The exchange activities of [Fe] hydrogenase (iron–sulfur-cluster-free hydrogenase) from methanogenic archaea in comparison with the exchange activities of [FeFe] and [NiFe] hydrogenases

[Fe] hydrogenase (iron–sulfur-cluster-free hydrogenase) catalyzes the reversible reduction of methenyltetrahydromethanopterin (methenyl-H4MPT+) with H2 to methylene-H4MPT, a reaction involved in methanogenesis from H2 and CO2 in many methanogenic archaea. The enzyme harbors an iron-containing cofactor, in which a low-spin iron is complexed by a pyridone, two CO and a cysteine sulfur. [Fe] hydrogenase is thus similar to [NiFe] and [FeFe] hydrogenases, in which a low-spin iron carbonyl complex, albeit in a dinuclear metal center, is also involved in H2 activation. Like the [NiFe] and [FeFe] hydrogenases, [Fe] hydrogenase catalyzes an active exchange of H2 with protons of water; however, this activity is dependent on the presence of the hydride-accepting methenyl-H4MPT+. In its absence the exchange activity is only 0.01% of that in its presence. The residual activity has been attributed to the presence of traces of methenyl-H4MPT+ in the enzyme preparations, but it could also reflect a weak binding of H2 to the iron in the absence of methenyl-H4MPT+. To test this we reinvestigated the exchange activity with [Fe] hydrogenase reconstituted from apoprotein heterologously produced in Escherichia coli and highly purified iron-containing cofactor and found that in the absence of added methenyl-H4MPT+ the exchange activity was below the detection limit of the tritium method employed (0.1 nmol min−1 mg−1). The finding reiterates that for H2 activation by [Fe] hydrogenase the presence of the hydride-accepting methenyl-H4MPT+ is essentially required. This differentiates [Fe] hydrogenase from [FeFe] and [NiFe] hydrogenases, which actively catalyze H2/H2O exchange in the absence of exogenous electron acceptors

Comparative Proteomic Analysis of Methanothermobacter themautotrophicus ΔH in Pure Culture and in Co-Culture with a Butyrate-Oxidizing Bacterium

To understand the physiological basis of methanogenic archaea living on interspecies H2 transfer, the protein expression of a hydrogenotrophic methanogen, Methanothermobacter thermautotrophicus strain ΔH, was investigated in both pure culture and syntrophic coculture with an anaerobic butyrate oxidizer Syntrophothermus lipocalidus strain TGB-C1 as an H2 supplier. Comparative proteomic analysis showed that global protein expression of methanogen cells in the model coculture was substantially different from that of pure cultured cells. In brief, in syntrophic coculture, although methanogenesis-driven energy generation appeared to be maintained by shifting the pathway to the alternative methyl coenzyme M reductase isozyme I and cofactor F420-dependent process, the machinery proteins involved in carbon fixation, amino acid synthesis, and RNA/DNA metabolisms tended to be down-regulated, indicating restrained cell growth rather than vigorous proliferation. In addition, our proteome analysis revealed that α subunits of proteasome were differentially acetylated between the two culture conditions. Since the relevant modification has been suspected to regulate proteolytic activity of the proteasome, the global protein turnover rate could be controlled under syntrophic growth conditions. To our knowledge, the present study is the first report on N-acetylation of proteasome subunits in methanogenic archaea. These results clearly indicated that physiological adaptation of hydrogenotrophic methanogens to syntrophic growth is more complicated than that of hitherto proposed

Function of H₂-forming methylenetetrahydromethanopterin dehydrogenase from Methanobacterium thermoautotrophicum in coenzyme F₄₂₀ reduction with H₂

