieper and A. Steinbüchel, FEMS Microbiol. Lett. 96:73-80, 1992). A recombinant strain of Escherichia coli XL1-Blue carrying the hybrid plasmid (pSKXA10*) with open reading frame 3 overexpressed the GA14 protein. The GA14 protein was subsequently purified in a three-step procedure including chromatography on DEAE-Sephacel, phenyl-Sepharose CL-4B, and Superose 12. Determination of the molecular weight by gel filtration as well as electron microscopic studies indicates that a tetrameric structure of the recombinant, native GA14 protein is most likely. Immunoelectron microscopy demonstrated a localization of the GA14 protein at the periphery of PHA granules as well as close to the cell membrane in R. ruber. Investigations of PHA-leaky and PHA-negative mutants of R. ruber indicated that expression of the GA14 protein depended strongly on PHA synthesis.     

Molecular characterization of three 3-ketosteroid-Δ(1)-dehydrogenase isoenzymes of Rhodococcus ruber strain Chol-4

Rhodococcus ruber strain Chol-4 isolated from a sewage sludge sample is able to grow on minimal medium supplemented with steroids, showing a broad catabolic capacity. This paper reports the characterization of three different 3-ketosteroid-Δ(1)-dehydrogenases (KstDs) in the genome of R. ruber strain Chol-4. The genome of this strain does not contain any homologues of a 3-keto-5α-steroid-Δ(4)-dehydrogenase (Kst4d or TesI) that appears in the genomes of Rhodococcus erythropolis SQ1 or Comamonas testosteroni. Growth experiments with kstD2 mutants, either a kstD2 single mutant, kstD2 double mutants in combination with kstD1 or kstD3, or the triple kstD1,2,3 mutant, proved that KstD2 is involved in the transformation of 4-androstene-3,17-dione (AD) to 1,4-androstadiene-3,17-dione (ADD) and in the conversion of 9α-hydroxy-4-androstene-3,17-dione (9OHAD) to 9α-hydroxy-1,4-androstadiene-3,17-dione (9OHADD). kstD2,3 and kstD1,2,3 R. ruber mutants (both lacking KstD2 and KstD3) did not grow in minimal medium with cholesterol as the only carbon source, thus demonstrating the involvement of KstD2 and KstD3 in cholesterol degradation. In contrast, mutation of kstD1 does not alter the bacterial growth on the steroids tested in this study and therefore, the role of this protein still remains unclear. The absence of a functional KstD2 in R. ruber mutants provoked in all cases an accumulation of 9OHAD, as a branch product probably formed by the action of a 3-ketosteroid-9α-hydroxylase (KshAB) on the AD molecule. Therefore, KstD2 is a key enzyme in the AD catabolism pathway of R. ruber strain Chol-4 while KstD3 is involved in cholesterol catabolism.     

Dihydrolipoamide dehydrogenase moonlighting activity as a DNA chelating agent

Dihydrolipoamide dehydrogenase (DLDH) is a mitochondrial enzyme that comprises an essential component of the pyruvate dehydrogenase complex. Lines of evidence have shown that many dehydrogenases possess unrelated actions known as moonlightings in addition to their oxidoreductase activity. As part of these activities, we have demonstrated that DLDH binds TiO₂ as well as produces reactive oxygen species (ROS). This ROS production capability was harnessed for cancer therapy via integrin‐mediated drug‐delivery of RGD‐modified DLDH (DLDH^RGD), leading to apoptotic cell death. In these experiments, DLDH^RGD not only accumulated in the cytosol but also migrated to the cell nuclei, suggesting a potential DNA‐binding capability of this enzyme. To explore this interaction under cell‐free conditions, we have analyzed DLDH binding to phage lambda (λ) DNA by gel‐shift assays and analytic ultracentrifugation, showing complex formation between the two, which led to full coverage of the DNA molecule with DLDH molecules. DNA binding did not affect DLDH enzymatic activity, indicating that there are neither conformational changes nor active site hindering in DLDH upon DNA‐binding. A Docking algorithm for prediction of protein‐DNA complexes, Paradoc, identified a putative DNA binding site at the C‐terminus of DLDH. Our finding that TiO₂‐bound DLDH failed to form a complex with DNA suggests partial overlapping between the two sites. To conclude, DLDH binding to DNA presents a novel moonlight activity which may be used for DNA alkylating in cancer treatment.     

