Nature of the Bonds Holding Pectic Substances in Nitella Cell Walls

Experimental evidence has been presented that the non-ionic, acid-labile and base-stable bonds involving pectic carboxylate groups, both oriented and not-oriented, play an important role in holding pectic substances in the cell wall of Nitella. Participation of hydroxyl groups in the bonds has been confirmed by the vapor phase deuteration method. Irreversible acid-degradation of the cell wall ultrastructure associated with the carboxylate groups has also been demonstrated by acid-base titrimetry.     

非核糖体肽Skyllamycin B生物合成中I型硫酯酶底物杂泛性的初步研究

[背景] Skyllamycins是一类从链霉菌中发现的具有血小板生长因子抑制和生物膜抑制作用的非核糖体肽类,其环肽环合反应是由非核糖体肽合成酶中的硫酯酶功能域催化完成。[目的] 克隆和表达Skyllamycin非核糖体肽合成酶最后一个模块中的硫酯酶（Skyxy-TE）基因,合成Skyxy-TE底物类似物,通过体外催化实验表征Skyxy-TE的底物杂泛性。[方法] 采用Ligation Independent Cloning（LIC）方法,从一株含有Skyllamycin B生物合成基因簇的链霉菌Streptomyces sp.PKU-MA01239中克隆和表达skyxy-TE,通过镍离子柱亲和层析纯化Skyxy-TE。运用固相多肽合成法合成2个底物类似物1和2,进行Skyxy-TE的体外催化实验。[结果] 通过对Skyxy-TE的表达纯化,获得了纯度较好的可溶性蛋白;通过固相多肽合成,得到了能够模拟Skyllamycin B底物类似物的化合物1和2,硫酯酶蛋白体外催化化合物1和2得到了化合物3和4,化合物3和4通过核磁共振和高分辨质谱确认为环肽。[结论] Skyllamycin B生物合成中Skyxy-TE表现出一定的底物杂泛性,可以识别底物类似物催化环化反应,该研究为将来利用化学-酶联法制备更多环肽类似物提供了依据。     

桃果实絮败与果胶质变化和细胞壁结构的关系

“白凤”桃果实在０ ℃下贮藏２０ ｄ 以后出现肉质发绵和干化的絮败现象。冷藏第１０ 天在１８ ℃下加温３８ ｈ 能有效减缓絮败的发生。连续冷藏１０ ｄ 以后,原果胶含量开始增多, 水溶性果胶含量变化不大。中途加温的果实原果胶含量变化不多,但水溶性果胶却不断增加。絮败果的果胶质粘度明显高于正常果。果肉出汁率是测定果实絮败程度的理想指标。絮败果肉细胞壁的明显特征是： 伴随胞间层的分解和胞间隙的扩大,出现大量凝胶状物质的沉积,初生壁结构变化不明显, 也没有细胞壁次生加厚的迹象。     

An ensemble of theta class glutathione transferases with novel catalytic properties generated by stochastic recombination of fragments of two mammalian enzymes

The correlation between sequence diversity and enzymatic function was studied in a library of Theta class glutathione transferases (GSTs) obtained by stochastic recombination of fragments of cDNA encoding human GST T1-1 and rat GST T2-2. In all, 94 randomly picked clones were characterized with respect to sequence, expression level, and catalytic activity in the conjugation reactions between glutathione and six alternative electrophilic substrates. Out of these six different compounds, dichloromethane is a selective substrate for human GST T1-1, whereas 1-menaphthyl sulfate and 1-chloro-2,4-dinitrobenzene are substrates for rat GST T2-2. The other three substances serve as substrates for both enzymes. Through this broad characterization, we have identified enzyme variants that have acquired novel activity profiles that differ substantially from those of the original GSTs. In addition, the expression levels of many clones were improved in comparison to the parental enzyme. A library of mutants can thus display a distribution of properties from which highly divergent evolutionary pathways may emerge, resembling natural evolutionary processes. From the GST library, a clone was identified that, by the point mutation N49D in the rat GST T2-2 sequence, has a 1700% increased activity with 1-menaphthyl sulfate and a 60% decreased activity with 4-nitrophenethyl bromide. Through the N49D mutation, the ratio of these activities has thus been altered 40-fold. An extensive characterization of a population of stochastically mutated enzymes can accordingly be used to find variants with novel substrate-activity profiles and altered catalytic properties. Recursive recombination of selected sequences displaying optimized properties is a strategy for the engineering of proteins for medical and biochemical applications. Such sequential design is combinatorial protein chemistry based on remodeling of existing structural scaffolds and has similarities to evolutionary processes in nature.     

