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Nitrogenase activity in Rhodospirillum rubrum is regulated by reversible ADP ribosylation of Arg-101 on a single subunit of the dinitrogenase reductase homodimer under the conditions of energy stress or nitrogen sufficiency. The transfer of the ADP-ribose moiety from NAD⁺ to dinitrogenase reductase is catalyzed by dinitrogenase reductase ADP-ribosyltransferase (DRAT) (4). Activation of dinitrogenase reductase via glycohydrolysis of the ADP-ribosyl protein linkage is catalyzed by dinitrogenase reductase-activating glycohydrolase (DRAG) (5, 6, 9). DRAG is a 32-kDa monomeric enzyme that requires Mg-ATP and free divalent metal for its activity with ADP-ribosylated dinitrogenase reductase as the substrate and has been shown to have a binuclear Mn(II) center at the active site (1). The role of the binuclear Mn(II) center in the deribosylation of the protein substrate ADP-ribosylated dinitrogenase reductase is not well understood. Therefore, based on DRAG's sequence similarity with DRAGs from other sources and with arginase (Fig. 1), which has a binuclear Mn(II) center at the active site, an attempt has been made to understand the role of D123, H142, H158, D243, and E279 residues by site-specific mutagenesis studies.

View larger version (90K): [in this window] [in a new window]   FIG. 1.   Sequence alignment of DRAG from R. rubrum (Rr) with DRAG from Azospirillum brasilense (Ab), Archaeoglobus fulgidus (Af), Methanococcus jannaschii (Mj), Aquifex aeolicus (Aa), Rhodobacter capsulatus (Rc), and arginase from Rattus norvegicus. The amino acid residues chosen for site-directed mutagenesis in this study are circled.

The Quick Change method (Stratagene, La Jolla, Calif.) was used according to the manufacturer's instructions to generate DRAG D123A, H142L, H158N, D243G, and E279R substitutions. The plasmid pYPZ148 containing a 3.3-kb PstI fragment of R. rubrum with draTGB cloned in pUC19 (2) was used as the template. After mutagenesis all draG mutations were confirmed by DNA sequence analysis. Plasmids containing mutagenezied draG were digested with BsaI and BstBI, and 1.1-kb fragments of draG were subcloned into pUX115 (2) to replace the wild-type region. In these plasmids draG is expressed from the strong nifH promoter and DRAG levels are about 100-fold higher than that of the wild type. Variants of DRAG were overexpressed in R. rubrum strain UR472, a draTGB deletion mutant.

Both the wild-type DRAG and the variants were purified following the published procedure (9). Wild-type DRAG was purified from an overexpressing R. rubrum strain, UR276. Cells were broken by the French press method. DRAG activity was measured as previously described by coupling the activity of dinitrogenase reductase with the reduction of acetylene by dinitrogenase (10). DRAG activity was calculated as nanomoles of ethylene produced per milligram of protein per minute.

The quantification of Mn(II) binding to DRAG variants was determined by electron paramagnetic resonance (EPR) (8) using a Varian E-3 spectrometer as follows. A Mn(II) standard curve was determined for the concentration range 25 to 500 µM in 50 mM MOPS (morpholinepropanesulfonic acid)-0.2 M NaCl-2 mM dithiothreitol, pH 7.0. The variants were then titrated with different concentrations of Mn(II), and the concentration of free Mn(II) was determined. Then, the concentration of bound Mn(II) was calculated from the difference between the total and free Mn(II). Finally, a Rosenthal-Scatchard plot was constructed by plotting bound Mn(II) versus free Mn(II) and the values of B_max (maximum amount of metal bound to the enzyme) were calculated. Low temperature X-band EPR spectra were recorded with a Varian E-15 EPR spectrometer equipped with an Oxford Instruments Cryostat. Samples for EPR spectroscopy were prepared by concentrating the enzyme with a Pall Filtron Microconcentrator to 10 to 16 mg/ml in 50 mM MOPS-0.2 M NaCl-2 mM dithiothreitol, pH 7.0. Then, MnCl₂ was added to a concentration of 0.5 mM to activate the enzyme.

