Mutational and crystallographic analyses of the active site residues of the Bacillus circulans xylanase.

Using site-directed mutagenesis we have investigated the catalytic residues in a xylanase from Bacillus circulans. Analysis of the mutants E78D and E172D indicated that mutations in these conserved residues do not grossly alter the structure of the enzyme and that these residues participate in the catalytic mechanism. We have now determined the crystal structure of an enzyme-substrate complex to 108 A resolution using a catalytically incompetent mutant (E172C). In addition to the catalytic residues, Glu 78 and Glu 172, we have identified 2 tyrosine residues, Tyr 69 and Tyr 80, which likely function in substrate binding, and an arginine residue, Arg 112, which plays an important role in the active site of this enzyme. On the basis of our work we would propose that Glu 78 is the nucleophile and that Glu 172 is the acid-base catalyst in the reaction.     

A 70-amino acid zinc-binding polypeptide fragment from the regulatory chain of aspartate transcarbamoylase causes marked changes in the kinetic mechanism of the catalytic trimer.

Interaction between a 70-amino acid and zinc-binding polypeptide from the regulatory chain and the catalytic (C) trimer of aspartate transcarbamoylase (ATCase) leads to dramatic changes in enzyme activity and affinity for active site ligands. The hypothesis that the complex between a C trimer and 3 polypeptide fragments (zinc domain) is an analog of R state ATCase has been examined by steady-state kinetics, heavy-atom isotope effects, and isotope trapping experiments. Inhibition by the bisubstrate ligand, N-(phosphonacetyl)-L-aspartate (PALA), or the substrate analog, succinate, at varying concentrations of substrates, aspartate, or carbamoyl phosphate indicated a compulsory ordered kinetic mechanism with carbamoyl phosphate binding prior to aspartate. In contrast, inhibition studies on C trimer were consistent with a preferred order mechanism. Similarly, 13C kinetic isotope effects in carbamoyl phosphate at infinite aspartate indicated a partially random kinetic mechanism for C trimer, whereas results for the complex of C trimer and zinc domain were consistent with a compulsory ordered mechanism of substrate binding. The dependence of isotope effect on aspartate concentration observed for the Zn domain-C trimer complex was similar to that obtained earlier for intact ATCase. Isotope trapping experiments showed that the compulsory ordered mechanism for the complex was attributable to increased "stickiness" of carbamoyl phosphate to the Zn domain-C trimer complex as compared to C trimer alone. The rate of dissociation of carbamoyl phosphate from the Zn domain-C trimer complex was about 10(-2) that from C trimer.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of spvR, the positive regulatory gene of Salmonella plasmid virulence genes

Abstract The regulation of the spvR promoter from the Salmonella dublin virulence plasmid was monitored using proter-reporter gene fusion constructs. Activity was dependent upon the presence of the spv region and was affected by the number of copies of the spv region present with the cell. Activity remained constant throughout exponential growth, and increased rapidly with the onset of stationary phase, under both aerobic and anaerobic conditions. Additionally, the level of spvR expression was controlled by the availability of iron, activity being greatest under low iron conditions in stationary phase. The spvA gene product negatively regulated spvR expression in a dose-dependent manner, indicating that SpvA provides a negative feedback mechanism for this operon.     

Investigation of the enantioselectivity and enantiomeric elution order of some piperidine-2,6-dione drugs on chiralcel OD column

The enantioselectivity and enantiomeric separation of five racemic piperidine-2,6-dione compounds, on the cellulose tris(3,5-dimethylphenyl carbamate) chiral stationary phase Chiralcel OD-CSP were investigated under the same chromatographic conditions. This class of drugs includes glutethimide, aminoglutethimide, cyclohexylaminoglutethimide, pyridoglutethimide, and phenglutarimide. The results revealed that chiral recognition and the binding sites of these drugs on the Chiralcel OD column are similar, regardless of the absolute configuration of the individual enantiomers. A possible chiral recognition mechanism(s) for this class of drugs and the CSP is presented. © 1994 Wiley-Liss, Inc.     

鸣鸣蝉（Oncotympana maculaticollis Motsch）发声肌中肌原纤维的双阵列结构及莫发声原初过程的分析

给出了鸣鸣蝉发声肌肌原纤维的双阵列结构,其肌纤维中并存两种不同阵列的“快”和“慢”动肌原纤维（ＦＳＭ和ＳＳＭ）．ＦＳＭ和ＳＳＭ虽然由粗肌丝构成相同的阵列骨架,但细肌丝对粗肌丝的比例（ＲＴＩＦ）不同,分别为３：１和５：１．明显区别于单音调鸣声的蝉类发声肌肌原纤维的ＲＴＩＦ为３：１的单阵列结构,即与鸣鸣蝉变音调声产生的原初机制相适应．     

Cloning and expression of distinct nitrite reductases in tobacco leaves and roots

Summary Three tobacco nitrite reductase (NiR) cDNA clones were isolated using spinach NiR cDNA as a probe. Sequence analysis and Southern blot hybridization revealed four genes in tobacco. Two of these genes presumably derived from the ancestral species Nicotiana tomentosiformis, the other two from the ancestor N. sylvestris. Northern blot analysis showed that one gene from each ancestral genome was expressed predominantly in leaves, whilst RNA from the other was detected mostly in roots. The accumulation of both leaf and root NiR mRNAs was induced by nitrate and repressed by nitrate- or ammonium-derived metabolites. In addition, the expression of the root NiR gene was detectable in leaves of a tobacco nitrate reductase (NR)-deficient mutant. Thus, the regulation of expression of tobacco NiR genes is comparable to the regulation of expression of barley NR genes.     

TheEscherichia coli mannitol permease as a model for transport via the bacterial phosphotransferase system

The bacterial phosphoenolpyruvate-dependent carbohydrate phosphotransferase system (PTS) consists of several proteins whose primary functions are to transport and phosphorylate their substrates. The complexity of the PTS undoubtedly reflects its additional roles in chemotaxis to PTS substrates and in regulation of other metabolic processes in the cell. The PTS permeases (Enzymes II) are the membrane-associated proteins of the PTS that sequentially recognize, transport, and phosphorylate their specific substrates in separate steps, and theEscherichia coli mannitol permease is one of the best studied of these proteins. It consists of two cytoplasmic domains (EIIA and EIIB) involved in mannitol phosphorylation and an integral membrane domain (EIIC) which is sufficient to bind mannitol, but which transports mannitol at a rate that is dependent on phosphorylation of the EIIA and EIIB domains. Recent results show that several residues in a hydrophilic, 85-residue segment of the EIIC domain are important for the binding, transport, and phosphorylation of mannitol. This segment may be at least partially exposed to the cytoplasm of the cell. A model is proposed in which this region of the EIIC domain is crucial in coupling phosphorylation of the EIIB domain to transport through the EIIC domain of the mannitol permease.     

Effect of oxygen abstraction on the peroxidase–luminol–perborate system: Relevance to the HRP enhanced chemiluminescence mechanism

Abstraction of oxygen from the HRP enhanced chemiluminescence system has no significant effect on the chemiluminescence generated. It is, therefore, proposed that in the peroxidase-luminol-perborate system at pH 7.3, chemiluminescence is generated by a direct reaction of diazaquinones with hydrogen peroxide and not, as generally assumed, from the reaction of luminol radicals with the molecular oxygen.