Simultaneous determination of intracellular nucleotides and coenzymes in Yarrowia lipolytica producing lipid and lycopene by capillary zone electrophoresis.

Yarrowia lipolytica is an oleaginous yeast with promise in producing terpenoids such as lycopene. Though methods for analyzing primary metabolic intermediates have been established, further work is needed to better analyze nucleotides and coenzymes. Here, we presented an optimized method for the separation of nucleotides and coenzymes in Y. lipolytica using the capillary electrophoresis. The separation of twelve metabolites including four coenzymes, five nucleotides and three nucleosides was achieved within 32min using a voltage of 15kV and 70mM sodium carbonate/hydrogencarbonate buffer with 1.0% ß-CD at pH 10. The results show that the concentrations of adenosine triphosphate and nicotinamide adenine dinucleotide phosphate changed significantly between lycopene producing strain and the control, indicating that these two metabolites may be closely related with lycopene production. The optimized method provides a useful approach for future metabolic analysis of fermentation process as well as industrial strain improvement.

Optimization of overexpression of a chaperone protein of steroid C25 dehydrogenase for biochemical and biophysical characterization.

Molybdenum is an essential nutrient for metabolism in plant, bacteria, and animals. Molybdoenzymes are involved in nitrogen assimilation and oxidoreductive detoxification, and bioconversion reactions of environmental, industrial, and pharmaceutical interest. Molybdoenzymes contain a molybdenum cofactor (Moco), which is a pyranopterin heterocyclic compound that binds a molybdenum atom via a dithiolene group. Because Moco is a large and complex compound deeply buried within the protein, molybdoenzymes are accompanied by private chaperone proteins responsible for the cofactor's insertion into the enzyme and the enzyme's maturation. An efficient recombinant expression and purification of both Moco-free and Moco-containing molybdoenzymes and their chaperones is of paramount importance for fundamental and applied research related to molybdoenzymes. In this work, we focused on a D1 protein annotated as a chaperone of steroid C25 dehydrogenase (S25DH) from Sterolibacterium denitrificans Chol-1S. The D1 protein is presumably involved in the maturation of S25DH engaged in oxygen-independent oxidation of sterols. As this chaperone is thought to be a crucial element that ensures the insertion of Moco into the enzyme and consequently, proper folding of S25DH optimization of the chaperon's expression is the first step toward the development of recombinant expression and purification methods for S25DH. We have identified common E. coli strains and conditions for both expression and purification that allow us to selectively produce Moco-containing and Moco-free chaperones. We have also characterized the Moco-containing chaperone by EXAFS and HPLC analysis and identified conditions that stabilize both forms of the protein. The protocols presented here are efficient and result in protein quantities sufficient for biochemical studies.

Assembly scaffold NifEN: A structural and functional homolog of the nitrogenase catalytic component.

NifEN is a biosynthetic scaffold for the cofactor of Mo-nitrogenase (designated the M-cluster). Previous studies have revealed the sequence and structural homology between NifEN and NifDK, the catalytic component of nitrogenase. However, direct proof for the functional homology between the two proteins has remained elusive. Here we show that, upon maturation of a cofactor precursor (designated the L-cluster) on NifEN, the cluster species extracted from NifEN is spectroscopically equivalent and functionally interchangeable with the native M-cluster extracted from NifDK. Both extracted clusters display nearly indistinguishable EPR features, X-ray absorption spectroscopy/extended X-ray absorption fine structure (XAS/EXAFS) spectra and reconstitution activities, firmly establishing the M-cluster-bound NifEN (designated NifEN(M)) as the only protein other than NifDK to house the unique nitrogenase cofactor. Iron chelation experiments demonstrate a relocation of the cluster from the surface to its binding site within NifEN(M) upon maturation, which parallels the insertion of M-cluster into an analogous binding site in NifDK, whereas metal analyses suggest an asymmetric conformation of NifEN(M) with an M-cluster in one αß-half and an empty cluster-binding site in the other αß-half, which led to the proposal of a stepwise assembly mechanism of the M-cluster in the two αß-dimers of NifEN. Perhaps most importantly, NifEN(M) displays comparable ATP-independent substrate-reducing profiles to those of NifDK, which establishes the M-cluster-bound αß-dimer of NifEN(M) as a structural and functional mimic of one catalytic αß-half of NifDK while suggesting the potential of this protein as a useful tool for further investigations of the mechanistic details of nitrogenase.

