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Microalgae are viewed as a potential future agricultural and biofuel feedstock and also provide an ideal biological means of carbon sequestration based on rapid growth rates and high biomass yields. Any potential improvement using high-yield microalgae to fix carbon will require additional fertilizer inputs to provide the necessary nitrogen required for protein and nucleotide biosynthesis. The free-living diazotroph Azotobacter vinelandii can fix nitrogen under aerobic conditions in the presence of reduced carbon sources such as sucrose or glycerol and is also known to produce a variety of siderophores to scavenge different metals from the environment. In this study, we identified two strains of green algae, Neochloris oleoabundans and Scenedesmus sp. BA032, that are able to utilize the A. vinelandii siderophore azotobactin as a source of nitrogen to support growth. When grown in a co-culture, S. sp. BA032 and N. oleoabundans obtained the nitrogen required for growth through the association with A. vinelandii. These results, indicating a commensalistic relationship, provide a proof of concept for developing a mutualistic or symbiotic relationship between these two species using siderophores as a nitrogen shuttle and might further indicate an additional fate of siderophores in the environment. [ABSTRACT FROM AUTHOR]

Nitrogenase catalyzes the reduction of N2 and protons to yield two NH3 and one H2. Substrate binding occurs at a complex organo-metallocluster called FeMo-cofactor (FeMo-co). Each catalytic cycle involves the sequential delivery of eight electrons/protons to this cluster, and this process has been framed within a kinetic scheme developed by Lowe and Thorneley. Rapid freezing of a modified nitrogenase under turnover conditions using diazene, methyldiazene (HN = N-CH3), or hydrazine as substrate recently was shown to trap a common S=½ intermediate, designated I. It was further concluded that the two N-atoms of N2 are hydrogenated alternately ("Alternating" (A) pathway). In the present work, Q-band CW EPR and 95Mo ESEEM spectroscopy reveal such samples also contain a common intermediate with FeMo-co in an integer-spin state having a ground-state "non-Kramers" doublet. This species, designated H, has been characterized by ESEEM spectroscopy using a combination of 14,15N isotopologs plus 1,2H isotopologs of methyldiazene. It is concluded that: H has NH2 bound to FeMo-co and corresponds to the penultimate intermediate of N2 hydrogenation, the state formed after the accumulation of seven electrons/protons and the release of the first NH3; I corresponds to the final intermediate in N2 reduction, the state formed after accumulation of eight electrons/protons, with NH3 still bound to FeMo-co prior to release and regeneration of resting-state FeMo-co. A proposed unification of the Lowe-Thorneley kinetic model with the "prompt" alternating reaction pathway represents a draft mechanism for N2 reduction by nitrogenase. [ABSTRACT FROM AUTHOR]

Nitrogenase reduces dinitrogen (N2) by six electrons and six protons at an active-site metallocluster called FeMo cofactor, to yield two ammonia molecules. Insights into the mechanism of substrate reduction by nitrogenase have come from recent successes in trapping and characterizing intermediates generated during the reduction of protons as well as nitrogenous and alkyne substrates by MoFe proteins with amino acid substitutions. Here, we describe an intermediate generated at a high concentration during reduction of the natural nitrogenase substrate, N2, by wild-type MoFe protein, providing evidence that it contains N2 bound to the active-site FeMo cofactor. When MoFe protein was frozen at 77 K during steady- state turnover with N2, the S = 3⁄2 EPR signal (g [4.3, 3.64, 2.00]) arising from the resting state of FeMo cofactor was observed to convert to a rhombic, S = ½, signal (g [2.08, 1.99, 1.97]). The intensity of the N2-dependent EPR signal increased with increasing N2 partial pressure, reaching a maximum intensity of approximately 20% of that of the original FeMo cofactor signal at ≥ 0.2 atm N2. An almost complete loss of resting FeMo cofactor signal in this sample implies that the remainder of the enzyme has been reduced to an EPR-silent intermediate state. The N2-dependent EPR signal intensity also varied with the ratio of Fe protein to MoFe protein (electron flux through nitrogenase), with the maximum signal intensity observed with a ratio of 2:1 (1:1 Fe protein:FeMo cofactor) or higher. The pH optimum for the signal was 7.1. The N2-dependent EPR signal intensity exhibited a linear dependence on the square root of the EPR microwave power in contrast to the nonlinear response of signal intensity observed for hydrazine-, diazene-, and methyldiazene-trapped states. 15N ENDOR spectroscopic analysis of MoFe protein captured during turnover with 15N2 revealed a 15N nuclear spin coupled to the FeMo cofactor with a hyperfine tensor A [0.9, 1.4, 0.45] MHz establishing that an N2-derived species was trapped on the FeMo cofactor. The observation of a single type of 15N-coupled nucleus from the field dependence, along with the absence of an associated exchangeable ¹H ENDOR signal, is consistent with an N2 molecule bound end-on to the FeMo cofactor. [ABSTRACT FROM AUTHOR]

