Molecular cloning of an alpha-glucosidase-like gene from Penicillium minioluteum and structure prediction of its gene product

The dexC cDNA, which is expressed in dextran-containing medium by the filamentous fungus Penicillium minioluteum, was cloned and sequence characterized. The cDNA sequence comprises 1859 bp plus a poly (A) tail, coding for a predicted protein of 597 amino acids. The genomic counterpart was isolated by PCR, finding three introns in its sequence. The dexC gene was located by Southern blot in the same 9-kb fragment that the previously isolated dextranase-encoding gene (dexA). Sequence analysis revealed that the deduced DexC protein belongs to glycosyl hydrolase family 13, showing a high sequence identity (58%) with Aspergillus parasiticus alpha-1,6-glucosidase. In addition, the high sequence identity (51%) between DexC protein and oligo-1,6-glucosidase of Bacillus cereus, with three-dimensional (3D) structure determined, leads us to proposed a 3D model for the structural core of DexC protein.     

Dextranase production from Penicillium funiculosum

Twenty-eight Penicillium cultures were screened for dextranase activity. Dextranase yield of about 2000 DU/ml was obtained with Penicillium funiculosum SH-5. Maximum dextranase concentration was attained only when cell mass decreased. The kinetics of the dextranase production was correlated with the cell mass by a two-parameter model. The optimum pH and temperature for dextranase were 5.0-5.5 and 55°C, respectively. Crude dextranase preparation was inhibitory to insoluble glucan formation by streptococcus mutans 6715 in vitro.     

Cloning and sequencing of a dextranase-encoding cDNA from Penicillium minioluteum

Abstract A cDNA from Penicillium minioluteum HI-4 encoding a dextranase (1,6-α-glucan hydrolase, EC 3.2.1.11) was isolated and characterized. cDNA clones corresponding to genes expressed in dextran-induced cultures were identified by differential hybridization. Southern hybridization and restriction mapping analysis of selected clones revealed four different groups of cDNAs. The dextranase cDNA was identified after expressing a cDNA fragment from each of the isolated groups of cDNA clones in the Escherichia coli T7 system. The expression of a 2 kb cDNA fragment in E. coli led to the production of a 67 kDa protein which was recognized by an anti-dextranase polyclonal antibody. The cDNA contains 2109 bp plus a poly(A) tail, coding for a protein of 608 amino acids, including 20 N-terminal amino acid residues which might correspond to a signal peptide. There was 29% sequence identity between the P. minioluteum dextranase and the dextranase from Arthrobacter sp. CB-8.     

Dextranase (alpha-1,6 glucan-6-glucanohydrolase) from Penicillium minioluteum expressed in Pichia pastoris: two host cells with minor differences in N-glycosylation

Differences in glycosylation between the natural alpha-1,6 glucan-6-glucanohydrolase from Penicillium minioluteum and the heterologous protein expressed in the yeast Pichia pastoris were analyzed. Glycosylation profiling was carried out using fluorophore-assisted carbohydrate electrophoresis and amine absorption high-performance liquid chromatography (NH(2)-HPLC) in combination with matrix-assisted laser desorption-time of flight-mass spectrometry. Both microorganisms produce only oligomannosidic type structures, but the oligosaccharide population differs in both enzymes. The native enzyme has mainly short oligosaccharide chains ranging from Man(5)GlcNAc(2) to Man(9)GlcNAc(2), of which Man(8)GlcNAc(2) was the most represented oligosaccharide. The oligosaccharides linked to the protein produced in P. pastoris range from Man(7)GlcNAc(2) up to Man(14)GlcNAc(2), with Man(8)GlcNAc(2) and Man(9)GlcNAc(2) being the most abundant structures. In both enzymes the first glycosylation site (Asn(5)) is always glycosylated. However, Asn(537) and Asn(540) are only partially glycosylated in an alternate manner.     

Isolation of a Pure Dextranase from Penicillium funiculosum

A dextranase, produced by Penicillium funiculosum, was purified 1,000-fold to yield the enzyme which was demonstrated by gel electrophoresis and electrofocusing to be a homogeneous protein. The purification method included acetone partition, ammonium sulfate fractionation, gel filtration, iron defecation and precipitation, and diethylaminoethyl-cellulose chromatography. The pure enzyme was also obtained by preparative gel electrophoresis. Gel-permeation chromatography indicates a molecular weight of 41,000. An isoelectric pH of 4.6 was established by electrofocusing. A 1-mg amount of the enzyme hydrolyzes a dextran substrate to yield 27,000 isomaltose reducing units in 2 hr.     