Identity of cofactor bound to mycothiol conjugate amidase (Mca) influenced by expression and purification conditions.

Mycothiol serves as the primary reducing agent in Mycobacterium species, and is also a cofactor for the detoxification of xenobiotics. Mycothiol conjugate amidase (Mca) is a metalloamidase that catalyzes the cleavage of MS-conjugates to form a mercapturic acid, which is excreted from the mycobacterium, and 1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside. Herein we report on the metal cofactor preferences of Mca from Mycobacterium smegmatis and Mycobacterium tuberculosis. Importantly, results from homology models of Mca from M. smegmatis and M. tuberculosis suggest that the metal binding site of Mca is identical to that of the closely related protein N-acetyl-1-D-myo-inosityl-2-amino-2-deoxy-α-D-glucopyranoside deacetylase (MshB). This finding is supported by results from zinc ion affinity measurements that indicate Mca and MshB have comparable K(D)(ZnII) values (~10-20 pM). Furthermore, results from pull-down experiments using Halo-Mca indicate that Mca purifies with (stoichiometric) Fe(2+) when purified under anaerobic conditions, and Zn(2+) when purified under aerobic conditions. Consequently, Mca is likely a Fe(2+)-dependent enzyme under physiological conditions; with Zn(2+)-Mca an experimental artifact that could become biologically relevant under oxidatively stressed conditions. Importantly, these findings suggest that efforts towards the design of Mca inhibitors should include targeting the Fe(2+) form of the enzyme.

Cofactor trapping, a new method to produce flavin mononucleotide.

We have purified flavin mononucleotide (FMN) from a flavoprotein-overexpressing Escherichia coli strain by cofactor trapping. This approach uses an overexpressed flavoprotein to trap FMN, which is thus removed from the cascade regulating FMN production in E. coli. This, in turn, allows the isolation of highly pure FMN.

Esmond E. Snell--the pathfinder of B vitamins and cofactors.

Esmond E. Snell (1914-2003) was a giant of B-vitamin and enzyme research. His early research in bacterial nutrition had lead to the discovery of vitamins such as lipoic acid and folic acid, and an anti-vitamin avidin. He developed microbiological assay methods for riboflavin and other vitamins and amino acids, which are still used today. He also investigated the metabolism of vitamins, discovered pyridoxal and pyridoxamine as the active forms of vitamin B(6) and revealed the mechanism of transamination and other reactions catalysed by vitamin B(6) enzymes. His research in later years on pyruvoyl-dependent histidine decarboxylase unveiled the biogenesis mechanism of this first built-in cofactor. Throughout his career, he was a great mentor of many people, all of whom are inspired by his philosophy of science.

Metal trafficking for nitrogen fixation: NifQ donates molybdenum to NifEN/NifH for the biosynthesis of the nitrogenase FeMo-cofactor.

The molybdenum nitrogenase, present in a diverse group of bacteria and archea, is the major contributor to biological nitrogen fixation. The nitrogenase active site contains an iron-molybdenum cofactor (FeMo-co) composed of 7Fe, 9S, 1Mo, one unidentified light atom, and homocitrate. The nifQ gene was known to be involved in the incorporation of molybdenum into nitrogenase. Here we show direct biochemical evidence for the role of NifQ in FeMo-co biosynthesis. As-isolated NifQ was found to carry a molybdenum-iron-sulfur cluster that serves as a specific molybdenum donor for FeMo-co biosynthesis. Purified NifQ supported in vitro FeMo-co synthesis in the absence of an additional molybdenum source. The mobilization of molybdenum from NifQ required the simultaneous participation of NifH and NifEN in the in vitro FeMo-co synthesis assay, suggesting that NifQ would be the physiological molybdenum donor to a hypothetical NifEN/NifH complex.

