Characterization of a Tryptophan 6-Halogenase from Streptomyces albus and Its Regioselectivity Determinants

Tryptophan halogenases are found in diverse organisms and catalyze regiospecific halogenation. They play an important role in the biosynthesis of halogenated indole alkaloids, which are biologically active and of therapeutic importance. Here, a tryptophan 6-halogenase (SatH) from Streptomyces albus was characterized by using a whole-cell reaction system in Escherichia coli. SatH showed substrate specificity for chloride and bromide ions, leading to regiospecific halogenation at the C6-position of l -tryptophan. In addition, SatH exhibited higher performance in bromination than that of previously reported tryptophan halogenases in the whole-cell reaction system. Through structure-based protein mutagenesis, it has been revealed that two consecutive residues, A78/V79 in SatH and G77/I78 in PyrH, are key determinants in the regioselectivity difference between tryptophan 6- and 5-halogenases. Substituting the AV with GI residues switched the regioselectivity of SatH by moving the orientation of tryptophan. These data contribute to an understanding of the key residues that determine the regioselectivity of tryptophan halogenases.     

Leader-following consensus of fractional-order multi-agent systems via adaptive control

This article focuses on the consensus problem of leader-following fractional-order multi-agent systems (MASs) with general linear and Lipschitz nonlinear dynamics. First, the distributed adaptive protocols for linear and nonlinear fractional-order MASs are constructed, respectively. We allow the control coupling gains to be time varying for each agent. Moreover, the adaptive modification schemes for the control gain are designed, which renders smaller control gains and thus requires smaller amplitude on the control input without sacrificing consensus convergence. Second, based on fractional-order Lyapunov stability theorem and Barbalat's lemma, two novel sufficient conditions in terms of linear matrix inequalities are provided to ensure that the leader-following consensus can be obtained in the case for any undirected connected communication graph. Furthermore, we show that the proposed algorithm also works for consensus of agents with intrinsic Lipschitz nonlinear dynamics. As a result, the proposed framework requires no global information and thus can be implemented in a fully distributed manner. Finally, the numerical simulations are given to demonstrate the effectiveness of obtained the theoretical results.     

Crossing the Border: From Keto- to Imine Reduction in Short-Chain Dehydrogenases/Reductases

The family of NAD(P)H-dependent short-chain dehydrogenases/reductases (SDRs) comprises numerous biocatalysts capable of C=O or C=C reduction. The highly homologous noroxomaritidine reductase (NR) from Narcissus sp. aff. pseudonarcissus and Zt_SDR from Zephyranthes treatiae, however, are SDRs with an extended imine substrate scope. Comparison with a similar SDR from Asparagus officinalis (Ao_SDR) exhibiting keto-reducing activity, yet negligible imine-reducing capability, and mining the Short-Chain Dehydrogenase/Reductase Engineering Database indicated that NR and Zt_SDR possess a unique active-site composition among SDRs. Adapting the active site of Ao_SDR accordingly improved its imine-reducing capability. By applying the same strategy, an unrelated SDR from Methylobacterium sp. 77 (M77_SDR) with distinct keto-reducing activity was engineered into a promiscuous enzyme with imine-reducing activity, thereby confirming that the ability to reduce imines can be rationally introduced into members of the “classical” SDR enzyme family. Thus, members of the SDR family could be a promising starting point for protein approaches to generate new imine-reducing enzymes.     

抗真菌蛋白Rs-AFP2基因定点突变及其对大肠杆菌中表达的影响

为了研究遗传密码子对表达调控的影响,利用PCR重叠延伸法,对萝卜抗真菌蛋白Rs-AFP2基因编码序列区的部分核苷酸进行沉默突变,构建突变体Rs-AFPm.序列分析表明,PCR产物全长240bp,有一个阅读框,编码的蛋白由29个氨基酸的信号肽和51个氨基酸的抗真菌蛋白组成.突变体与突变前的Rs-AFP2基因相比,在编码区第3号氨基酸Lys相差一个碱基(TTG→TTA),第5号氨基酸Gln相差一个碱基(CAG→CAA),第6号稀有密码子Arg相差两个碱基(CAG→CGA).重新合成引物,将切除信号肽的Rs-AFP2基因和Rs-AFPm基因与原核表达载体pET-21b(+)分别重组到大肠杆菌BL21菌株.IPTG诱导后,二者均得到了表达.软件分析显示,突变前pETAFPo表达产物占全菌蛋白的3%,突变后pETAFPm的表达产物占全菌蛋白含量的8%;表达蛋白主要以包涵体的形式存在,包涵体经超声波破碎后,蛋白质复性,抑菌结果表明,pETAFPm表达产物的抑菌半径大于pETAFP2表达产物的抑菌半径.这些都说明改造后的Rs-AFPm基因与Rs-AFP2基因相比,已有效地提高表达量.     

