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

In Rhodospirillum rubrum, dinitrogenase reductase ADP-ribosyltransferase (DRAT) is responsible for the ADP-ribosylation of dinitrogenase reductase in response to the addition of NH(+)(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.