Occurrence of 5'-deoxyadenosylcobalamin and its physiological function as the coenzyme of methylmalonyl-CoA mutase in a marine eukaryotic microorganism, Schizochytrium limacinum SR21.

A marine eukaryotic microorganism, Schizochytrium limacinum SR21, had the ability to absorb and accumulate exogenous cobalamin, which was converted to the cobalamin coenzymes 5'-deoxyadenosylcobalamin (20.1%) and methylcobalamin (29.6%). A considerably high activity (about 38 mU/mg protein) of 5'-deoxyadenosylcobalamin-dependent methylmalonyl-CoA mutase (EC 5.4.99.2) involved in amino acid and odd-chain fatty acid metabolism was found in the cell homogenate of S. limacinum SR21. The enzyme was purified to homogeneity and characterized.

Protein engineering of formate dehydrogenase.

NAD+-dependent formate dehydrogenase (FDH, EC 1.2.1.2) is one of the best enzymes for the purpose of NADH regeneration in dehydrogenase-based synthesis of optically active compounds. Low operational stability and high production cost of native FDHs limit their application in commercial production of chiral compounds. The review summarizes the results on engineering of bacterial and yeast FDHs aimed at improving their chemical and thermal stability, catalytic activity, switch in coenzyme specificity from NAD+ to NADP+ and overexpression in Escherichia coli cells.

Navigating the chaperone network: an integrative map of physical and genetic interactions mediated by the hsp90 chaperone.

Physical, genetic, and chemical-genetic interactions centered on the conserved chaperone Hsp90 were mapped at high resolution in yeast using systematic proteomic and genomic methods. Physical interactions were identified using genome-wide two hybrid screens combined with large-scale affinity purification of Hsp90-containing protein complexes. Genetic interactions were uncovered using synthetic genetic array technology and by a microarray-based chemical-genetic screen of a set of about 4700 viable yeast gene deletion mutants for hypersensitivity to the Hsp90 inhibitor geldanamycin. An extended network, consisting of 198 putative physical interactions and 451 putative genetic and chemical-genetic interactions, was found to connect Hsp90 to cofactors and substrates involved in a wide range of cellular functions. Two novel Hsp90 cofactors, Tah1 (YCR060W) and Pih1 (YHR034C), were also identified. These cofactors interact physically and functionally with the conserved AAA(+)-type DNA helicases Rvb1/Rvb2, which are key components of several chromatin remodeling factors, thereby linking Hsp90 to epigenetic gene regulation.

Extracellular RNA is a natural cofactor for the (auto-)activation of Factor VII-activating protease (FSAP).

FSAP (Factor VII-activating protease) is a new plasma-derived serine protease with putative dual functions in haemostasis, including activation of coagulation Factor VII and generation of urinary-type plasminogen activator (urokinase). The (auto-)activation of FSAP is facilitated by polyanionic glycosaminoglycans, such as heparin or dextran sulphate, whereas calcium ions stabilize the active form of FSAP. In the present study, extracellular RNA was identified and characterized as a novel FSAP cofactor. The conditioned medium derived from various cell types such as smooth muscle cells, endothelial cells, osteosarcoma cells or CHO (Chinese-hamster ovary) cells contained an acidic factor that initiated (auto-)activation of FSAP. RNase A, but not other hydrolytic enzymes (proteases, glycanases and DNase), abolished the FSAP cofactor activity, which was subsequently isolated by anion-exchange chromatography and unequivocally identified as RNA. In purified systems, as well as in plasma, different forms of natural RNA (rRNA, tRNA, viral RNA and artificial RNA) were able to (auto-)activate FSAP into the two-chain enzyme form. The specific binding of FSAP to RNA (but not to DNA) was shown by mobility-shift assays and UV crosslinking, thereby identifying FSAP as a new extracellular RNA-binding protein, the K(D) estimated to be 170-350 nM. Activation of FSAP occurred through an RNA-dependent template mechanism involving a nucleic acid size of at least 100 nt. In a purified system, natural RNA augmented the FSAP-dependent Factor VII activation several-fold (as shown by subsequent Factor Xa generation), as well as the FSAP-mediated generation of urokinase. Our results provide evidence for the first time that extracellular RNA, present at sites of cell damage or vascular injury, can serve an important as yet unrecognized cofactor function in haemostasis by inducing (auto-)activation of FSAP through a novel surface-dependent mechanism.