Anti-inflammatory spiroaxane and drimane sesquiterpenoids from Talaromyces minioluteus (Penicillium minioluteum)

A new spiroaxane sesquiterpenoid talaminoid A (1) and two drimane sesquiterpenoid talaminoids B and C (2 and 3), together with four known compounds (4–7), were isolated from the solid culture broth of fungus Talaromyces minioluteum. The structures were determined by extensive 1D and 2D NMR and HRESIMS spectroscopic data analyses, and the absolute configuration of these new compounds were undoubtedly confirmed by X-ray crystal diffrations. Compound 1 is a rare spiroaxane sesquiterpenoid and the absolute configuration of spiroaxane sesquiterpenoid was determined for the first time. Compound 2 is the first drimane-type sesquiterpenoid containing both amino acid residue and butanediol group. Compounds 1, 4, and 5 showed significant suppressive effect on the production of NO on LPS induced BV-2 cells, with IC₅₀ values ranging from 4.97 to 7.81 μM. In addition, 1, 4, and 5 exhibited significant anti-inflammatory activities against the production of TNF-α and IL-6. Further immunofluorescence experiments revealed the mechanism of action to be inhibitory the NF- κB-activated pathway.     

Amine free crystal structure: the crystal structure of d(CGCGCG)2 and methylamine complex crystal

We succeeded in the crystallization of d(CGCGCG)2 and methylamine Complex. The crystal was clear and of sufficient size to collect the X-ray crystallographic data up to 1.0 A resolution using synchrotron radiation. As a result of X-ray crystallographic analysis of 2Fo-Fc map was much clear and easily traced. It is the first time monoamine co-crystallizes with d(CGCGCG)2. However, methylamine was not found from the complex crystal of d(CGCGCG)2 and methylamine. Five Mg ions were found around d(CGCGCG)2 molecules. These Mg ions neutralized the anion of 10 values of the phosphate group of DNA with five Mg2+. DNA stabilized only by a metallic ion and there is no example of analyzing the X-ray crystal structure like this. Mg ion stabilizes the conformation of Z-DNA. To use monoamine for crystallization of DNA, we found that we can get only d(CGCGCG)2 and Mg cation crystal. Only Mg cation can stabilize the conformation of Z-DNA. The method of using the monoamine for the crystallization of DNA can be applied to the crystallization of DNA of long chain of length in the future like this.     

刺五加内生青霉鲨烯合酶基因的克隆与序列分析

【目的】克隆刺五加内生青霉Penicillium minioluteum P116-1a的鲨烯合酶(Squalene synthase,SS)基因。【方法】采用cDNA 5末端快速扩增(Rapid Amplification ofcDNA 5 Ends,5 RACE)技术扩增P.minioluteum P116-1a SS基因的全长cDNA序列和DNA序列;运用生物信息学方法对该基因进行分析,预测其编码蛋白的结构与功能;并通过RT-PCR法和SDS-PAGE法检测SS的表达情况。【结果】P.minioluteum P116-1a的SS基因含有4个外显子和3个内含子,开放阅读框长1 416 bp,编码471个氨基酸,预测蛋白含67.73%的α螺旋,5.31%的延伸链,2.97%的β折叠,23.99%的无规则卷曲,含有鲨烯合酶和八氢番茄红素合成酶的特异性识别区域,定位于内质网膜。与P.marneffei和Talaromyces stipitatus中SS蛋白的氨基酸同源性达90%以上。不同温度下SS的表达情况不同。【结论】首次在刺五加内生青霉P.minioluteum P116-1a中克隆到SS基因,为进一步研究P.minioluteum P116-1a提高刺五加皂苷含量的机制奠定基础。     

3-methyladenine-DNA glycosylase II: the crystal structure of an AlkA-hypoxanthine complex suggests the possibility of product inhibition

Escherichia coli (E. coli) protein 3-methyladenine-DNA glycosylase II (AlkA) functions primarily by removing alkylation damage from duplex and single stranded DNA. A crystal structure of AlkA was refined to 2.0 A resolution. This structure in turn was used to refine an AlkA-hypoxanthine (substrate) complex structure to 2.4 A resolution. The complex structure shows hypoxanthine located in AlkA's active site stacked between residues W218 and Y239. The structural analysis of the AlkA and AlkA-hypoxanthine structures indicate that free hypoxanthine binding in the active site may inhibit glycosylase activity.     

