Lipoyl synthase (LipA) catalyzes the formation of the lipoyl cofactor, which is employed by several multienzyme complexes for the oxidative decarboxylation of various alpha-keto acids, as well as the cleavage of glycine into CO(2) and NH(3), with concomitant transfer of its alpha-carbon to tetrahydrofolate, generating N(5),N(10)-methylenetetrahydrofolate. In each case, the lipoyl cofactor is tethered covalently in an amide linkage to a conserved lysine residue located on a designated lipoyl-bearing subunit of the complex. Genetic and biochemical studies suggest that lipoyl synthase is a member of a newly established class of metalloenzymes that use S-adenosyl-l-methionine (AdoMet) as a source of a 5'-deoxyadenosyl radical (5'-dA(*)), which is an obligate intermediate in each reaction. These enzymes contain iron-sulfur clusters, which provide an electron during the cleavage of AdoMet, forming l-methionine in addition to the primary radical. Recently, one substrate for lipoyl synthase has been shown to be the octanoylated derivative of the lipoyl-bearing subunit (E(2)) of the pyruvate dehydrogenase complex [Zhao, S., Miller, J. R., Jian, Y., Marletta, M. A., and Cronan, J. E., Jr. (2003) Chem. Biol. 10, 1293-1302]. Herein, we show that the octanoylated derivative of the lipoyl-bearing subunit of the glycine cleavage system (H-protein) is also a substrate for LipA, providing further evidence that the cofactor is synthesized on its target protein. Moreover, we show that the 5'-dA(*) acts directly on the octanoyl substrate, as evidenced by deuterium transfer from [octanoyl-d(15)]H-protein to 5'-deoxyadenosine. Last, our data indicate that 2 equiv of AdoMet are cleaved irreversibly in forming 1 equiv of [lipoyl]H-protein and are consistent with a model in which two LipA proteins are required to synthesize one lipoyl group.

Quinolinic acid is an intermediate in the biosynthesis of nicotinamide-containing redox cofactors. The ultimate step in the formation of quinolinic acid in prokaryotes is the condensation of iminosuccinate and dihydroxyacetone phosphate, which is catalyzed by the product of the nadA gene in Escherichia coli. A combination of UV-vis, Mössbauer, and EPR spectroscopies, along with analytical methods for the determination of iron and sulfide, demonstrates for the first time that anaerobically purified quinolinate synthetase (NadA) from E. coli contains one [4Fe-4S] cluster per polypeptide. The protein is active, catalyzing the formation of quinolinic acid with a Vmax [ET]-1 of 0.01 s-1.

Quinolinate synthase (NadA) catalyzes a unique condensation reaction between iminoaspartate and dihydroxyacetone phosphate, affording quinolinic acid, a central intermediate in the biosynthesis of nicotinamide adenine dinucleotide (NAD). Iminoaspartate is generated via the action of L-aspartate oxidase (NadB), which catalyzes the first step in the biosynthesis of NAD in most prokaryotes. NadA from Escherichia coli was hypothesized to contain an iron-sulfur cluster as early as 1991, because of its observed labile activity, especially in the presence of hyperbaric oxygen, and because its primary structure contained a CXXCXXC motif, which is commonly found in the [4Fe-4S] ferredoxin class of iron-sulfur (Fe/S) proteins. Indeed, using analytical methods in concert with Mössbauer and electron paramagnetic resonance spectroscopies, the protein was later shown to harbor a [4Fe-4S] cluster. Recently, the X-ray structure of NadA from Pyrococcus horikoshii was solved to 2.0 Å resolution [H. Sakuraba, H. Tsuge, K. Yoneda, N. Katunuma, and T. Ohshima, (2005) J. Biol. Chem. 280, pp. 26645-26648]. This protein does not contain a CXXCXXC motif, and no Fe/ S cluster was observed in the structure or even mentioned in the report. Moreover, rates of quinolinic acid production were reported to be 2.2 μmol min -1 mg -1 , significantly greater than that of E. coli NadA containing an Fe/S cluster (0.10 μmol min -1 mg -1 ), suggesting that the [4Fe-4S] cluster of E. coli NadA may not be necessary for catalysis. In the study described herein, nadA genes from both Mycobacterium tuberculosis and Pyrococcus horikoshii were cloned, and their protein products

Velocity dispersion of compressional stress waves in cylindrical rods of 61S-T6 aluminum alloy has been investigated experimentally. Electronically generated pulses of the desired shape and carrier frequency were used to drive the barium titanate and quartz transducers. Both the pulse echo and pulse transmission methods were employed. It was found that the group velocity depends strongly on the ratio of a, the radius of the specimen, to λ, the wavelength of the stress disturbance, when the value of a/λ is less than 2.5. For instance, in the neighborhood of a/λ equal to unity, the group velocity approaches that of Rayleigh surface waves which is 0.95 times that of the shear wave velocity when Poisson's ratio equals 0.29. As a/λ is increased, the group velocity also increases and approaches the bulk velocity as a limit. When a/λ is greater than 2.5, the group velocity does not deviate appreciably from the bulk velocity. This dispersion effect disappears when pulsed shear waves are used. The experimental error is estimated to be less than 2 percent.

In this paper a brief theory is given about the amplitude and probability density distribution of a narrow band shot noise. It is found that the probability density distribution follows the normal law and the amplitude distribution is the integral of the normal function. However, if the noise voltage is applied across a nonlinear impedance such as a type 1N34 crystal diode, both the amplitude and probability density distributions are changed. The change depends upon the current-voltage characteristic of the nonlinear impedance. An experimental method is described which can be used to measure the amplitude and the probability density distribution. The noise source was a photomultiplier tube followed by a narrow band amplifier centered at 16 kc. The measured results agree with that obtained by calculations.