FUNCTIONAL-ANALYSIS OF AN INTERSPECIES CHIMERA OF ACYL CARRIER PROTEINS INDICATES A SPECIALIZED DOMAIN FOR PROTEIN RECOGNITION

The nodulation protein NodF of Rhizobium shows 25% identity to acyl ca rrier protein (ACP) from Escherichia coli (encoded by the gene acpP). However, NodF cannot be functionally replaced by AcpP. We have investi gated whether NodF is a substrate for various E. coli enzymes which ar e involved in the synthesis of fatty acids. NodF is a substrate for th e addition of the 4'-phosphopantetheine prosthetic group by holo-ACP s ynthase. The K-m value for NodF is 61 mu M, as compared to 2 mu M for AcpP. The resulting holo-NodF serves as a substrate for coupling of ma lonate by malonyl-CoA:ACP transacylase (MCAT) and for coupling of palm itic acid by acyl-ACP synthetase. NodF is not a substrate for beta-ket o-acyl ACP synthase III (KASIII), which catalyses the initial condensa tion reaction in fatty acid biosynthesis. A chimeric gene was construc ted comprising part of the E.coli acpP gene and part of the nodF gene. Circular dichroism studies of the chimeric AcpP-NodF (residues 1-33 o f AcpP fused to amino acids 43-93 of NodF) protein encoded by this gen e indicate a similar folding pattern to that of the parental proteins. Enzymatic analysis shows that AcpP-NodF is a substrate for the enzyme s holo-ACP synthase, MCAT and acyl-ACP synthetase, Biological compleme ntation studies show that the chimeric AcpP-NodF gene is able function ally to replace NodF in the root nodulation process in Vicia sativa. W e therefore conclude that NodF is a specialized acyl carrier protein w hose specific features are encoded in the C-terminal region of the pro tein. The ability to exchange domains between such distantly related p roteins without affecting conformation opens exciting possibilities fo r further mapping of the functional domains of acyl carrier proteins ( i. e., their recognition sites for many enzymes).