New function of the amino group of thiamine diphosphate in thiamine catalysis

In this work, we investigated the rate of formation of the central intermediate of the transketolase reaction with thiamine diphosphate (ThDP) or 4′-methylamino-ThDP as cofactors and its stability using stopped-flow spectroscopy and circular dichroism (CD) spectroscopy. The intermediates of the transketolase reaction were analyzed by NMR spectroscopy. The kinetic stability of the intermediate was shown to be dependent on the state of the amino group of the coenzyme. The rates of the intermediate formation were the same in the case of the native and methylated ThDP, but the rates of the protonation or oxidation of the complex in the ferricyanide reaction were significantly higher in the complex with methylated ThDP. A new negative band was detected in the CD spectrum of the complex transketolase—4′-methylamino-ThDP corresponding to the protonated dihydroxyethyl-4′-methylamino-ThDP released from the active sites of the enzyme. These data suggest that transketolase in the complex with the NH²-methylated ThDP exhibits dihydroxyethyl-4′-methylamino-ThDP-synthase activity. Thus, the 4′-amino group of the coenzyme provides kinetic stability of the central intermediate of the transketolase reaction, dihydroxyethyl-ThDP.     

A new perspective on Listeria monocytogenes evolution

Listeria monocytogenes is a model organism for cellular microbiology and host-pathogen interaction studies and an important food-borne pathogen widespread in the environment, thus representing an attractive model to study the evolution of virulence. The phylogenetic structure of L. monocytogenes was determined by sequencing internal portions of seven housekeeping genes (3,288 nucleotides) in 360 representative isolates. Fifty-eight of the 126 disclosed sequence types were grouped into seven well-demarcated clonal complexes (clones) that comprised almost 75% of clinical isolates. Each clone had a unique or dominant serotype (4b for clones 1, 2 and 4, 1/2b for clones 3 and 5, 1/2a for clone 7, and 1/2c for clone 9), with no association of clones with clinical forms of human listeriosis. Homologous recombination was extremely limited (r/m<1 for nucleotides), implying long-term genetic stability of multilocus genotypes over time. Bayesian analysis based on 438 SNPs recovered the three previously defined lineages, plus one unclassified isolate of mixed ancestry. The phylogenetic distribution of serotypes indicated that serotype 4b evolved once from 1/2b, the likely ancestral serotype of lineage I. Serotype 1/2c derived once from 1/2a, with reference strain EGDe (1/2a) likely representing an intermediate evolutionary state. In contrast to housekeeping genes, the virulence factor internalin (InlA) evolved by localized recombination resulting in a mosaic pattern, with convergent evolution indicative of natural selection towards a truncation of InlA protein. This work provides a reference evolutionary framework for future studies on L. monocytogenes epidemiology, ecology, and virulence.     

A new perspective on clinical technology management

In 1996 Intermountain Health Care (IHC) launched an endeavor for the development of a strategy that would provide a systematic management of its assets, specifically, clinical equipment. The scope would be enterprise wide and would focus on the 6 diverse elements of the "Equipment Life Cycle." These elements include: planning, assessment, acquisition, utilization, maintenance, and disposition. This endeavor resulted in the deployment of Clinical Technology Management (CTM). This article addresses the development of the concepts and methods used to organize CTM in 1996 and the subsequent refinements for the past 6 years and in its entirety, this article is a mere summary of thousands of hours of work for 6 years.     

A stochastic attractor participates in chymotrypsin catalysis. A new facet of enzyme catalysis

The previously observed discrete levels of vibrational parameters of chymotrypsin and tosyl-chymotrypsin were analyzed by the methods of non-linear dynamics in order to investigate their origin. The fractal dimensionality of the step sequence was determined using the correlation function and the Farey-tree method. These methods yielded the same value and indicated the presence of a "Devil's Staircase", i.e. the existence of a stochastic attractor. The latter was assigned to the substrate-mobilizable conformation. The attractor dimension of the catalytic site in the acylated state was found to be indistinguishable from that of the substrate mobilizable conformation. A "Devil's Staircase" signals the transition from the regular to the stochastic regime. This transition is shared by critical phenomena and may be a prerequisite for catalysis.     