Nitrogenase catalyzes the sequential addition of six electrons and six protons to a N2 that is bound to the active site metal cluster FeMo-cofactor, yielding two ammonia molecules. The nature of the intermediates bound to FeMo-cofactor along this reduction pathway remains unknown, although it has been suggested that there are intermediates at the level of reduction of diazene (HN=NH, also called diimide) and hydrazine (H2N-NH2). Through in situ generation of diazene during nitrogenase turnover, we show that diazene is a substrate for the wild-type nitrogenase and is reduced to NH3. Diazene reduction, like N2 reduction, is inhibited by H2. This contrasts with the absence of H2 inhibition when nitrogenase reduces hydrazine. These results support the existence of an intermediate early in the N2 reduction pathway at the level of reduction of diazene. Freeze-quenching a MoFe protein variant with α-195His substituted by Gln and α-70Val substituted by Ala during steady-state turnover with diazene resulted in conversion of the S = ¾ resting state FeMo-cofactor to a novel S = ½ state with g1 = 2.09,g2 = 2.01, and g3 ∼ 1.98. 15N- and 1H-ENDOR establish that this state consists of a diazene-derived [-NHx] moiety bound to FeMo-cofactor. This moiety is indistinguishable from the hydrazine-derived [-NHx] moiety bound to FeMo-cofactor when the same MoFe protein is trapped during turnover with hydrazine. These observations suggest that diazene joins the normal N2-reduction pathway, and that the diazene- and hydrazine-trapped turnover states represent the same intermediate in the normal reduction of N2 by nitrogenase. Implications of these findings for the mechanism of N2 reduction by nitrogenase are discussed. [ABSTRACT FROM AUTHOR]

A major obstacle to understanding the reduction of N2 to NH3 by nitrogenase has been the impossibility of synchronizing electron delivery to the MoFe protein for generation of specific enzymatic intermediates. When an intermediate is trapped without synchronous electron delivery, the number of electrons, n, it has accumulated is unknown. Consequently, the intermediate is untethered from kinetic schemes for reduction, which are indexed by n. We show that a trapped intermediate itself provides a "synchronously prepared" initial state, and its relaxation to the resting state at 253 K, conditions that prevent electron delivery to MoFe protein, can be analyzed to reveal n and the nature of the relaxation reactions. The approach is applied to the "H+/H- intermediate" (A) that appears during turnover both in the presence and absence of N2 substrate. A exhibits an S = 1/2 EPR signal from the active-site iron-molybdenum cofactor (FeMo-co) to which are bound at least two hydrides/protons. A undergoes two-step relaxation to the resting state (C): A → B → C, where B has an S = 3/2 FeMo-co. Both steps show large solvent kinetic isotope effects: KIE ≈ 3-4 (85% D2O). In the context of the Lowe-Thorneley kinetic scheme for N2 reduction, these results provide powerful evidence that H2 is formed in both relaxation steps, that A is the catalytically central state that is activated for N2 binding by the accumulation of n = 4 electrons, and that B has accumulated n = 2 electrons. [ABSTRACT FROM AUTHOR]

