Isolation and characterization of draT mutants that have altered regulatory properties of dinitrogenase reductase ADP-ribosyltransferase in Rhodospirillum rubrum

Abstract

In Rhodospirillum rubrum, dinitrogenase reductase ADP-ribosyltransferase (DRAT) is responsible for the ADP-ribosylation of dinitrogenase reductase in response to the addition of NH M 4 or removal from light, resulting in a decrease in nitrogenase activity. DRAT is itself subject to post-translational regulation ; to investigate the mechanism for the regulation of DRAT activity, random PCR mutagenesis of draT (encoding DRAT) was performed and mutants with altered DRAT regulation were screened. Two mutants (with substitutions of K103E and N248D) were obtained in which DRAT showed activity under conditions where wild-type DRAT (DRAT-WT) did not. These mutants showed lower nitrogenase activity and a higher degree of ADP-ribosylation of dinitrogenase reductase under N 2 -fixing conditions than was seen in a wild-type control strain. DRAT-K103E was overexpressed and purified. DRAT-K103E displayed a much weaker affinity for an Affi-gel Blue matrix than did DRAT-WT, suggestive of a fairly striking biochemical change. However, there was no significant difference in kinetic constants, such as K m for NAD and V max , between DRAT-K103E and DRAT-WT. Like DRAT-WT, DRAT-K103E also modified reduced dinitrogenase reductase poorly. The biochemical properties of these variants are rationalized with respect to their behaviour in vivo.

The poor growth of Rhodospirillum rubrum mutants lacking PII proteins is due to an excess of glutamine synthetase activity

“…As a control, UR2 (wild type) was also inoculated into SMN. After growth for 2–3 days, a 5 ml culture was quickly collected in three 2 ml Eppendorf tubes by microcentrifugation for 20 s at 16 000 g at 4°C and washed with cold N‐free (MN – ) medium (Zhang et al ., 2001b). After another quick centrifugation, pellets were frozen in liquid nitrogen, and resuspended in 1 ml of cold 250 mM Tris‐HCl (pH 6.8), and broken by sonication.…”

Characterization of altered regulation variants of dinitrogenase reductase‐activating glycohydrolase from Rhodospirillum rubrum

“…mutagenesis The random PCR mutagenesis followed a published protocol [15]. Higher concentrations (1 mM) of the deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP) and lower concentrations (0.2 mM) of the deoxyguanosine triphosphate (dGTP), deoxyadenosine triphosphate (dATP), and 0.5 mM MnCl 2 and native Taq DNA polymerase (Fisher Scienti¢c, USA) were used to increase the mutation frequency in the PCR mixture.…”

Post-Translational Regulation of Nitrogenase in Photosynthetic Bacteria

“…Substituting arginine-101 of dinitrogenase reductase affected the reaction with NAD + , but did not preclude the formation of a complex of the two proteins, suggesting that this residue is not absolutely required for either complex formation or NAD + binding, but is needed for NAD + cleavage (Ma and Ludden, 2001). Using DRAT mutants identified glutamate-112 in dinitrogenase reductase, along with lysine-103 and asparagine-248 in DRAT, as important in the regulation and activity of DRAT (Kim et al, 1999;Zhang et al, 2001a). The major unknown component in this model is the molecule that is postulated to affect DRAT, presumably as an inhibitor.…”

“…The two genes encoding DRAT and DRAG are the draT and the draG gene, respectively, which in R. rubrum probably are co-transcribed together with an ORF downstream of draG encoding a protein of 15 kDa (Fitzmaurice et al, 1989;Liang et al, 1991). The ORF has been named draB but the role of the gene product has not been elucidated (Zhang et al, 2001a). The draTGB region is localized 402 bp from the nifHDK genes, but is transcribed in the opposite direction.…”