Structural Studies on Flavin Reductase PheA2 Reveal Binding of NAD in an Unusual Folded Conformation and Support Novel Mechanism of Action

The catabolism of toxic phenols in the thermophilic organism Bacillus thermoglucosidasius A7 is initiated by a two-component enzyme system. The smaller flavin reductase PheA2 component catalyzes the NADH-dependent reduction of free FAD according to a ping-pong bisubstrate-biproduct mechanism. The reduced FAD is then used by the larger oxygenase component PheA1 to hydroxylate phenols to the corresponding catechols. We have determined the x-ray structure of PheA2 containing a bound FAD cofactor (2.2 Angstrom), which is the first structure of a member of this flavin reductase family. We have also determined the x-ray structure of reduced holo-PheA2 in complex with oxidized NAD (2.1 Angstrom). PheA2 is a single domain homodimeric protein with each FAD-containing subunit being organized around a six-stranded beta-sheet and a capping alpha-helix. The tightly bound FAD prosthetic group (K-d=10 nM) binds near the dimer interface, and the re face of the FAD isoalloxazine ring is fully exposed to solvent. The addition of NADH to crystalline PheA2 reduced the flavin cofactor, and the NAD product was bound in a wide solvent-accessible groove adopting an unusual folded conformation with ring stacking. This is the first observation of an enzyme that is very likely to react with a folded compact pyridine nucleotide. The PheA2 crystallographic models strongly suggest that reactive exogenous FAD substrate binds in the NADH cleft after release of NAD product. Nanoflow electrospray mass spectrometry data indeed showed that PheA2 is able to bind one FAD cofactor and one FAD substrate. In conclusion, the structural data provide evidence that PheA2 contains a dual binding cleft for NADH and FAD substrate, which alternate during catalysis

Evolution of Neutron-Star, Carbon-Oxygen White-Dwarf Binaries

At least one, but more likely two or more, eccentric neutron-star, carbon-oxygen white-dwarf binaries with an unrecycled pulsar have been observed. According to the standard scenario for evolving neutron stars which are recycled in common envelope evolution we expect to observe \gsim 50 such circular neutron star-carbon oxygen white dwarf binaries, since their formation rate is roughly equal to that of the eccentric binaries and the time over which they can be observed is two orders of magnitude longer, as we shall outline. We observe at most one or two such circular binaries and from that we conclude that the standard scenario must be revised. Introducing hypercritical accretion into common envelope evolution removes the discrepancy by converting the neutron star into a black hole which does not emit radio waves, and therefore would not be observed.Comment: 25 pages, 1 figure, accepted in Ap

A Survey of Best Monotone Degree Conditions for Graph Properties

We survey sufficient degree conditions, for a variety of graph properties, that are best possible in the same sense that Chvatal's well-known degree condition for hamiltonicity is best possible.Comment: 25 page

An Unusual Spectral State of an Ultra-luminous Very Soft X-ray Source during Outburst

We report the results of Chandra and XMM-Newton observations of a new outburst of an ultra-luminous X-ray source in M101. During a Chandra monitoring observation of M101, M101 ULX-1 was found to be in outburst in 2004 December, the second outburst in 2004. The peak bolometric luminosity is about 3e40 erg/s (7e39 erg/s in 0.3-7 keV). The outburst spectra are very soft and can generally be fitted with a blackbody model with temperatures of 40-80 eV, similar to supersoft X-ray sources in the Milky Way and in the Magellanic Clouds. In one Chandra observation, the source spectrum appears to be harder with a temperature of 150 eV. Such a spectral state is rarely seen in M101 ULX-1 and no X-ray source in the Milky Way shows this kind of spectrum. However, such an unusual spectral state very likely belongs to a new class of X-ray sources, quasisoft X-ray sources, recently discovered in nearby galaxies. M101 ULX-1 returned to supersoft state in a subsequent XMM-Newton observation. Based on the two outbursts in 2004, the extremely high luminosity (L_bol=1e40-1e41 erg/s), very soft X-ray spectra (kT=40-150 eV), transient behavior, and state transition provide strong evidence that M101 ULX-1 harbors an intermediate-mass black hole.Comment: 4 pages, 2 figures, accepted for publication in ApJ

The "Twin Jet" Planetary Nebula M2-9

We present a model for the structure, temporal behavior, and evolutionary status of the bipolar nebula M2-9. According to this model the system consists of an AGB or post-AGB star and a hot white dwarf companion, with an orbital period of about 120 years. The white dwarf has undergone a symbiotic nova eruption about 1200 years ago, followed by a supersoft x-ray source phase. The positional shift of the bright knots in the inner nebular lobes is explained in terms of a revolving ionizing source. We show that the interaction between the slow, AGB star's wind, and a collimated fast wind from the white dwarf clears a path for the ionizing radiation in one direction, while the radiation is attenuated in others. This results in the mirror-symmetric (as opposed to the more common point-symmetric) shift in the knots. We show that M2-9 provides an important evolutionary link among planetary nebulae with binary central stars, symbiotic systems, and supersoft x-ray sources.Comment: 13 pages + 2 figures. Submitted to Ap

Real-time monitoring of enzymatic DNA hydrolysis by electrospray ionization mass spectrometry