Methyldiazene (HNNCH3) isotopomers labeled with 15N at the terminal or internal nitrogens or with 13C or ²H were used as substrates for the nitrogenase α-195Gln-substituted MoFe protein. Freeze quenching under turnover traps an S = 1/2 state that has been characterized by EPR and ¹H-, 15N-, and 13C-electron nuclear double resonance spectroscopies. These studies disclosed the following: (i) a methyldiazene-derived species is bound to the active-site FeMo cofactor; (ii) this species binds through an [-NHx] fragment whose N derives from the methyldiazene terminal N; and (iii) the internal N from methyldiazene probably does not bind to FeMo cofactor. These results constrain possible mechanisms for reduction of methyldiazene. In the Chatt-Schrock mechanism for N2 reduction, H atoms sequentially add to the distal N before NN bond cleavage (d-mechanism). In a d-mechanism for methyldiazene reduction, a bound [-NHx] fragment only occurs after reduction by three electrons, which leads to NN bond cleavage and the release of the first NH3. Thus, the appearance of bound [-NHx] is compatible with the d-mechanism only if it represents a late stage in the reduction process. In contrast are mechanisms where H atoms add alternately to distal and proximal nitrogens before NN cleavage (a-mechanism) and release of the first NH3 after reduction by five electrons. An [-NHx] fragment would be bound at every stage of methyldiazene reduction in an a-mechanism. Although current information does not rule out the d-mechanism, the a-mechanism is more attractive because proton delivery to substrate has been specifically compromised in α-195Gln-substituted MoFe protein. [ABSTRACT FROM AUTHOR]

The article focuses on a study conducted by two scientists, R. Kalscheuer and G.M. Rodrioguez related to analysis of a biosynthetic route to volatile esters and acetate esters in reference to biodiesels. According to the research, neutral lipid-accumulating bacteria have natural pathways to produce esters using wax ester synthase or acyl-coenzyme A (WS/DGAT) from Acinetobacter baylyi with enzymes derived from bacterial species Zymomonas mobilis and fatty acyl reductases (RARs).

The article presents the first report of a high-population intermediate trapped during an early stage of N2 reduction by nitrogenase. Three nitrogenase turnover systems were freeze-quenched during steady-state enzymatic turnover. The observation of distinct splittings is suggested that three different states represent distinct stages of N2 reduction. However, as each intermediate is formed in a different MoFe protein variant, one must consider whether environmental differences have induced differences in an otherwise equivalent common state that accumulates during turnover of all three substrates.

Wax esters, ester-linked fatty acids and long-chain alcohols, are important energy storage compounds in select bacteria. The synthesis of wax esters from fatty acids is proposed to require the action of a four-enzyme pathway. An essential step in the pathway is the reduction of a fatty aldehyde to the corresponding fatty alcohol, although the enzyme responsible for catalyzing this reaction has yet to be identified in bacteria. We report here the purification and characterization of an enzyme from the wax ester-accumulating bacterium Marinobacter aquaeolei VT8, which is a proposed fatty aldehyde reductase in this pathway. The enzyme, a 57-kDa monomer, was expressed in Escherichia coli as a fusion protein with the maltose binding protein on the N terminus and was purified to near homogeneity by using amylose affinity chromatography. The purified enzyme was found to reduce a number of long-chain aldehydes to the corresponding alcohols coupled to the oxidation of NADPH. The highest specific activity was observed for the reduction of decanal (85 nmol decanal reduced/mm/mg). Short-chain and aromatic aldehydes were not substrates. The enzyme showed no detectable catalysis of the reverse reaction, the oxidation of decanol by NADP[sup+]. The mechanism of the enzyme was probed with several site-specific chemical probes. The possible uses of this enzyme in the production of wax esters are discussed. [ABSTRACT FROM AUTHOR]

Importance: Our results demonstrate increased extracellular ammonium release in the endophyte plant growth-promoting bacterium Gluconacetobacter diazotrophicus . Strains were constructed in a manner that leaves no antibiotic markers behind, such that these strains contain no transgenes. Levels of ammonium achieved by cultures of modified G. diazotrophicus strains reached concentrations of approximately 18 mM ammonium, while wild-type G. diazotrophicus remained much lower (below 50 µM). These findings demonstrate a strong potential for further improving the biofertilizer potential of this important microbe., Competing Interests: Provisional patents have been applied for, but no current licensing arrangements are in place.