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

Departments of Biochemistry¹ and Bacteriology² and the Center for the Study of Nitrogen Fixation³, University of Wisconsin-Madison, Madison WI 53706, USA

Author for correspondence: Gary P. Roberts. Tel: +1 608 262 3567. Fax: +1 608 262 9865. e-mail: groberts{at}bact.wisc.edu

In Rhodospirillum rubrum, dinitrogenase reductase ADP-ribosyltransferase (DRAT) is responsible for the ADP-ribosylation of dinitrogenase reductase in response to the addition of 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 N₂-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.

Keywords: nitrogen fixation, regulation, ADP-ribosylation, random PCR mutagenesis

Abbreviations: DRAG, dinitrogenase reductase activating glycohydrolase; DRAT, dinitrogenase reductase ADP-ribosyltransferase; DRAT-WT, DRAT from wild-type

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