A fast and direct method for the monitoring of enzymatic DNA hydrolysis was developed using electrospray ionization mass spectrometry. We incorporated the use of a robotic chip-based electrospray ionization source for increased reproducibility and throughput. The mass spectrometry method allows the detection of DNA fragments and intact non-covalent protein–DNA complexes in a single experiment. We used the method to monitor in real-time single-stranded (ss) DNA hydrolysis by colicin E9 DNase and to characterize transient non-covalent E9 DNase–DNA complexes present during the hydrolysis reaction. The mass spectra showed that E9 DNase interacts with ssDNA in the absence of a divalent metal ion, but is strictly dependent on Ni(2+) or Co(2+) for ssDNA hydrolysis. We demonstrated that the sequence selectivity of E9 DNase is dependent on the ratio protein:ssDNA or the ssDNA concentration and that only 3′-hydroxy and 5′-phosphate termini are produced. It was also shown that the homologous E7 DNase is reactive with Zn(2+) as transition metal ion and that this DNase displays a different sequence selectivity. The method described is of general use to analyze the reactivity and specificity of nucleases

Double Neutron Star Systems and Natal Neutron Star Kicks

We study the four double neutron star systems found in the Galactic disk in terms of the orbital characteristics of their immediate progenitors and the natal kicks imparted to neutron stars. Analysis of the effect of the second supernova explosion on the orbital dynamics, combined with recent results from simulations of rapid accretion onto neutron stars lead us to conclude that the observed systems could not have been formed had the explosion been symmetric. Their formation becomes possible if kicks are imparted to the radio-pulsar companions at birth. We identify the constraints imposed on the immediate progenitors of the observed double neutron stars and calculate the ranges within which their binary characteristics (orbital separations and masses of the exploding stars) are restricted. We also study the dependence of these limits on the magnitude of the kick velocity and the time elapsed since the second explosion. For each of the double neutron stars, we derive a minimum kick magnitude required for their formation, and for the two systems in close orbits we find it to exceed 200km/s. Lower limits are also set to the center-of-mass velocities of double neutron stars, and we find them to be consistent with the current proper motion observations.Comment: 25 pages, 6 figs (9 parts), 4 tables, AASTeX, Accepted in Ap

Mutant mitochondrial elongation factor G1 and combined oxidative phosphorylation deficiency

Although most components of the mitochondrial translation apparatus are encoded by nuclear genes, all known molecular defects associated with impaired mitochondrial translation are due to mutations in mitochondrial DNA. We investigated two siblings with a severe defect in mitochondrial translation, reduced levels of oxidative phosphorylation complexes containing mitochondrial DNA (mtDNA)–encoded subunits, and progressive hepatoencephalopathy. We mapped the defective gene to a region on chromosome 3q containing elongation factor G1 (EFG1), which encodes a mitochondrial translation factor. Sequencing of EFG1 revealed a mutation affecting a conserved residue of the guanosine triphosphate (GTP)–binding domain. These results define a new class of gene defects underlying disorders of oxidative phosphorylation

Optical Detection of Two Intermediate Mass Binary Pulsar Companions

We report the detection of probable optical counterparts for two Intermediate Mass Binary Pulsar (IMBP) systems, PSR J1528-3146 and PSR J1757-5322. Recent radio pulsar surveys have uncovered a handful of these systems with putative massive white dwarf companions, thought to have an evolutionary history different from that of the more numerous class of Low Mass Binary Pulsars (LMBPs) with He white dwarf companions. The study of IMBP companions via optical observations offers us several new diagnostics: the evolution of main sequence stars near the white-dwarf-neutron star boundary, the physics of white dwarfs close to the Chandrasekhar limit, and insights into the recycling process by which old pulsars are spun up to high rotation frequencies. We were unsuccessful in our attempt to detect optical counterparts of PSR J1141-6545, PSR J1157-5112, PSR J1435-6100, and PSR J1454-5846.Comment: 9 pages, 2 figures, accepted for publication in ApJ

The Role of Helium Stars in the Formation of Double Neutron Stars

We have calculated the evolution of 60 model binary systems consisting of helium stars in the mass range of M_He= 2.5-6Msun with a 1.4Msun neutron star companion to investigate the formation of double neutron star systems.Orbital periods ranging from 0.09 to 2 days are considered, corresponding to Roche lobe overflow starting from the helium main sequence to after the ignition of carbon burning in the core. We have also examined the evolution into a common envelope phase via secular instability, delayed dynamical instability, and the consequence of matter filling the neutron star's Roche lobe. The survival of some close He-star neutron-star binaries through the last mass transfer episode (either dynamically stable or unstable mass transfer phase) leads to the formation of extremely short-period double neutron star systems (with P<~0.1days). In addition, we find that systems throughout the entire calculated mass range can evolve into a common envelope phase, depending on the orbital period at the onset of mass transfer. The critical orbital period below which common envelope evolution occurs generally increases with M_He. In addition, a common envelope phase may occur during a short time for systems characterized by orbital periods of 0.1-0.5 days at low He-star masses (~ 2.6-3.3Msun). The existence of a short-period population of double neutron stars increases the predicted detection rate of inspiral events by ground-based gravitational-wave detectors and impacts their merger location in host galaxies and their possible role as gamma-ray burst progenitors. We use a set of population synthesis calculations and investigate the implications of the mass-transfer results for the orbital properties of DNS populations.Comment: 30 pages, Latex (AASTeX), 1 table, 8 figures. To appear in ApJ, v592 n1 July 20, 200