Wild-type DRAG and the variants were purified according to the published procedure, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of purified variants showed a single band that migrated with the wild-type DRAG at a molecular mass of 32 kDa. The purity of all protein samples was estimated as greater than 95%. The in vitro activity, Mn(II) binding, and EPR properties of the variants are summarized in Table 1. The variants DRAG-H142L and DRAG-E279R had in vitro activities similar to that of the wild-type, whereas the DRAG-D123A variant was only 70% active. The variant DRAG-H158N had only 2% of the activity of the wild-type enzyme, and the DRAG-D243G variant did not show any in vitro DRAG activity.

To demonstrate that the catalytically active wild type and the variants were structurally intact, their secondary structures were characterized by circular dichroism spectroscopy. Although small differences were observed in the circular dichroism spectra of the variants compared to that of the wild type, the intensity differences did not indicate significant changes in the secondary structures (data not shown).

The Mn binding properties of the altered forms of DRAG were studied by EPR. The large six-line EPR signal due to free Mn(II) is quenched upon binding to DRAG, and this property was used to quantitate Mn(II) binding to DRAG variants. EPR quantitation of Mn(II) binding revealed binding ratios of 0.4, 2, and 1.2 mol/mol for the variants DRAG-D123A, DRAG-H142L, and DRAG-E279R, respectively. The variants DRAG-H158N and DRAG-D243G did not bind any detectable manganese. The EPR spectra of DRAG-H142L and DRAG-E279R were very similar to that of the wild type (Fig. 2 [compare the spectra F, E, and D]) and showed the characteristic 11-line ⁵⁵Mn hyperfine spectra centered at 1,400 and 2,800 G with the 45-G spacing typical of exchange-coupled Mn(II) pairs.

View larger version (12K): [in this window] [in a new window]   FIG. 2.   Low-temperature EPR spectra of DRAG-D243G (A), DRAG-H158N (B), DRAG-D123A (C), DRAG-E279R (D), and DRAG-H142L (E) variants and wild-type DRAG (F). Spectra were recorded at 4.2 K. Each sample contains 200 µl of enzyme solution (10 to 16 mg/ml) in 50 mM MOPS-0.2 M NaCl, pH 7.0. The spectral features characteristic of a ligand-bridged binuclear Mn(II)-Mn(II) cluster centered at 1,400 and 2,800 G are indicated by arrows.

The variant DRAG-E279R with full in vitro DRAG activity bound only 1.2 mol of Mn(II)/mol of enzyme. EPR analysis of the sample without adding exogenous Mn(II) did not show the presence of any bound Mn(II); thus, DRAG-E279R does not contain tightly bound Mn as isolated. The variant DRAG-D123A bound only 0.4 mol of Mn(II)/mol of enzyme although exhibiting 70% in vitro DRAG activity. Addition of ADP-ribosylated dinitrogenase reductase (the substrate) to the sample failed either to increase the affinity for Mn(II) or to restore the binuclear metal signal. The ambiguity in metal binding properties of these two variants could be due to (i) nonspecific binding of some other metal at the active site or (ii) differences in the experimental conditions used in the activity assay and the metal binding experiments, or it could be due to both. The amino acid residues H142 and E279 cannot be critical for the coordination to binuclear Mn(II) center or for catalysis since the variants DRAG-H142L and DRAG-E279R retained both catalytic and EPR spectral properties similar to that of the wild-type enzyme. Since DRAG-H158N and DRAG-D243G variants lack the ability to bind Mn(II) or to catalyze hydrolysis of the substrate, the ADP-ribosylated dinitrogenase reductase, we propose that residues H158 and D243 could be involved in the coordination of the binuclear Mn(II) center in DRAG from R. rubrum. These observations suggest that although DRAG has some sequence similarity with arginase, for which the structure (3) is known, the environment of the binuclear Mn site in the two enzymes is very different.

Although DRAG can also be activated in vitro by Fe²⁺ (2.5 mM), Mg²⁺, and Co³⁺ (>20 mM) (7), Mn²⁺ (1 mM) is still thought to be the physiological factor since Mn²⁺ binds more tightly to the enzyme compared to other metals. However, neither the nature of metal used in vivo nor the role of the binuclear metal center in the hydrolytic deribosylation reaction of the protein substrate ADP-ribosylated dinitrogenase reductase is understood. Structure-function analysis of the binuclear metal center of DRAG enzyme is the focus of our ongoing crystallographic studies.