Studies on Substrate Specificity at PR/p3 Cleavage Site of HTLV-1 Protease

Substrate specificities for recognition at the PR/p3 site of HTLV-1 protease were clarified using small libraries of substrate peptides. Specificities at P₁ and P₁′ positions were examined by parallel synthesis/digestion of synthetic peptides covering the PR/p3 site (KGPPVILPIQA). Specificities at P₂ to P₄ positions were examined by split and mix syntheses of olefin-peptide libraries containing the substrate sequence (PPVILPIQ). The solid-phase Horner-Emmons reaction was successfully applied to syntheses of multi-component substrates for library preparation. From the digestion of substrate peptides by a chemically synthesized mutant of HTLV-1 protease (C2A HTLV-1 PR), it was found for the first time that the preference for Pro at the P₁′ position and for Ile at the P₂ position is unique for this enzyme. We dedicate this article to Prof. Bruce Merrifield for his great role and impact on solid-phase chemistry.     

Synthesis of dipeptides by suspension-to-suspension conversion via thermolysin catalysis: from analytical to preparative scale

When using proteases in direct reversal of their normal hydrolytic function, the equilibrium position is very important in limiting the attainable yield in equilibrium-controlled enzymic peptide synthesis. Analysis of the equilibrium position reveals a favourable shift towards the peptide product if starting materials are largely undissolved in the reaction medium and the product precipitates. This approach enabled us to obtain high peptide yields in thermolysin-catalysed reactions in high-density aqueous media with an equimolar supply of substrates. The easy scale-up (up to mol-scale) of this approach is demonstrated by two examples. Z-His-Phe- NH₂ and Z-Asp-Phe-OMe, precursors for cyclo-[-His-Phe-] and the low-calorie sweetener Aspartame, respectively, were synthesized in preparative yields of 84–88%. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

Purification and specificity of two alpha-glucosidase isoforms of the parasitic protist Trichomonas vaginalis

Two isoforms of alpha-glucosidase were purified from the parasitic protist Trichomonas vaginalis. Both consisted of 103 kDa subunits, but differed in pH optimum and substrate specificity. Isoform 1 had a pH optimum around 4.5 and negligible activity on glucose oligomers other than maltose, while isoform 2 with a pH optimum of 5.5 hydrolyzed also such substrates at considerable rates. Neither had activity on glycogen or starch. Isoform 1 had a specific activity for hydrolysis of maltose of 30 U/mg protein and isoform 2 101 U/mg protein. The Km values were 0.4 mM and 2.0 mM, respectively. Isoform 2 probably corresponds to the activity detected on the cell surface.     