Identification and characterization of the Rhizobium sp. strain GIN611 glycoside oxidoreductase resulting in the deglycosylation of ginsenosides

Using enrichment culture, Rhizobium sp. strain GIN611 was isolated as having activity for deglycosylation of a ginsenoside, compound K (CK). The purified heterodimeric protein complex from Rhizobium sp. GIN611 consisted of two subunits with molecular masses of 63.5 kDa and 17.5 kDa. In the genome, the coding sequence for the small subunit was located right after the sequence for the large subunit, with one nucleotide overlapping. The large subunit showed CK oxidation activity, and the deglycosylation of compound K was performed via oxidation of ginsenoside glucose by glycoside oxidoreductase. Coexpression of the small subunit helped soluble expression of the large subunit in recombinant Escherichia coli. The purified large subunit also showed oxidation activity against other ginsenoside compounds, such as Rb1, Rb2, Rb3, Rc, F2, CK, Rh2, Re, F1, and the isoflavone daidzin, but at a much lower rate. When oxidized CK was extracted and incubated in phosphate buffer with or without enzyme, (S)-protopanaxadiol [PPD(S)] was detected in both cases, which suggests that deglycosylation of oxidized glucose is spontaneous.     

Preparative isolation of lipid inclusions from Rhodococcus opacus and Rhodococcus ruber and identification of granule-associated proteins

Triacylglycerol granules synthesized and accumulated by Rhodococcus opacus and Rhodococcus ruber were isolated by glycerol density gradient centrifugation. Whereas only one type of granule could be isolated from R. opacus, two types of granules with different specific densities were isolated from R. ruber. Both types of R. ruber granules showed a similar content of triacylglycerols and poly(3-hydroxybutyrate- co-3-hydroxyvalerate), but the protein profiles of both types were significantly different. The granules with the lower specific density were colorless; the granules with the higher specific density had a deep orange pigmentation. Solubilization studies revealed three different groups of granule-associated proteins: (1) unspecifically bound proteins, (2) relatively weakly associated proteins, and (3) proteins that resisted solubilization by treatment with 2 M NaCl, 2% (w/v) Triton X-114, 6 M guanidinium hydrochloride, up to 8% (w/v) SDS, and proteolytic digestion. The strong association of proteins of the last group suggested that these may play a specific role in the synthesis or mobilization of storage lipids or in the structure of the granules. The N-terminal amino acid sequences of the most tightly bound proteins were obtained. Proteins of low molecular weight with striking sequence similarity to the ribosomal protein L7 from various actinomycetes were always copurified with the granules.     

Identification, cloning and sequence analysis of the poly(3-hydroxyalkanoic acid) synthase gene of the Gram-positive bacterium Rhodococcus ruber

The first polyhydroxyalkanoic acid (PHA) synthase gene (phbCRr) of a Gram-positive bacterium was cloned from a genomic library of Rhodococcus ruber in the broad-host-range plasmid vector pRK404. The hybrid plasmid harboring phbCRr allowed the expression of polyhydroxybutyric acid (PHB) synthase activity and restored the ability of PHB synthesis in a PHB-negative mutant of Alcaligenes eutrophus. Nucleotide sequence analysis of phbCRr revealed an open reading frame of 1686 bp starting with the rare codon TTG and encoding a protein of relative molecular mass 61,371. The deduced amino acid sequence of phbCRr exhibited homologies to the primary structures of the PHA synthases of A. eutrophus and Pseudomonas oleovorans. Preparation of PHA granules by discontinuous density gradient centrifugation of crude cellular extracts revealed four major bands in an SDS polyacrylamide gel. A Mr 61,000 protein was identified as the PHA synthase of R. ruber by N-terminal amino acid sequence determination.     

对诺卡氏菌AS4.1186和AS4.1187保藏菌株的重新分类研究

重新对念球状诺卡氏菌 (Nocardianostocoides)AS4 1 1 86和鲑色诺卡氏菌桔橙变种(Nocardiasalmonicolorvar.aurantiaca)AS4 1 1 87进行的多相分类研究表明 ,菌株AS4 1 1 86与德克萨斯糖丝菌 (Saccharothrixtexasensis)NRRLB 1 6 1 3 4 T 关系密切 ,它们的 1 6SrDNA序列相似性为 99 3 % ,DNA同源性为 77 6 % ;菌株AS4 1 1 87与赤红球菌 (Rhodococcusruber)DSM 43 3 3 8T 之间有着密切的关系 ,其 1 6SrDNA序列相似性为 99 5 % ,DNA同源性为 82 9%。胞壁组分、枝菌酸、甲基萘醌、磷酸类脂和DNAG +Cmol%测定等化学分类结果支持了上述结论。在形态和生理生化特性上 ,菌株AS4 1 1 86与NRRLB 1 6 1 3 4 T,菌株AS4 1 1 87与DSM 43 3 3 8T 之间也表现出非常相似的性状。根据系统发育分析、DNA同源性值、化学分类、形态和生理生化特性等研究结果 ,我们对菌株AS4 1 1 86和…     