Enantio convergent biotransformation of O,O-dimethyl-4-oxoazetidin-2-ylphosphonate using fungal cells of Penicillium minioluteum and purified enzymes

This report presents the bioconversion of O,O-dimethyl-4-oxoazetidin-2-ylphosphonate 1 performed in two ways: with the enzymatic system of P. minioluteum and with the application of purified enzymes: penicillinase and two proteases of different origin. Recorded NMR spectra allowed confirming the reaction progress and also postulating possible mechanism of conversion. The path of bioconversion was defined as enantio convergent process for both modes of applied biocatalysts. This means that kinetically driven resolution of racemic mixture of the substrate leads to the one enantiomer of the product. The bioconversion started from ester bond hydrolysis (equally in both enantiomers) with the conversion degree from 30% (whole-cell) to 35% (isolated enzymes) and with the production of optically pure monoester (compound 2; 100% of e.e). For whole-cell bioprocess it was the initiative step for the enantioselective amide bond hydrolysis, what resulted in synthesis of desired product 3-amino-3-phosphonopropanoic acid 4. However, the most effective enzymatic hydrolysis of ester bond performed with penicillinase from Enterobacter cloacae led only to the monoester product 2.     

Synthesis, characterization and crystal structure of manganese (IV) complex derived from salicylic acid

The binuclear manganese (IV) [Mn₂(Hsal)₄(OH)₄] (H₂sal = salicylic acid) complex has been obtained from a complex reaction mixture in methanol consisting of Mn(II)(OAc)₂ · 4H₂O, GS ( a reagent obtained by refluxing glycine and salicylaldehyde in 1:1 molar ratio in methanol), monosodium salicylate and pyridine. The compound contains a distorted octahedral MnO₆ coordination unit of potential importance to high oxidation state manganese bimolecules.     

Salinity stress resistance offered by endophytic fungal interaction between Penicillium minioluteum LHL09 and glycine max. L

Endophytic fungi are little known for their role in gibberellins (GAs) synthesis and abiotic stress resistance in crop plants. We isolated 10 endophytes from the roots of field-grown soybean and screened their culture filtrates (CF) on the GAs biosynthesis mutant rice line - Waito-C. CF bioassay showed that endophyte GMH-1B significantly promoted the growth of Waito-C compared with controls. GMH-1B was identified as Penicillium minioluteum LHL09 on the basis of ITS regions rDNA sequence homology and phylogenetic analyses. GC/MS-SIM analysis of CF of P. minioluteum revealed the presence of bioactive GA(4) and GA(7). In endophyte-soybean plant interaction, P. minioluteum association significantly promoted growth characteristics (shoot length, shoot fresh and dry biomasses, chlorophyll content, and leaf area) and nitrogen assimilation, with and without sodium chloride (NaCl)-induced salinity (70 and 140 mM) stress, as compared with control. Field-emission scanning electron microcopy showed active colonization of endophyte with host plants before and after stress treatments. In response to salinity stress, low endogenous abscisic acid and high salicylic acid accumulation in endophyte-associated plants elucidated the stress mitigation by P. minioluteum. The endophytic fungal symbiosis of P. minioluteum also increased the daidzein and genistein contents in the soybean as compared with control plants, under salt stress. Thus, P. minioluteum ameliorated the adverse effects of abiotic salinity stress and rescued soybean plant growth by influencing biosynthesis of the plant's hormones and flavonoids.     

The first lanthanide carboxylate complex constructed from hydroxyl bridging bimetallic units: Hydrothermal synthesis, crystal structure and luminescent properties

One novel terbium complex [Tb₂(Dinic)₂(phen)₂(OH)₂(H₂O)₂]_n · 3nH₂O (1) has been synthesized by hydrothermal method, where Dinic = 3,4-pyridinedicarboxylate and phen = 1,10-phenanthroline. X-ray crystallography reveals that complex 1 possesses a one-dimensional chain structure constructed from OH⁻ bridging binuclear units, and hydrogen bonding between chains result in a 3D network. The fluorescent properties of complex 1 were investigated in the crystal state.     

