Reductive activation of the methyl-tetrahydromethanopterin: coenzyme M methyltransferase from Methanobacterium thermoautotrophicum strain ΔH

The enzymatic conversion of formaldehyde to CH₃S-CoM in crude extracts of Methanobacterium thermoautotrophicum was used as a means to investigate the methyl-tetrahydromethanopterin: HS-CoM methyltransferase reaction. All components necessary for formaldehyde conversion were shown to be present in a soluble protein fraction. This soluble cell fraction still contained a major amount of corrinoids. Apart from tetrahydromethanopterin no other soluble cofactors were required for formaldehyde conversion. The dependence of the system on catalytic amounts of ATP was shown to be specific. Several nucleoside triphosphates or ADP were unable to substitute for ATP. Remarkably, various strong reducing systems, especially titanium(III)citrate could replace ATP to a large extent. The ATP-dependent formaldehyde conversion to CH₃S-CoM was inhibited in the presence of nitrous oxide, detergents or 2,3-dialdehyde-ATP. The results support a role for a corrinoid protein in the methyl-tetrahydromethanopterin: HS-CoM methyltransferase reaction at which ATP is involved in the activation of this protein, probably in the conversion of inactive B_12a or B_12r to active B_12s.Abbreviations HS-CoM Coenzyme M, 2-mercaptoethanesulfonate - CH₃S-CoM methylcoenzyme M, 2-(methylthio)ethanesulfonate - H₄MPT 5,6,7,8-tetrahydromethanopterin - BES 2-bromoethanesulfonate - BCE boiled cell-free extract - DTT dithiothreitol - TCS 3,3,4,5-tetrachlorosalicylanilide - DNTB 2,2-dinitro-5,5-dithiobenzoic acid - TES N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - PIPES piperazine-N,N-bis[2-ethanesulfonic acid] - AMP-PNP 5-adenylyl imidophosphate     

Dehalogenation of trichlorofluoromethane (CFC-11) by Methanosarcina barkeri

Methanobacterium barkeri was found to catalyze the reductive dehalogenation of trichlorofluoromethane (CFC-11), also known as FREON 11. Products detected were CHFCl2, CH2FCl, CO and fluoride.     

一株河流污泥中产甲烷杆菌的生物学特性研究

目的:筛选产甲烷菌并对其进行鉴定和特性分析.方法:利用亨盖特厌氧操作技术从河流污泥中分离出的一株产甲烷杆菌.通过形态特征、生理生化、16S rRNA基因序列分析确定菌株的分类地位,利用气相色谱仪测定甲烷含量.结果:该菌株杆状,产芽孢,极端严格厌氧,宽约0.36μm,长约3.20μm,有时观察成链状,在液体培养的过程中,聚集成絮状沉积在管底.能够利用H2/CO2和甲钠盐生长,不利用甲醇、三甲胺、乙酸和二级醇类.最适生长温度范围为37℃,最适pH为7.5 ～8.0,能在0～1％盐浓度范围内生长.结论:菌株AG的最适温度较高,最适pH偏碱,在沼气发酵中具有重要应用前景.     

Structure and molecular dynamics simulation of archaeal prefoldin: the molecular mechanism for binding and recognition of nonnative substrate proteins

Prefoldin (PFD) is a heterohexameric molecular chaperone complex in the eukaryotic cytosol and archaea with a jellyfish-like structure containing six long coiled-coil tentacles. PFDs capture protein folding intermediates or unfolded polypeptides and transfer them to group II chaperonins for facilitated folding. Although detailed studies on the mechanisms for interaction with unfolded proteins or cooperation with chaperonins of archaeal PFD have been performed, it is still unclear how PFD captures the unfolded protein. In this study, we determined the X-ray structure of Pyrococcus horikoshii OT3 PFD (PhPFD) at 3.0 Å resolution and examined the molecular mechanism for binding and recognition of nonnative substrate proteins by molecular dynamics (MD) simulation and mutation analyses. PhPFD has a jellyfish-like structure with six long coiled-coil tentacles and a large central cavity. Each subunit has a hydrophobic groove at the distal region where an unfolded substrate protein is bound. During MD simulation at 330 K, each coiled coil was highly flexible, enabling it to widen its central cavity and capture various nonnative proteins. Docking MD simulation of PhPFD with unfolded insulin showed that the β subunit is essentially involved in substrate binding and that the α subunit modulates the shape and width of the central cavity. Analyses of mutant PhPFDs with amino acid replacement of the hydrophobic residues of the β subunit in the hydrophobic groove have shown that βIle107 has a critical role in forming the hydrophobic groove.     

Complete genome sequence of Thermanaerovibrio acidaminovorans type strain (Su883)

Thermanaerovibrio acidaminovorans (Guangsheng et al. 1997) Baena et al. 1999 is the type species of the genus Thermanaerovibrio and is of phylogenetic interest because of the very isolated location of the novel phylum Synergistetes. T. acidaminovorans Su883(T) is a Gram-negative, motile, non-spore-forming bacterium isolated from an anaerobic reactor of a sugar refinery in The Netherlands. Here we describe the features of this organism, together with the complete genome sequence, and annotation. This is the first completed genome sequence from a member of the phylum Synergistetes. The 1,848,474 bp long single replicon genome with its 1765 protein-coding and 60 RNA genes is part of the Genomic Encyclopedia of Bacteria and Archaea project.     