In most methanogenic archaea, two hydrogenase systems that can catalyze the reduction of coenzyme F-420 (F-420) with H-2 are present: (1) the F-420-reducing hydrogenase, which is a nickel iron-sulfur flavoprotein composed of three different subunits, and (2) the N-5,N-10-methylenetetrahydromethanopterin dehydrogenase system, which is composed of H-2-forming methylenetetrahydromethanopterin dehydrogenase and F-420-dependent methylenetetrahydromethanopterin dehydrogenase, both metal-free proteins without an apparent prosthetic group. We report here that in nickel-limited chemostat cultures of Methanobacterium thermoautotrophicum, the specific activity of the F-420-reducing Ni/Fe-hydrogenase was essentially zero, whereas that of the H-2-forming methylenete trahydromethanopterin dehydrogenase was six times higher, and that of the F-420-dependent methylenetetrahydromethanopterin dehydrogenase was four times higher than in cells grown under non-nickel-limited conditions. This evidence supports the hypothesis that when M. thermoautotrophicum grows under conditions of nickel limitation, the reduction of F-420 with H-2 is catalyzed by the metal-free methylenetetrahydromethanopterin dehydrogenase system

Regulation of the synthesis of H₂-forming methylenetetrahydromethanopterin dehydrogenase (Hmd) and of HmdII and HmdIII in Methanothermobacter marburgensis

Recently it was found that the specific activity of H-2-forming methylenetetrahydromethanopterin dehydrogenase (Hmd) in Methanothermobacter marburgensis (formerly Methanobacterium thermoautotrophicum strain Marburg) increased six-fold when the hydrogenotrophic archaeon was grown in chemostat culture under nickel-limited conditions. We report here that the increase is due, at least in part, to increased expression of the hmd gene. This was demonstrated by Northern and Western blot analysis. These techniques were also used to show that hmd expression in growing M. marburgensis is not under the control of the H-2 concentration. Studies with monoclonal antibodies on the effect of growth conditions on the expression of hmdII and hmdIII, which have been proposed to encode Hmd isoenzymes, were also carried out. The results indicate that the expression of these two genes is regulated by H-2 rather than by nickel, and that HmdII and HmdIII most probably do not exhibit Hmd activity

Quantitative importance of non-skeletal-muscle <i>N</i>τ-methylhistidine and creatine in human urine

The excretion of N tau-methylhistidine and creatinine was determined in a totally paralysed patient wih neither macroscopic nor microscopic detectable skeletal-muscle tissue. In this subject, it was possible for the first time to measure the basal non-skeletal-muscle-dependent excretion of N tau-methylhistidine and creatinine per 24 h and per kg of non-muscular body weight, 1.15 mumol (N tau-methylhistidine) and 35 mumol (creatinine) respectively. For the calculation of myofibrillar protein breakdown and skeletal-muscle mass on the basis of N tau-methylhistidine and creatinine excretion, the values have to be corrected for non-muscular sources. Our data show that skeletal-muscle tissue is the major contributor of N tau-methylhistidine in urine, since it contributes as much as 75% to the urinary excretion.</jats:p

Minimal-invasive 3D laser printing of microimplants in organismo

Multi-photon 3D laser printing has gathered much attention in recent years as a means of manufacturing biocompatible scaffolds that can modify and guide cellular behavior in vitro. However, in vivo tissue engineering efforts have been limited so far to the implantation of beforehand 3D printed biocompatible scaffolds and in vivo bioprinting of tissue constructs from bioinks containing cells, biomolecules, and printable hydrogel formulations. Thus, a comprehensive 3D laser printing platform for in vivo and in situ manufacturing of microimplants raised from synthetic polymer-based inks is currently missing. Here, a platform for minimal-invasive manufacturing of microimplants directly in the organism is presented by one-photon photopolymerization and multi-photon 3D laser printing. Employing a commercially available elastomeric ink giving rise to biocompatible synthetic polymer-based microimplants, first applicational examples of biological responses to in situ printed microimplants are demonstrated in the teleost fish Oryzias latipes and in embryos of the fruit fly Drosophila melanogaster. This provides a framework for future studies addressing the suitability of inks for in vivo 3D manufacturing. The platform bears great potential for the direct engineering of the intricate microarchitectures in a variety of tissues in model organisms and beyond