Engineering the acceptor specificity of trehalose phosphorylase for the production of trehalose analogs

Trehalose (α‐D ‐glucopyranosyl‐(1,1)‐α‐D ‐glucopyranoside) is widely used in the food industry, thanks to its protective effect against freezing and dehydration. Analogs of trehalose have the additional benefit that they are not digested and thus do not contribute to our caloric intake. Such trehalose analogs can be produced with the enzyme trehalose phosphorylase, when it is applied in the reverse, synthetic mode. Despite the enzyme's broad acceptor specificity, its catalytic efficiency for alternative monosaccharides is much lower than for glucose. For galactose, this difference is shown here to be caused by a lower K_m whereas the k_cat for both substrates is equal. Consequently, increasing the affinity was attempted by enzyme engineering of the trehalose phosphorylase from Thermoanaerobacter brockii, using both semirational and random mutagenesis. While a semirational approach proved unsuccessful, high‐throughput screening of an error‐prone PCR library resulted in the discovery of three beneficial mutations that lowered K_m two‐ to three‐fold. In addition, it was found that mutation of these positions also leads to an improved catalytic efficiency for mannose and fructose, suggesting their involvement in acceptor promiscuity. Combining the beneficial mutations did not further improve the affinity, and even resulted in a decreased catalytic activity and thermostability. Therefore, enzyme variant R448S is proposed as new biocatalyst for the industrial production of lactotrehalose (α‐D ‐glucopyranosyl‐(1,1)‐α‐D ‐galactopyranoside). © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2012     

Altered substrate specificity of the gluco‐oligosaccharide oxidase from Acremonium strictum

A gluco‐oligosaccharide oxidase (GOOX) from Acremonium strictum type strain CBS 346.70 was cloned and expressed in Pichia pastoris. The recombinant protein, GOOX‐VN, contained fifteen amino acid substitutions compared with the previously reported A. strictum GOOX. These two enzymes share 97% sequence identity; however, only GOOX‐VN oxidized xylose, galactose, and N‐acetylglucosamine. Besides monosaccharides, GOOX‐VN oxidized xylo‐oligosaccharides, including xylobiose and xylotriose with similar catalytic efficiency as for cello‐oligosaccharides. Of three mutant enzymes that were created in GOOX‐VN to improve substrate specificity, Y300A and Y300N doubled k_cat values for monosaccharide and oligosaccharide substrates. With this novel substrate specificity, GOOX‐VN and its variants are particularly valuable for oxidative modification of cello‐ and xylo‐oligosaccharides. Biotechnol. Bioeng. 2011;108: 2261–2269. © 2011 Wiley Periodicals, Inc.     

Construction of cellobiose phosphorylase variants with broadened acceptor specificity towards anomerically substituted glucosides

The general application of glycoside phosphorylases such as cellobiose phosphorylase (CP) for glycoside synthesis is hindered by their relatively narrow substrate specificity. We have previously reported on the creation of Cellulomonas uda CP enzyme variants with either modified donor or acceptor specificity. Remarkably, in this study it was found that the donor mutant also displays broadened acceptor specificity towards several β‐glucosides. Triple mutants containing donor (T508I/N667A) as well as acceptor mutations (E649C or E649G) also display a broader acceptor specificity than any of the parent enzymes. Moreover, further broadening of the acceptor specificity has been achieved by site‐saturation mutagenesis of residues near the active site entrance. The best enzyme variant contains the additional N156D and N163D mutations and is active towards various alkyl β‐glucosides, methyl α‐glucoside and cellobiose. In comparison with the wild‐type C. uda CP enzyme, which cannot accept anomerically substituted glucosides at all, the obtained increase in substrate specificity is significant. The described CP enzyme variants should be useful for the synthesis of cellobiosides and other glycosides with prebiotic and pharmaceutical properties. Biotechnol. Bioeng. 2010;107: 413–420. © 2010 Wiley Periodicals, Inc.     