Prophenoloxidase (proPO) activation in Manduca sexta: an analysis of molecular interactions among proPO, proPO-activating proteinase-3, and a cofactor.

Proteolytic activation of prophenoloxidase (proPO) is an integral part of the insect immune system against pathogen and parasite infection. This reaction is mediated by a proPO-activating proteinase (PAP) and its cofactor in the tobacco hornworm, Manduca sexta (Proc. Natl. Acad. Sci. USA 95 (1998) 12220; J. Biol. Chem. 278 (2003) 3552; Insect Biochem. Mol. Biol. 33 (2003) 1049). The cofactor consists of two serine proteinase homologs (SPHs), which associate with immulectin-2, a calcium-dependent lectin that binds to lipopolysaccharide (Insect Biochem. Mol. Biol. 33 (2003) 197). In order to understand the auxiliary effect of SPH-1 and SPH-2 in proPO activation, we started to investigate the molecular interactions among proPO, PAP-3, and the proteinase-like proteins. M. sexta SPH-1 and SPH-2 were purified from hemolymph of prepupae by hydroxylapatite, gel filtration, lectin-affinity, and ion exchange chromatography. They existed as non-covalent oligomers with an average molecular mass of about 790 kDa. MALDI-TOF mass fingerprint analysis revealed a new cleavage site in SPH-1 before Asp85. The PAP cofactor did not significantly alter Michaelis constant (KM) or kcat of PAP-3 towards a synthetic substrate, acetyl-Ile-Glu-Ala-Arg-p-nitroanilide, but greatly enhanced proPO activation by PAP-3. The apparent KM for proPO was determined to be about 9.4 microg/ml, close to its estimated concentration in larval hemolymph. In the presence of excess proPO and a set amount of PAP-3, increasing levels of phenoloxidase (PO) activity were detected as more SPHs were added. Half of the maximum proPO activation occurred when the molar ratio of PAP-3 to SPH was 1:1.4. Gel filtration experiments suggested that proPO, PAP-3, and the cofactor formed a ternary complex.

Coenzyme precursor-assisted cooperative overexpression of an active pyridoxine 4-oxidase from Microbacterium luteolum.

The overexpression system of the active pyridoxine 4-oxidase from Microbacterium luteolum was developed. When chaperonin GroEL/ES genes in plasmid pKY206 were coexpressed, the pyridoxine 4-oxidase gene cloned in the vector pTrc99A was overexpressed in Escherichia coli JM109 cultured in LB medium containing 50microM riboflavin, the precursor of coenzyme (FAD) of the enzyme, under the cold stress at 23 degrees C. The crude extract from the cotransformant cells showed 88-fold higher specific activity than that from M. luteolum. The chaperonins, cold stress, and the riboflavin cooperatively served to increase the soluble form of the enzyme. A significant correlation between the specific activity and percentage of the soluble form in the total expressed enzyme was found. The overexpressed pyridoxine 4-oxidase was easily purified to homogeneity with two steps of the conventional column chromatography.

Occurrence of coenzyme forms of vitamin B12 in a cultured purple laver (Porphyla yezoensis).

Porphyra yezoensis (Susabinori, an edible purple laver), which was cultured aseptically for 12 weeks and then lyophilized, contained 50+/-2 microg/g of vitamin B(12) per 100 g dry weight. Coenzyme forms of vitamin B(12) (about 60% of the total vitamin B(12)) were found in the cultured purple laver aseptically, which may have the ability to biosynthesize the coenzymes.

Subsecond separation of cellular flavin coenzymes by microchip capillary electrophoresis with laser-induced fluorescence detection.

