Adaptive laboratory evolution recruits the promiscuity of succinate semialdehyde dehydrogenase to repair different metabolic deficiencies.

Promiscuous enzymes often serve as the starting point for the evolution of novel functions. Yet, the extent to which the promiscuity of an individual enzyme can be harnessed several times independently for different purposes during evolution is poorly reported. Here, we present a case study illustrating how NAD(P)-dependent succinate semialdehyde dehydrogenase of Escherichia coli (Sad) is independently recruited through various evolutionary mechanisms for distinct metabolic demands, in particular vitamin biosynthesis and central carbon metabolism.

A pH-dependent shift of redox cofactor specificity in a benzyl alcohol dehydrogenase of aromatoleum aromaticum EbN1.

We characterise a reversible bacterial zinc-containing benzyl alcohol dehydrogenase (BaDH) accepting either NAD or NADP as a redox cofactor. Remarkably, its redox cofactor specificity is pH-dependent with the phosphorylated cofactors favored at lower and the dephospho-forms at higher pH. BaDH also shows different steady-state kinetic behavior with the two cofactor forms.

Structural and mechanistic basis of the central energy-converting methyltransferase complex of methanogenesis.

Methanogenic archaea inhabiting anaerobic environments play a crucial role in the global biogeochemical material cycle. The most universal electrogenic reaction of their methane-producing energy metabolism is catalyzed by -methyl-tetrahydromethanopterin: coenzyme M methyltransferase (MtrABCDEFGH), which couples the vectorial Na transport with a methyl transfer between the one-carbon carriers tetrahydromethanopterin and coenzyme M via a vitamin B derivative (cobamide) as prosthetic group. We present the 2.

Cytosolic sorting platform complexes shuttle type III secretion system effectors to the injectisome in Yersinia enterocolitica.

Bacteria use type III secretion injectisomes to inject effector proteins into eukaryotic target cells. Recruitment of effectors to the machinery and the resulting export hierarchy involve the sorting platform. These conserved proteins form pod structures at the cytosolic interface of the injectisome but are also mobile in the cytosol.

Dying transplanted neural stem cells mediate survival bystander effects in the injured brain.

Neural stem and progenitor cell (NSPC) transplants provide neuroprotection in models of acute brain injury, but the underlying mechanisms are not fully understood. Here, we provide evidence that caspase-dependent apoptotic cell death of NSPCs is required for sending survival signals to the injured brain. The secretome of dying NSPCs contains heat-stable proteins, which protect neurons against glutamate-induced toxicity and trophic factor withdrawal in vitro, and from ischemic brain damage in vivo.

Adaptation of CM MSU 529 to Aerobic Growth: A Proteomic Approach.

The study describes the effect of aerobic conditions on the proteome of homofermentative lactic acid bacterium CM MSU 529 grown in a batch culture. Aeration caused the induction of the biosynthesis of 43 proteins, while 14 proteins were downregulated as detected by label-free LC-MS/MS. Upregulated proteins are involved in oxygen consumption (Pox, LctO, pyridoxine 5'-phosphate oxidase), xylulose 5-phosphate conversion (Xfp), pyruvate metabolism (PdhD, AlsS, AlsD), reactive oxygen species (ROS) elimination (Tpx, TrxA, Npr), general stress response (GroES, PfpI, universal stress protein, YqiG), antioxidant production (CysK, DkgA), pyrimidine metabolism (CarA, CarB, PyrE, PyrC, PyrB, PyrR), oligopeptide transport and metabolism (OppA, PepO), and maturation and stability of ribosomal subunits (RbfA, VicX).

In Vitro Biosynthesis of the [Fe]-Hydrogenase Cofactor Verifies the Proposed Biosynthetic Precursors.

In the FeGP cofactor of [Fe]-hydrogenase, low-spin Fe is in complex with two CO ligands and a pyridinol derivative; the latter ligates the iron with a 6-acylmethyl substituent and the pyridinol nitrogen. A guanylylpyridinol derivative, 6-carboxymethyl-3,5-dimethyl-4-guanylyl-2-pyridinol (3), is produced by the decomposition of the FeGP cofactor under irradiation with UV-A/blue light and is also postulated to be a precursor of FeGP cofactor biosynthesis. HcgC and HcgB catalyze consecutive biosynthesis steps leading to 3.