Structural basis for juvenile hormone biosynthesis by the juvenile hormone acid methyltransferase

Juvenile hormone (JH) acid methyltransferase (JHAMT) is a rate-limiting enzyme that converts JH acids or inactive precursors of JHs to active JHs at the final step of JH biosynthesis in insects and thus presents an excellent target for the development of insect growth regulators or insecticides. However, the three-dimensional properties and catalytic mechanism of this enzyme are not known. Herein, we report the crystal structure of the JHAMT apoenzyme, the three-dimensional holoprotein in binary complex with its cofactor S-adenosyl-l-homocysteine, and the ternary complex with S-adenosyl-l-homocysteine and its substrate methyl farnesoate. These structures reveal the ultrafine definition of the binding patterns for JHAMT with its substrate/cofactor. Comparative structural analyses led to novel findings concerning the structural specificity of the progressive conformational changes required for binding interactions that are induced in the presence of cofactor and substrate. Importantly, structural and biochemical analyses enabled identification of one strictly conserved catalytic Gln/His pair within JHAMTs required for catalysis and further provide a molecular basis for substrate recognition and the catalytic mechanism of JHAMTs. These findings lay the foundation for the mechanistic understanding of JH biosynthesis by JHAMTs and provide a rational framework for the discovery and development of specific JHAMT inhibitors as insect growth regulators or insecticides.     

Novel product specificity toward erlose and panose exhibited by multisite engineered mutants of amylosucrase

A computer‐aided engineering approach recently enabled to deeply reshape the active site of N. polysaccharea amylosucrase for recognition of non‐natural acceptor substrates. Libraries of variants were constructed and screened on sucrose allowing the identification of 17 mutants able to synthesize molecules from sole sucrose, which are not synthesized by the parental wild‐type enzyme. Three of the isolated mutants as well as the new products synthesized were characterized in details. Mutants contain between 7 and 11 mutations in the active site and the new molecules were identified as being a sucrose derivative, named erlose (α‐d ‐glucopyranosyl‐(1→4)‐α‐d ‐glucopyranosyl‐(1→2)‐β‐d ‐Fructose), and a new malto‐oligosaccharide named panose (α‐d ‐glucopyranosyl‐(1→6)‐α‐d ‐glucopyranosyl‐(1→4)‐α‐d ‐Glucose). These product specificities were never reported for none of the amylosucrases characterized to date, nor their engineered variants. Optimization of the production of these trisaccharides of potential interest as sweeteners or prebiotic molecules was carried out. Molecular modelling studies were also performed to shed some light on the molecular factors involved in the novel product specificities of these amylosucrase variants.     

蓝藻硫酯酶催化多肽环化适用性分析

硫酯酶(thioesterase, TE)具有区域定向性（regiospecific）、化学定向性（chemospecific）及立体定向性（stereospecific）的特点。这些特性决定了TE作为生物催化剂（biocatalysis）在工业生产中具有较高的应用价值和广阔的应用前景。McyC-TE (microcystin thioesterase, McyC TE)来自铜绿微囊藻（microcystis aeruginosa）NRPS/PKS生物合成基因簇。我们利用正交试验提高McyC TE表达量,得到稳定的诱导表达条件,并结合成熟的线性多肽化学合成法对其底物适用性做了进一步研究。得到的最佳诱导表达条件为：诱导时机2 h,诱导剂异丙基-β-D-硫代半乳糖苷（isopropyl-β-D-thiogalactopyranoside, IPTG）浓度0.75 mmol/L,诱导时间6 h,诱导转速210 r/min,诱导温度20 ℃,使TE的表达量由8.75 mg/L提高至22.15 mg/L,时间缩短了6.5 h。TE表达量的大幅度提升和表达时间的缩短为将来酶的结构及催化机制研究奠定了基础。TE底物适用性研究结果发现：McyC TE并不遵循“4 n + 2原则”;底物中转角过多不仅不利于环肽的形成,更可能形成卷曲影响环化;无D型氨基酸亦可通过加入其它位阻较小较灵活的Gly或者自带天然转角Pro的可弱化肽链的刚性,促进催化反应;含苯环的Phe的引入在一定程度上阻碍了环化;底物无肽链氨基酸数目奇偶性的选择;延长多肽链长度也可环化,McyC-TE的底物容忍度较大,使天然多肽药物筛选范围增大,也为增强天然多肽药物药效增加了改良方案,为进一步研究McyC TE的催化功能提供了实验基础。     