Comparative analysis of thermoadaptation within the archaeal glyceraldehyde-3-phosphate dehydrogenases from mesophilic Methanobacterium bryantii and thermophilic Methanothermus fervidus

To gain insight into the molecular determinants of thermoadaptation within the family of archaeal glyceraldehyde-3-phosphate dehydrogenases (GAPDH), a homology-based 3-D model of the mesophilic GAPDH from Methanobacterium bryantii was built and compared with the crystal structure of the thermophilic GAPDH from Methanothermus fervidus. The homotetrameric model of the holoenzyme was initially assembled from identical subunits completed with NADP molecules. The structure was then refined by energy minimization and simulated-annealing procedures. PROCHECK and the 3-D profile method were used to appraise the model reliability. Striking molecular features underlying the difference in stability between the enzymes were deduced from their structural comparison. First, both the increase in hydrophobic contacts and the decrease in accessibility to the protein core were shown to discriminate in favor of the thermophilic enzyme. Besides, but to a lesser degree, the number of ion pairs involved in cooperative clusters appeared to correlate with thermostability. Finally, the decreased stability of the mesophilic enzyme was also predicted to proceed from both the lack of charge-dipole interactions within alpha-helices and the enhanced entropy of unfolding due to an increase in chain flexibility. Thus, archaeal GAPDHs appear to be governed by thermoadaptation rules that differ in some aspects from those previously observed within their eubacterial counterparts.     

Adaptation of methane formation and enzyme contents during growth of Methanobacterium thermoautotrophicum (strain ΔH) in a fed-batch fermentor

During growth of Methanobacterium thermoautotrophicum in a fed-batch fermentor, the cells are confronted with a steady decrease in the concentration of the hydrogen energy supply. In order to investigate how the organism responds to these changes, cells collected during different growth phases were examined for their methanogenic properties. Cellular levels of the various methanogenic isoenzymes and functionally equivalent enzymes were also determined. Cells were found to maintain the rates of methanogenesis by lowering their affinity for hydrogen: the apparent K _m ^H2 decreased in going from the exponential to the stationary phase. Simultaneously, the maximal specific methane production rate changed. Levels of H₂-dependent methenyl-tetrahydromethanopterin dehydrogenase (H₂-MDH) and methyl coenzyme M reductase isoenzyme II (MCR II) decreased upon entry of the stationary phase. Cells grown under conditions that favored MCR II expression had higher levels of MCR II and H₂-MDH, whereas in cells grown under conditions favoring MCR I, levels of MCR II were much lower and the cells had an increased affinity for hydrogen throughout the growth cycle. The use of thiosulfate as a medium reductant was found to have a negative effect on levels of MCR II and H₂-MDH. From these results it was concluded that M. thermoautotrophicum responds to variations in hydrogen availability and other environmental conditions (pH, growth temperature, medium reductant) by altering its physiology. The adaptation includes, among others, the differential expression of the MDH and MCR isoenzymes.     

Thermodynamic analysis of growth of Methanobacterium thermoautotrophicum

Growth of Methanobacterium thermoautotrophicum, an anaerobic archaebacterium using methanogenesis as the catabolic pathway, is characterized by large heat production rates, up to 13 W g⁻¹, and low biomass yields, in the order of 0.02 C‐mol mol⁻¹ H₂ consumed. These values, indicating a possibly “inefficient” growth mechanism, warrant a thermodynamic analysis to obtain a better understanding of the growth process. The growth‐associated heat production (Δ_rH) and the growth‐associated Gibbs energy dissipation per mol biomass formed (Δ_rG) were −3730 kJ C‐mol⁻¹ and −802 kJ C‐mol⁻¹, respectively. The Gibbs energy change found in this study is indeed unusually high as compared to aerobic methylotrophes, but not untypical for methanogens grown on CO₂. It explains the low biomass yield. Based on the information available on the energetic metabolism and on an ATP balance, the biomass yield can be predicted to be approximately in the range of the experimentally determined value. The fact that the exothermicity exceeds vastly even the Gibbs energy change can be explained by a dramatic entropy decrease of the catabolic reaction. Microbial growth characterized by entropy reduction and correspondingly by unusually large heat production may be called entropy‐retarded growth. © 1999 John Wiley & Sons, Inc. Biotechnol Bioeng 64: 74–81, 1999.     

Naturally occurring 1,3,4,6-hexanetetracarboxylic acid has meso stereochemistry

1,3,4,6-Hexanetetracarboxylic acid was first identified as a natural product during the structural characterization of methanofuran, one of several coenzymes involved in methanogenesis. A combination of high resolution ¹H NMR, ¹³C NMR, and gas chromatography-mass spectrometry has been used to establish that the naturally occurring diastereomer of 1,3,4,6-hexanetetracarboxylic acid in methanogenic Archaea is meso. © 1996 Wiley-Liss, Inc.     

两种鸣禽发声控制核团体积的比较研究

用焦油紫染色,图像分析及统计学方法比较研究了两种鸣禽栗巫鸟和燕雀发声控制核团的体积差异．结果表明,在发声活动中起重要作用的核团前脑ＨＶｃ、ＲＡ 和Ｘ区的体积存在显著的种间差异：发声技巧较高的雄性栗巫鸟的相应核团均相对大于雄性燕雀;而在发声中作用较小的ｎⅫｔｓ及雌性鸣禽的相应所有核团均无显著种间差异． 这一结果表明,发声能力的高低与前脑发声核团的体积直接相关．