A new perspective on lakes: Kawanabe's latest achievements

In October 1996 the Lake Biwa Museum opened in Oroshimo, Japan after 10 years of planing and construction. It is a most ambitious concept and a very impressive facility. By June of 1997 it had attracted almost 800 000 visitors – almost twice the at tendance predicted for the entire first year of operation.The founding Director General of the Lake Biwa Museum is Hiroya Kawanabe. After a distinguished career as professor of ecology at Kyoto University, Hiroya Kawanabe founded the Centre for Ecological Research at Otsu. After retirement from the university he moved to his new position at the Lake Biwa Museum. Kawanabe-san is internationally recognized for his pioneering and collaborative research on the ayu, Pleoglossus altivelis, Lake Biwa and the ecology of Lake Tanganyika fishes (Kawanabe 1996, Kawanabe et al. 1997). The Lake Biwa Museum is not only the pinnacle of his personal and professional achievements, but also the physical embodiment of his philosophy of science and life.     

A new formal perspective on ‘Cambrian explosions’

The ‘Cambrian explosion’ 500 Myr ago saw a relatively sudden proliferation of organism Bauplan and ecosystem niche structure that continues to haunt evolutionary biology. Here, adapting standard methods from information theory and statistical mechanics, we model the phenomenon as a noise-driven phase transition, in the context of deep-time relaxation of current path-dependent evolutionary constraints. The result is analogous to recent suggestions that multiple ‘explosions’ of increasing complexity in the genetic code were driven by rising intensities of available metabolic free energy. In the absence of severe path-dependent lock-in, ‘Cambrian explosions’ are standard features of blind evolutionary process, representing outliers in the ongoing routine of evolutionary punctuated equilibrium.     

A new perspective on thermal inactivation kinetics of human serum butyrylcholinesterase

Butyrylcholinesterase purified from human serum as 6600-fold was heated at 37°, 40°, 45°, and 50°C for 24 hr. It was observed that the enzyme heated at 45°C for 24 hr converted to a stabilized form and followed Michaelis-Menten kinetics, whereas the enzyme samples, heated at the other temperatures for 24 hr, shown negative cooperativity with respect to its substrate, butyrylthiocholine. Even the sample heated at 45°C for 12 hr shown negative cooperativity. On the contrary to the heated enzyme at 40°C for 24 hr, the heated enzyme at 45°C for 24 hr could not be reactivated when it was kept at 4°C for 24 hr. In the kinetic studies, it was found that substrate analogs choline and benzoylcholine inhibited both the native enzyme and the enzyme heated at 45°C for 24 hr competitively, whereas succinylcholine was the partial competitive inhibitor of native enzyme but the pure competitive inhibitor of the heated enzyme.     

Action of the thiamine antagonist bacimethrin on thiamine biosynthesis

Bacimethrin is an analog of the 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP) moiety of thiamine and inhibits the growth of Salmonella enterica serovar Typhimurium on a defined medium. Two classes of mutants that had increased bacimethrin resistance were isolated and characterized. Results showed that overexpression of the thi operon or specific lesions in thiD resulted in a bacimethrin-resistant phenotype. Phenotypic analyses of the thiD mutants suggested that they had a specific defect in one of the two kinase activities associated with this gene product and, further, that ThiD and not PdxK was primarily responsible for salvage of HMP from the medium.     

A new principle of enzyme catalysis: coupled vibrations facilitate conformational changes

The coupling between the molecular vibrations in chymotrypsinogen, alpha-chymotrypsin and tosyl-alpha-chymotrypsin, as expressed by the temperature factors of individual amino acid sidechains and by a flexibility parameter calculated from the masses and co-ordinates of the atoms, has been analyzed by calculation of the integral correlation coefficient, the autocorrelation coefficient, the Poincaré projection, the first Liapunov coefficient and the power spectra. The agreement between the results obtained with the temperature factors and the flexibility parameter as well as the correct display by the latter of known structural features support the validity of the approach. The localization and extent of the conformational change in the enzyme following its binding of a specific substrate is detected in the difference plot between the enzyme and the acylenzyme of the distribution of the flexibility parameter over the peptide chain. As many as about 70% of the aminoacids participate in this rearrangement. An attractor of low dimensionality, two, i.e. a limit cycle, is detected both in the total enzyme and in its domain which is mobilized by the specific substrate. A simple model based on a known prominent structural feature, which is common to the trypsin family of serine proteases, two extensive coaxial halfcylinders of beta-sheets, to which previously no mechanistic function could be assigned, is proposed to account for the role of the attractor in the catalytic process: (1) control of the entry of a specific substrate to the catalytic site by co-ordinated disentanglement of the interlocking sidechains; (2) correct positioning of the functional groups in the active site; (3) lowering of the activation energy of the formation of the transition state complex.     

