Characterization of the NADP(+)-dependent malic enzyme of Sinorhizobium (Rhizobium) meliloti and investigations into the requirements of malate uptake and malic enzyme activity in bacteroids

Abstract

<p>Malic enzymes are responsible for the conversion of malate to pyruvate with the concomitant reduction of a nicotinamide cofactor. Previous experiments revealed that Sinorhizobium ( Rhizobium ) meliloti has both a diphosphopyridine (NAD + ) dependent enzyme (DME), which is essential for nitrogen fixation, and a triphosphopyridine (NADP + ) dependent enzyme (TME), which is not required for symbiotic N 2 fixation. Sequence analysis of tme revealed that it encodes a protein of 761 amino acids, which is much larger than previously described prokaryotic malic enzymes. The elongated structure comprises an unusual chimeric organization with the N-terminal region of the protein showing similarity to other malic enzymes, including six conserved domains implicated in malic enzyme function. The C-terminal region shows similarity to phosphotransacetylase enzymes (PTA) and deletion of this PTA-like domain results in a decrease, but not an abolishment of malic enzyme activity, indicating that this region is not essential for the conversion of malate to pyruvate. To investigate the requirements of the malic enzyme in symbiotic nitrogen fixation several metabolic engineering experiments were carried out. The NAD + -dependent malic enzyme (SfcA) from E. coli , which lacks the PTA-like extension, was expressed in S. meliloti dme mutant strains. The S. meliloti dme - , sfcA derivatives formed nodules that fixed N 2 at rates indistinguishable from those formed by wild type S. meliloti . Hence, the SfcA protein can functionally replace DME. Other metabolic engineering attempts were made by overexpressing dme in S. meliloti bacteroids. The resulting increase in DME levels in bacteroids did not result in increased symbiotic N 2 fixation or improved plant growth. In order to determine if cofactor specificity is responsible for the requirement of DME during symbiosis attempts were made to alter the cofactor requirement of TME. Regions of dme corresponding to the putative co-factor binding site were exchanged for the corresponding regions of tme to produce chimeric proteins. To investigate the carbon source utilized during symbiosis a malate specific permease from S. bovis was expressed in S. meliloti strains lacking the dicarboxylic acid transporter DctA. When expressed as a single copy construct on the chromosome using either the dme or dctA promoter no nitrogen fixation was detected. When the constructs were expressed from a high copy number plasmid the resulting plants showed 40% nitrogen fixation and 25% plant dry weights relative to wild type inoculated plants. (Abstract shortened by UMI.)</p>