In this article, it was demonstrated that a subsecond separation of cellular metabolites such as riboflavin (RF), flavin mononucleotide (FMN), and flavin-adenine dinucleotide (FAD) was achieved using microchip capillary electrophoresis with laser-induced fluorescence detection. The influences of crucial parameters that governed analysis time (e.g., channel length and electric field for separation) and separation resolution (e.g., sample size) were investigated, both in theoretical aspects and experimental practice. Quantitative analyses were performed that exhibited linear dynamic range of two orders of magnitude, with calculated detection limits of 34, 201, and 127 nM for RF, FAD, and FMN, respectively. To test the validity of the method, it was successfully applied to characterize several recombinant flavin-binding domains in a human neuronal nitric oxide synthase.

Characterization of pyridine nucleotide coenzymes in the hyperthermophilic archaeon Pyrococcus furiosus.

Pyridine-type nucleotides were identified in cell-free extracts of the hyperthermophilic archaeon Pyrococcus furiosus by their ability to replace authentic nicotinamide adenine dinucleotide (phosphate) [NAD(P)] in assays using pure P. furiosus enzymes. The nucleotides were purified using a combination of ion-exchange and reverse-phase chromatography. They were identified as NAD and NADP by analyses using liquid chromatography-mass spectrometry and high performance liquid chromatography. Their intracellular concentrations were measured in P. furiosus grown using maltose and peptides as the carbon sources. The concentrations decreased during the lag phase but remained constant during the exponential phase at approximately 0.17 and 0.13 mM, respectively. The amount of NAD was significantly lower (more than four-fold lower) than that in mesophilic bacteria, although the NADP concentration was comparable. The internal concentrations of NADH and NADPH in P. furiosus were determined to be 0.14 mM and 0.04 mM, respectively. The overall cellular concentration of NAD(P)(H) in P. furiosus (0.48 mM) is about half the value in the mesophiles. The NAD(H)/NADP(H) ratio in P. furiosus is consistent with the preferred use of NADP by several catabolic enzymes that have been purified from this organism. The mechanisms by which hyperthermophiles stabilize these thermally labile nicotinamide nucleotides are not known.

Expression and purification of an active, full-length hepatitis C viral NS4A.

The nonstructural protein 3 (NS3) of the hepatitis C virus (HCV) is a bifunctional protein with protease and helicase activities. Nonstructural protein 4A (NS4A) is preceded by NS3 and augments the proteolytic activity of NS3 through protein-protein interaction. The central domain of NS4A has been shown to be sufficient for the enhancement of the NS3 protease activity. However, investigations on the roles of the N-terminal and the C-terminal regions of NS4A have been hampered by the difficulty of purification of full-length NS4A, a polypeptide that contains highly hydrophobic amino acid residues. Here we report a procedure by which one can produce and purify an active, full-length NS4A using maltose-binding protein fusion method. The full-length NS4A fused to the maltose binding protein is soluble and maintains its NS3 protease-enhancing activity.

Pyrroloquinoline quinone: a novel vitamin?

Pyrroloquinoline quinone (PQQ), otherwise known as methoxatin, is a water-soluble, redox-cycling orthoquinone that was initially isolated from cultures of methylotropic bacteria. It has been found to be a cofactor of some bacterial alcohol dehydrogenases, and is present in many animal tissues. It may be a novel vitamin because it has been shown to be essential for normal growth and development. The redox-cycling ability of PQQ enables it to scavenge or generate superoxide. When fed to animals as a supplement, PQQ prevents oxidative changes that would ordinarily occur. It has been reported to inhibit glutamate decarboxylase activity and protect against N-methyl-D-aspartate (NMDA) receptor-mediated neurotoxicity in the brain. It appears that in the whole animal, however, PQQ does not cross the blood-brain barrier. Furthermore, it increases nerve growth factor (NGF) synthesis in mouse astroglial cells, but has to be bound to glycine to penetrate and exert this effect in whole brain. It may therefore be regarded as a "Janus faced" molecule, with its potential for a therapeutic role in the brain still in question.