对胞必佳生产菌株的重新鉴定

主要是从形态学观察、菌株脂肪酸成分和16S rRNA基因全序列3个方面出发,重新对胞必佳生产菌株红色诺卡氏菌(Nocardia rubra)进行鉴定。结果表明,该菌株并非诺卡氏菌属中的红色诺卡氏菌,而属于红球菌属。16S rRNA序列相似性比较和系统进化树进一步说明,该菌株与Rhodococcus ruber(AY114117.1)的同源性最高,是1株红色红球菌。     

OlpB, a new outer layer protein of Clostridium thermocellum, and binding of its S-layer-like domains to components of the cell envelope.

Several proteins of Clostridium thermocellum possess a C-terminal triplicated sequence related to bacterial cell surface proteins. This sequence was named the SLH domain (for S-layer homology), and it was proposed that it might serve to anchor proteins to the cell surface (A. Lupas, H. Engelhardt, J. Peters, U. Santarius, S. Volker, and W. Baumeister, J. Bacteriol. 176:1224-1233, 1994). This hypothesis was investigated by using the SLH-containing protein ORF1p from C. thermocellum as a model. Subcellular fractionation, immunoblotting, and electron microscopy of immunocytochemically labeled cells indicated that ORF1p was located on the surface of C. thermocellum. To detect C. thermocellum components interacting with the SLH domains of ORF1p, a probe was constructed by grafting these domains on the C terminus of the MalE protein of Escherichia coli. The SLH domains conferred on the chimeric protein (MalE-ORF1p-C) the ability to bind noncovalently to the peptidoglycan of C. thermocellum. In addition, 125I-labeled MalE-ORF1p-C was shown to bind to SLH-bearing proteins transferred onto nitrocellulose, and to a 26- to 28-kDa component of the cell envelope. These results agree with the hypothesis that SLH domains contribute to the binding of exocellular proteins to the cell surface of bacteria. The gene carrying ORF1 and its product, ORF1p, are renamed olpB and OlpB (for outer layer protein B), respectively.     

Binding of the major phasin, PhaP1, from Ralstonia eutropha H16 to poly(3-hydroxybutyrate) granules

The surface of polyhydroxybutyrate (PHB) storage granules in bacteria is covered mainly by proteins referred to as phasins. The layer of phasins stabilizes the granules and prevents coalescence of separated granules in the cytoplasm and nonspecific binding of other proteins to the hydrophobic surfaces of the granules. Phasin PhaP1(Reu) is the major surface protein of PHB granules in Ralstonia eutropha H16 and occurs along with three homologues (PhaP2, PhaP3, and PhaP4) that have the capacity to bind to PHB granules but are present at minor levels. All four phasins lack a highly conserved domain but share homologous hydrophobic regions. To identify the region of PhaP1(Reu) which is responsible for the binding of the protein to the granules, N-terminal and C-terminal fusions of enhanced green fluorescent protein with PhaP1(Reu) or various regions of PhaP1(Reu) were generated by recombinant techniques. The fusions were localized in the cells of various recombinant strains by fluorescence microscopy, and their presence in different subcellular protein fractions was determined by immunodetection of blotted proteins. The fusions were also analyzed to determine their capacities to bind to isolated PHB granules in vitro. The results of these studies indicated that unlike the phasin of Rhodococcus ruber, there is no discrete binding motif; instead, several regions of PhaP1(Reu) contribute to the binding of this protein to the surface of the granules. The conclusions are supported by the results of a small-angle X-ray scattering analysis of purified PhaP1(Reu), which revealed that PhaP1(Reu) is a planar, triangular protein that occurs as trimer. This study provides new insights into the structure of the PHB granule surface, and the results should also have an impact on potential biotechnological applications of phasin fusion proteins and PHB granules in nanobiotechnology.     