Structural Rearrangements of a Dodecameric Ketol-Acid Reductoisomerase Isolated from a Marine Thermophilic Methanogen.

Ketol-acid reductoisomerase (KARI) orchestrates the biosynthesis of branched-chain amino acids, an elementary reaction in prototrophic organisms as well as a valuable process in biotechnology. Bacterial KARIs belonging to class I organise as dimers or dodecamers and were intensively studied to understand their remarkable specificity towards NADH or NADPH, but also to develop antibiotics. Here, we present the first structural study on a KARI natively isolated from a methanogenic archaea.

Three-megadalton complex of methanogenic electron-bifurcating and CO-fixing enzymes.

The first reaction of the methanogenic pathway from carbon dioxide (CO) is the reduction and condensation of CO to formyl-methanofuran, catalyzed by formyl-methanofuran dehydrogenase (Fmd). Strongly reducing electrons for this reaction are generated by heterodisulfide reductase (Hdr) in complex with hydrogenase or formate dehydrogenase (Fdh) using a flavin-based electron-bifurcation mechanism. Here, we report enzymological and structural characterizations of Fdh-Hdr-Fmd complexes from .

Crystal structure of a key enzyme for anaerobic ethane activation.

Ethane, the second most abundant hydrocarbon gas in the seafloor, is efficiently oxidized by anaerobic archaea in syntrophy with sulfate-reducing bacteria. Here, we report the 0.99-angstrom-resolution structure of the proposed ethane-activating enzyme and describe the specific traits that distinguish it from methane-generating and -consuming methyl-coenzyme M reductases.

Multiple Drug-Induced Stress Responses Inhibit Formation of Escherichia coli Biofilms.

In most ecosystems, bacteria exist primarily as structured surface-associated biofilms that can be highly tolerant to antibiotics and thus represent an important health issue. Here, we explored drug repurposing as a strategy to identify new antibiofilm compounds, screening over 1,000 compounds from the Prestwick Chemical Library of approved drugs for specific activities that prevent biofilm formation by Most growth-inhibiting compounds, which include known antibacterial but also antiviral and other drugs, also reduced biofilm formation. However, we also identified several drugs that were biofilm inhibitory at doses where only a weak effect or no effect on planktonic growth could be observed.

Mobilization and Cellular Distribution of Phosphate in the Diatom .

Unicellular organisms that live in marine environments must cope with considerable fluctuations in the availability of inorganic phosphate (P). Here, we investigated the extracellular P concentration-dependent expression, as well as the intracellular or extracellular localization, of phosphatases and phosphate transporters of the diatom . We identified P-regulated plasma membrane-localized, ER-localized, and secreted phosphatases, in addition to plasma membrane-localized, vacuolar membrane-localized, and plastid-surrounding membrane-localized phosphate transporters that were also regulated in a P concentration-dependent manner.

Peroxisomal targeting of a protein phosphatase type 2C via mitochondrial transit.

Correct intracellular distribution of proteins is critical for the function of eukaryotic cells. Certain proteins are targeted to more than one cellular compartment, e.g.

Drosophila carboxypeptidase D (SILVER) is a key enzyme in neuropeptide processing required to maintain locomotor activity levels and survival rate.

Neuropeptides are processed from larger preproproteins by a dedicated set of enzymes. The molecular and biochemical mechanisms underlying preproprotein processing and the functional importance of processing enzymes are well-characterised in mammals, but little studied outside this group. In contrast to mammals, Drosophila melanogaster lacks a gene for carboxypeptidase E (CPE), a key enzyme for mammalian peptide processing.

Two Different Quinohemoprotein Amine Dehydrogenases Initiate Anaerobic Degradation of Aromatic Amines in Aromatoleum aromaticum EbN1.