Average consensus in networks of dynamic agents with switching topologies and multiple time-varying delays

In this paper, we discuss average consensus problem in undirected networks of dynamic agents with fixed and switching topologies as well as multiple time-varying communication delays. By employing a linear matrix inequality method, we prove that all the nodes in the network achieve average consensus asymptotically for appropriate communication delays if the network topology is connected. Particularly, several feasible linear matrix inequalities are established to determine the maximal allowable upper bound of time-varying communication delays. Numerical examples are given to demonstrate the effectiveness and the sharpness of the theoretical results.     

异构空间信息的分级共识模型研究

Internet上异构、分布的空间信息因各自独立、相对封闭而无法相互沟通和协作,从而形成了空间信息孤岛。论文利用计算机的知识表示理论和Ontology技术能够明确表达相关领域共识的特点,探索性地提出了异构空间信息的分级共识模型。该模型为解决上述问题提供了一条切实可行的新思路。     

Parameters affecting the frequencies of transformation and co-transformation with synthetic oligonucleotides in yeast.

Factors influencing the direct transformation of the yeast Saccharomyces cerevisiae with synthetic oligonucleotides were investigated by selecting for cyc1 transformants that contained at least partially functional iso-1-cytochrome c. Approximately 3 x 10(4) transformants, constituting 0.1% of the cells, were obtained by using 1 mg of oligonucleotide in the reaction mixture. Carrier, such as heterogeneous oligonucleotides, enhanced transformation frequencies. Transformation frequencies were dramatically reduced if the oligonucleotides had a large number of mismatches or had terminally located mismatches. Transformation with oligonucleotides, but not with linearized double-strand plasmid, was efficient in a rad52- strain, suggesting that the pathway for transformation with oligonucleotides is different from that with linearized double-strand plasmid. We describe a procedure of co-transformation with two oligonucleotides, one correcting the cyc1 defect of the target allele in the host strain, and the other producing a desired amino acid alteration elsewhere in the iso-1-cytochrome c molecule; approximately 20% of the transformants obtained by co-transformation contained these desired second alterations.     

6-Phospho-{beta}-galactosidases of Gram-positive and 6-phospho-{beta}-glucosidase B of Gram-negative bacteria: comparison of structure and function by kinetic and immunological methods and mutagenesis of the lacG gene of staphylococcus aureus

The 6-phospho-ß-galactosidase of Staphylococcus aureus,Lactococcus lactis and Lactobacillus casei and 6-phospho-ßglucosidaseB of Escherichia coli build a subfamily inside a greater enzymefamily, named the glycosal hydrolase family 1, which, hi addition,contains nine ß-glycosidases of different origins.Kinetic and immunological evidence is provided in this reportwhich strengthens the relationship of the four 6-phospho-ß-glycosidases.It is shown that the 6-phospho-ß-galactosidases and6-phospho-ß-glucosidase B are able to split aromaticß-galactoside phosphates and ß-glucosidephosphates. The turnover numbers of hydrolysis of substrateswith different epimerization at C-4 of the glycon vary up to15-fold only. Two polydonal antisera, one derived against thenative 6-phospho-ß-galactosidase from S.aureus andthe other derived against the 6-phospho-ß-glucosidaseB, cross-reacted with both enzymes. Peptides of the proteinswere separated by reverse phase HPLC. The cross-reacting peptideswere sequenced and shown to be localized at almost the sameposition in the aligned primary structures of both enzymes.An insertion of nine amino adds near these antigenic domainsis unique for the 6-phospho-ß-glycosidases and missingwithin the sequences of the ß-glycoside-specific membersof the family. The lacG gene of a 6-phospho-ß-galactosidasenegative S.aureus mutant was doned into E.coli and sequenced.In the totally inactive mutant protein only the glycine at position332 was changed to an arginine. This amino acid is part of thesequence insertion near the antigenic domain reacting with bothantisera. These data support the assumption that the regionis of great importance for the function of the enzymes and thatit is possible it determines the specificity of the phosphorylatedform of the substrates. In addition, the 6-phospho-ß-galactosidaseof S.aureus was modified by sitedirected mutagenesis of thecorresponding lacG gene hi order to replace residues Glul60and Glu375, which were suspected of being involved hi the generalacid catalysis of substrate hydrolysis, with glutamine residues.The mutant protein 160EQ retained some catalytic activity whilethe protein 375EQ was totally inactive. Glu375 is the activesite nudeophile of the 6-phospho-ß-galactosidase ofS.aureus. It is located in the sequence motif ENG where Glu358was identified as the catalytkally active nudeophile hi theß-glucosidase of Agrobacterium.