A biophysical perspective on the cellulosome: new opportunities for biomass conversion

The cellulosome is a multiprotein complex, produced primarily by anaerobic microorganisms, which functions to degrade lignocellulosic materials. An important topic of current debate is whether cellulosomal systems display greater ability to deconstruct complex biomass materials (e.g. plant cell walls) than nonaggregated enzymes, and in so doing would be appropriate for improved, commercial bioconversion processes. To sufficiently understand the complex macromolecular processes between plant cell wall polymers, cellulolytic microbes, and their secreted enzymes, a highly concerted research approach is required. Adaptation of existing biophysical techniques and development of new science tools must be applied to this system. This review focuses on strategies likely to permit improved understanding of the bacterial cellulosome using biophysical approaches, with emphasis on advanced imaging and computational techniques.     

Copper-containing amine oxidases. Biogenesis and catalysis; a structural perspective

This review will focus on how X-ray crystallographic studies of copper-containing amine oxidases have complemented the solution, kinetic, and spectroscopic research on this ubiquitous class of enzymes. These enzymes not only contain a copper ion at the active site, but also a unique organic cofactor, 2,4,5-trihydroxyphenylalanine quinone (TPQ), which is absolutely required for catalysis. Structural data have not only shed light on the catalytic mechanism of the enzyme, which converts primary amines, using molecular oxygen, to aldehydes, ammonia, and hydrogen peroxide, but also on biogenesis of the cofactor. The cofactor is derived from a tyrosine in the enzyme amino acid sequence and requires only the addition of copper(II) and molecular oxygen in a self-processing event.     

Antibiofilm agents: A new perspective for antimicrobial strategy

Biofilms are complex microbial architectures that attach to surfaces and encase microorganisms in a matrix composed of self-produced hydrated extracellular polymeric substances (EPSs). In biofilms, microorganisms become much more resistant to antimicrobial treatments, harsh environmental conditions, and host immunity. Biofilm formation by microbial pathogens greatly enhances survival in hosts and causes chronic infections that result in persistent inflammation and tissue damages. Currently, it is believed over 80% of chronic infectious diseases are mediated by biofilms, and it is known that conventional antibiotic medications are inadequate at eradicating these biofilm-mediated infections. This situation demands new strategies for biofilm-associated infections, and currently, researchers focus on the development of antibiofilm agents that are specific to biofilms, but are nontoxic, because it is believed that this prevents the development of drug resistance. Here, we review the most promising antibiofilm agents undergoing intensive research and development.     

A new perspective on developmental plasticity and the principles of adaptive morph determination

Organisms can have divergent paths of development leading to alternative phenotypes, or morphs. The choice of developmental path may be set by environmental cues, the individual's genotype, or a combination of the two. Using individual-based simulation and analytical investigation, we explore the idea that from the viewpoint of a developmental switch, genetic morph determination can sometimes be regarded as adaptive developmental plasticity. We compare the possibilities for the evolution of environmental and genetic morph determination and combinations of the two in situations with spatial variation in conditions. We find that the accuracy of environmental cues in predicting coming selective conditions is important for environmental morph determination, in accordance with previous results, and that genetic morph determination is favored in a similar way by the accuracy of genetic cues, in the form of selectively maintained gene frequency differences between local populations. Restricted gene flow and strong selection acting on the phenotypic alternatives produce clearer gene frequency differences and lead to greater accuracy of genetic cues. For combined environmental and genetic morph determination, we show that the developmental machinery can evolve toward efficiently combining information in environmental and genetic cues for the purpose of predicting coming selective conditions.