Pretransition state and apo structures of the filament-forming enzyme SgrAI elucidate mechanisms of activation and substrate specificity

Enzyme filamentation is a widespread phenomenon that mediates enzyme regulation and function. For the filament-forming sequence-specific DNA endonuclease SgrAI, the process of filamentation both accelerates its DNA cleavage activity and expands its DNA sequence specificity, thus allowing for many additional DNA sequences to be rapidly cleaved. Both outcomes—the acceleration of DNA cleavage and the expansion of sequence specificity—are proposed to regulate critical processes in bacterial innate immunity. However, the mechanistic bases underlying these events remain unclear. Herein, we describe two new structures of the SgrAI enzyme that shed light on its catalytic function. First, we present the cryo-EM structure of filamentous SgrAI bound to intact primary site DNA and Ca²⁺ resolved to ∼2.5 Å within the catalytic center, which represents the trapped enzyme–DNA complex prior to the DNA cleavage reaction. This structure reveals important conformational changes that contribute to the catalytic mechanism and the binding of a second divalent cation in the enzyme active site, which is expected to contribute to increased DNA cleavage activity of SgrAI in the filamentous state. Second, we present an X-ray crystal structure of DNA-free (apo) SgrAI resolved to 2.0 Å resolution, which reveals a disordered loop involved in DNA recognition. Collectively, these multiple new observations clarify the mechanism of expansion of DNA sequence specificity of SgrAI, including the indirect readout of sequence-dependent DNA structure, changes in protein–DNA interactions, and the disorder-to-order transition of a crucial DNA recognition element.     

Engineering the substrate specificity of Escherichia coli asparaginase. II. Selective reduction of glutaminase activity by amino acid replacements at position 248

The use of Escherichia coli asparaginase II as a drug for the treatment of acute lymphoblastic leukemia is complicated by the significant glutaminase side activity of the enzyme. To develop enzyme forms with reduced glutaminase activity, a number of variants with amino acid replacements in the vicinity of the substrate binding site were constructed and assayed for their kinetic and stability properties. We found that replacements of Asp248 affected glutamine turnover much more strongly than asparagine hydrolysis. In the wild-type enzyme, N248 modulates substrate binding to a neighboring subunit by hydrogen bonding to side chains that directly interact with the substrate. In variant N248A, the loss of transition state stabilization caused by the mutation was 15 kJ mol(-1) for L-glutamine compared to 4 kJ mol(-1) for L-aspartic beta-hydroxamate and 7 kJ mol(-1) for L-asparagine. Smaller differences were seen with other N248 variants. Modeling studies suggested that the selective reduction of glutaminase activity is the result of small conformational changes that affect active-site residues and catalytically relevant water molecules.     

Formation of Fine Oil-in-Water Emulsions by the Gel Emulsification Method with Saccharide and Protein

An emulsification method using a gel-like phase of a saccharide and protein mixture has been developed. In the method, which is called a gel emulsification method, an oil is added to the highly concentrated saccharide solution containing protein to form a clear gel-like phase, which followed by dilution with water to form a fine oil-in-water emulsion. This emulsion was investigated as to its emulsifying activity and emulsion stability as compared with that obtained by high-shear equipment, which was called a homomixer method. The emulsifying activity of the emulsions prepared by the gel emulsification method was much higher than that of the emulsions prepared by the homomixer method.

The emulsions prepared by both methods were highly stable in terms of the stability against coalescence. On the other hand, the stability against creaming of the emulsions prepared by the gel emulsification method was much higher than that of the emulsions prepared by the homomixer method.

The surface hydrophobicity of the protein and the unfreezable water content in the highly concentrated saccharide solution containing protein were not correlated to the emulsifying properties of the emulsions prepared by the gel emulsification method, which appeared to be dependent on the viscosity of the highly concentrated saccharide solution containing protein.     