A novel integrin specificity exemplified by binding of the alpha v beta 5 integrin to the basic domain of the HIV Tat protein and vitronectin

Several studies have addressed the interaction of the HIV Tat protein with the cell surface. Our analysis of the cell attachment-promoting activity of Tat and peptides derived from it revealed that the basic domain of Tat, not the arg-gly-asp (RGD) sequence, is required for cell attachment to Tat. Affinity chromatography with Tat peptides and immunoprecipitation with various anti-integrin antibodies suggest that the vitronectin-binding integrin, alpha v beta 5, is the cell surface protein that binds to the basic domain of Tat. The Tat basic domain contains the sequence RKKRRQRRR. A related sequence, KKQRFRHRNRKG, present in the heparin-binding domain of an alpha v beta 5 ligand, vitronectin, also bound alpha v beta 5 in affinity chromatography and, in combination with an RGD peptide, was an inhibitor of cell attachment to vitronectin. The alpha v beta 5 interaction with these peptides was not solely due to high content of basic amino acids in the ligand sequences; alpha v beta 5 did not bind substantially to peptides consisting entirely of arginine or lysine, whereas a beta 1 integrin did bind to these peptides. The interaction of alpha v beta 5 with Tat is atypical for integrins in that the binding to Tat is divalent cation independent, whereas the binding of the same integrin to an RGD- containing peptide or to vitronectin requires divalent cations. These data define an auxiliary integrin binding specificity for basic amino acid sequences. These basic domain binding sites may function synergistically with the binding sites that recognize RGD or equivalent sequences.     

Isolation of a gene encoding a glycosylated cytokinin oxidase from maize

The major cytokinin oxidase in immature maize kernels was purified to homogeneity. Selected tryptic peptides were used to design degenerate oligonucleotide primers for PCR isolation of a fragment of the oxidase gene. Hybridization of the PCR fragment to a maize genomic library allowed isolation of a full-length cytokinin oxidase gene (ckx1). The gene encodes a protein of approximately 57 kDa that possesses a signal peptide, eight consensus N-glycosylation sequences and a consensus FAD binding sequence. Expression of ckx1 in Pichia caused secretion of active glycosylated cytokinin oxidase that contains a substrate-reducible FAD. The gene displays sequence homology with a putative oxidoreductase from Arabidopsis thaliana and with the fas5 gene from Rhodococcus fascians.     

Primary structure of the CFA1 fimbrial protein from human enterotoxigenic Escherichia coli strains

The complete amino acid sequence of the structural protein that constitutes the subunit of the CFA1 fimbria has been elucidated. The protein was fragmented by cyanogen bromide cleavage, and by enzymatic cleavage with trypsin. Secondary cleavage of the resulting peptides was performed with chymotrypsin, Staphylococcus aureus protease, and thermolysin. Sequential Edman degradation was performed manually. The CFA1 protein comprises 147 amino acid residues, with a molecular weight of 15058.     

Peptides of the liver stage antigen-1 (LSA-1) of Plasmodium falciparum bind to human hepatocytes

Synthetic peptides from the liver stage antigen-1 (LSA-1) antigen sequence were used in HepG2 cell and erythrocyte binding assays to identify regions that could be involved in parasite invasion. LSA-1 protein peptides 20630 ((21)INGKIIKNSEKDEIIKSNLRY(40)), 20637 ((157)KEKLQGQQSDSEQERRAY(173)), 20638 ((174)KEKLQEQQSDLEQERLAY(190)) and 20639 (191KEKLQEQQSDLEQERRAY(207)) had high binding activity in HepG2 assays. Were located in immunogenic regions; peptide cell binding was saturable. Peptide 20630 bound specifically to 48kDa HepG2 membrane surface protein. LSA-1 peptides 20630 ((21)INGKIIKNSEKDEIIKSNLRY(40)) and 20633 ((81)DKELTMSNVKNVSQTNFKSLY(100)) showed specific erythrocyte binding activity and inhibited merozoite invasion of erythrocytes in vitro. A monkey serum prepared against LSA-1 20630 peptide analog (CGINGKNIKNAEKPMIIKSNLRGC) inhibited merozoite invasion in vitro. The data suggest LSA-1 "High Activity Binding Peptides" could play a possible role in hepatic cell invasion as well as merozoite invasion of erythrocytes.     

产顺式环氧琥珀酸水解酶的红球菌M1菌株的分离鉴定及其产酶条件优化

从土壤中筛选到一株产顺式环氧琥珀酸水解酶（ESH）的菌株,经生理生化、Biolog碳源利用试验和16S rDNA序列分析系统发育研究,菌株可能为赤红球菌(Rhodococcus ruber) M1。摇瓶试验确定了最佳碳源、氮源、顺式环氧琥珀酸二钠添加时间和添加量。正交优化试验的最佳培养基和培养时间为：葡萄糖12%,硫酸铵06%,酵母膏05%;顺式环氧琥珀酸二钠投加时间为30h,投加量为358%;菌体培养时间70h。摇瓶试验ESH酶活达750U/g湿细胞。目前该菌株已经应用于固定化细胞连续生产L(+)酒石酸。