Aromatic amines like 2-phenylethylamine (2-PEA) and benzylamine (BAm) have been identified as novel growth substrates of the betaproteobacterium EbN1, which degrades a wide variety of aromatic compounds in the absence of oxygen under denitrifying growth conditions. The catabolic pathway of these amines was identified, starting with their oxidative deamination to the corresponding aldehydes, which are then further degraded via the enzymes of the phenylalanine or benzyl alcohol metabolic pathways. Two different periplasmic quinohemoprotein amine dehydrogenases involved in 2-PEA or BAm metabolism were identified and characterized.

Characterization of an Aldehyde Oxidoreductase From the Mesophilic Bacterium EbN1, a Member of a New Subfamily of Tungsten-Containing Enzymes.

The biochemical properties of a new tungsten-containing aldehyde oxidoreductase from the mesophilic betaproteobacterium EbN1 (AOR ) are presented in this study. The enzyme was purified from phenylalanine-grown cells of an overexpressing mutant lacking the gene for an aldehyde dehydrogenase normally involved in anaerobic phenylalanine degradation. AOR catalyzes the oxidation of a broad variety of aldehydes to the respective acids with either viologen dyes or NAD as electron acceptors.

The Bacterial [Fe]-Hydrogenase Paralog HmdII Uses Tetrahydrofolate Derivatives as Substrates.

[Fe]-hydrogenase (Hmd) catalyzes the reversible hydrogenation of methenyl-tetrahydromethanopterin (methenyl-H MPT ) with H . H MPT is a C1-carrier of methanogenic archaea. One bacterial genus, Desulfurobacterium, contains putative genes for the Hmd paralog, termed HmdII, and the HcgA-G proteins.

The multicatalytic compartment of propionyl-CoA synthase sequesters a toxic metabolite.

Cells must cope with toxic or reactive intermediates formed during metabolism. One coping strategy is to sequester reactions that produce such intermediates within specialized compartments or tunnels connecting different active sites. Here, we show that propionyl-CoA synthase (PCS), an ∼ 400-kDa homodimer, three-domain fusion protein and the key enzyme of the 3-hydroxypropionate bi-cycle for CO fixation, sequesters its reactive intermediate acrylyl-CoA.

The Ustilago maydis repetitive effector Rsp3 blocks the antifungal activity of mannose-binding maize proteins.

To cause disease in maize, the biotrophic fungus Ustilago maydis secretes a large arsenal of effector proteins. Here, we functionally characterize the repetitive effector Rsp3 (repetitive secreted protein 3), which shows length polymorphisms in field isolates and is highly expressed during biotrophic stages. Rsp3 is required for virulence and anthocyanin accumulation.

Mating-Induced Differential Peptidomics of Neuropeptides and Protein Hormones in Agrotis ipsilon Moths.

In many insects, mating induces drastic changes in male and female responses to sex pheromones or host-plant odors. In the male moth Agrotis ipsilon, mating induces a transient inhibition of behavioral and neuronal responses to the female sex pheromone. As neuropeptides and peptide hormones regulate most behavioral processes, we hypothesize that they could be involved in this mating-dependent olfactory plasticity.

Feeding-induced changes in allatostatin-A and short neuropeptide F in the antennal lobes affect odor-mediated host seeking in the yellow fever mosquito, Aedes aegypti.

Aedes aegypti is a model species in which the endogenous regulation of odor-mediated host seeking behavior has received some attention. Sugar feeding and host seeking in female A. aegypti are transiently inhibited following a blood meal.

Phylogenetic and Structural Comparisons of the Three Types of Methyl Coenzyme M Reductase from Methanococcales and Methanobacteriales.

The phylogenetically diverse family of methanogenic archaea universally use methyl coenzyme M reductase (MCR) for catalyzing the final methane-forming reaction step of the methanogenic energy metabolism. Some methanogens of the orders and contain two isoenzymes. Comprehensive phylogenetic analyses on the basis of all three subunits grouped MCRs from and into three distinct types: (i) MCRs from , (ii) MCRs from and , and (iii) MCRs from The first and second types contain MCR isoenzymes I and II from , respectively; therefore, they were designated MCR type I and type II and accordingly; the third one was designated MCR type III.