Identification and Characterization of IgdE,a Novel IgG-degrading Protease of Streptococcus suis with Unique Specificity for Porcine IgG

Streptococcus suis is a major endemic pathogen of pigs causing meningitis, arthritis, and other diseases. Zoonotic S. suis infections are emerging in humans causing similar pathologies as well as severe conditions such as toxic shock-like syndrome. Recently, we discovered an IdeS family protease of S. suis that exclusively cleaves porcine IgM and represents the first virulence factor described, linking S. suis to pigs as their natural host. Here we report the identification and characterization of a novel, unrelated protease of S. suis that exclusively targets porcine IgG. This enzyme, designated IgdE for immunoglobulin G-degrading enzyme of S. suis, is a cysteine protease distinct from previous characterized streptococcal immunoglobulin degrading proteases of the IdeS family and mediates efficient cleavage of the hinge region of porcine IgG with a high degree of specificity. The findings that all S. suis strains investigated possess the IgG proteolytic activity and that piglet serum samples contain specific antibodies against IgdE strongly indicate that the protease is expressed in vivo during infection and represents a novel and putative important bacterial virulence/colonization determinant, and a thus potential therapeutic target.     

Synthesis of acceptor substrate analogs for the study of glycosyltransferases involved in the second step of the biosynthesis of O-antigen repeating units

O-antigens of Gram negative bacteria are polysaccharides covalently attached to lipopolysaccharides (LPS) that have roles as virulence factors. Due to the lack of defined substrates for in vitro assays only a few of the enzymes involved in the biosynthesis of O-antigens have been studied. Many O-antigens have GlcNAc at the reducing end of the oligosaccharide chain linked to pyrophosphate-lipid. We therefore designed and synthesized a series of GlcNAc-pyrophosphate-lipid analogs of the natural GlcNAc-pyrophosphate-undecaprenol acceptor substrate for studies of the acceptor specificities of O-antigen biosynthetic enzymes. We synthesized analogs with modifications of the pyrophosphate bond as well as the lipid chain. These compounds will be useful for the specificity studies of many bacterial glycosyltransferases. Knowledge of the substrate specificities is the basis for the development of specific glycosyltransferase inhibitors that could block O-antigen biosynthesis.     

Impact of remote mutations on metallo-beta-lactamase substrate specificity: implications for the evolution of antibiotic resistance

Metallo-beta-lactamases have raised concerns due to their ability to hydrolyze a broad spectrum of beta-lactam antibiotics. The G262S point mutation distinguishing the metallo-beta-lactamase IMP-1 from IMP-6 has no effect on the hydrolysis of the drugs cephalothin and cefotaxime, but significantly improves catalytic efficiency toward cephaloridine, ceftazidime, benzylpenicillin, ampicillin, and imipenem. This change in specificity occurs even though residue 262 is remote from the active site. We investigated the substrate specificities of five other point mutants resulting from single-nucleotide substitutions at positions near residue 262: G262A, G262V, S121G, F218Y, and F218I. The results suggest two types of substrates: type I (nitrocefin, cephalothin, and cefotaxime), which are converted equally well by IMP-6, IMP-1, and G262A, but even more efficiently by the other mutants, and type II (ceftazidime, benzylpenicillin, ampicillin, and imipenem), which are hydrolyzed much less efficiently by all the mutants. G262V, S121G, F218Y, and F218I improve conversion of type I substrates, whereas G262A and IMP-1 improve conversion of type II substrates, indicating two distinct evolutionary adaptations from IMP-6. Substrate structure may explain the catalytic efficiencies observed. Type I substrates have R2 electron donors, which may stabilize the substrate intermediate in the binding pocket. In contrast, the absence of these stabilizing interactions with type II substrates may result in poor conversion. This observation may assist future drug design. As the G262A and F218Y mutants confer effective resistance to Escherichia coli BL21(DE3) cells (high minimal inhibitory concentrations), they are likely to evolve naturally.