The crystal structure of polyhydroxybutyrate depolymerase from Penicillium funiculosum provides insights into the recognition and degradation of biopolyesters

Polyhydroxybutyrate is a microbial polyester that can be produced from renewable resources, and is degraded by the enzyme polyhydroxybutyrate depolymerase. The crystal structures of polyhydroxybutyrate depolymerase from Penicillium funiculosum and its S39 A mutant complexed with the methyl ester of a trimer substrate of (R)-3-hydroxybutyrate have been determined at resolutions of 1.71 A and 1.66 A, respectively. The enzyme is comprised of a single domain, which represents a circularly permuted variant of the alpha/beta hydrolase fold. The catalytic residues Ser39, Asp121, and His155 are located at topologically conserved positions. The main chain amide groups of Ser40 and Cys250 form an oxyanion hole. A crevice is formed on the surface of the enzyme, to which a single polymer chain can be bound by predominantly hydrophobic interactions with several hydrophobic residues. The structure of the S39A mutant-trimeric substrate complex reveals that Trp307 is responsible for the recognition of the ester group adjacent to the scissile group. It is also revealed that the substrate-binding site includes at least three, and possibly four, subsites for binding monomer units of polyester substrates. Thirteen hydrophobic residues, which are exposed to solvent, are aligned around the mouth of the crevice, forming a putative adsorption site for the polymer surface. These residues may contribute to the sufficient binding affinity of the enzyme for PHB granules without a distinct substrate-binding domain.     

Beauvericin and divalent cations: crystal structure of the barium complex.

The molecular structure of the 2:2 complex of the cyclohexadepsipeptide antibiotic beauvericin with barium picrate has been determined by X-ray crystallography. The structure serves to confirm previous observations on the bimolecular behavior of beauvericin and of the ions transported by beauvercin. The intimate involvement of the anions in the coordination of the barium also explains observations that the cation specificity of beauvericin in membrane transport depends on the species of anions present.     

Putting two and two together: crystal structure of the FGF-receptor complex

The recently determined crystal structure of an FGF-receptor complex reveals a surprising architecture and a novel mode of receptor dimerization. The structure also elucidates the role of heparan sulfate proteoglycans in receptor activation, showing significant differences from previously proposed models.     

Implications for the ubiquitination reaction of the anaphase-promoting complex from the crystal structure of the Doc1/Apc10 subunit

The anaphase-promoting complex (APC) is a multi-subunit E3 protein ubiquitin ligase that is responsible for the metaphase to anaphase transition and the exit from mitosis. One of the subunits of the APC that is required for its ubiquitination activity is Doc1/Apc10, a protein composed of a Doc1 homology domain that has been identified in a number of diverse putative E3 ubiquitin ligases. Here, we present the crystal structure of Saccharomyces cerevisiae Doc1/Apc10 at 2.2A resolution. The Doc1 homology domain forms a beta-sandwich structure that is related in architecture to the galactose-binding domain of galactose oxidase, the coagulation factor C2 domain and a domain of XRCC1. Residues that are invariant amongst Doc1/Apc10 sequences, including a temperature-sensitive mitotic arrest mutant, map to a beta-sheet region of the molecule, whose counterpart in galactose oxidase, the coagulation factor C2 domains and XRCC1, mediate bio-molecular interactions. This finding suggests the identification of the functionally important and conserved region of Doc1/Apc10 and, since invariant residues of Doc1/Apc10 colocalise with conserved residues of other Doc1 homology domains, we propose that the Doc1 homology domains perform common ubiquitination functions in the APC and other E3 ubiquitin ligases.     

Synthesis, crystal structure and electroluminescent properties of a Schiff base zinc complex

A new complex of zinc with a Schiff base, zinc(N,N′-bis(salicylidene)-3, 6-dioxa-1, 8-diaminooctane monohydrate) (ZnBSO · H₂O), was synthesized and characterized by means of elemental analyses, IR spectra and DTA-TG. Its structure was determined by X-ray single crystal analysis. It was demonstrated that the zinc atom is coordinated by the two oxygen atoms in phenolate and two nitrogen atoms in imine of the ligand in a slightly distorted tetrahedral geometry, while the two oxygen atoms from the oxa-alkyl chain are not coordinated to Zn(II) atom. The energy levels of the HOMO, LUMO and the electrochemical band gap were determined by cyclic voltammeter. The electroluminescent devices with the complex as the emitter showed bright blue emission with a peak at 450 nm, which is same as the fluorescence of the complex in both solution and solid states.