Functional analysis of an interspecies chimera of acyl carrier proteins indicates a specialized domain for protein recognition

Abstract

The nodulation protein NodF of Rhizobium shows 25% identity to acyl carrier protein (ACP) from Escherichia coli (encoded by the gene acpP). However, NodF cannot be functionally replaced by AcpP. We have investigated whether NodF is a substrate for various E. coli enzymes which are involved in the synthesis of fatty acids. NodF is a substrate for the addition of the 4'-phosphopantetheine prosthetic group by holo-ACP synthase. The Km value for NodF is 61 microM, as compared to 2 microM for AcpP. The resulting holo-NodF serves as a substrate for coupling of malonate by malonyl-CoA:ACP transacylase (MCAT) and for coupling of palmitic acid by acyl-ACP synthetase. NodF is not a substrate for beta-keto-acyl ACP synthase III (KASIII), which catalyses the initial condensation reaction in fatty acid biosynthesis. A chimeric gene was constructed 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 of AcpP fused to amino acids 43-93 of NodF) protein encoded by this gene indicate a similar folding pattern to that of the parental proteins. Enzymatic analysis shows that AcpP-NodF is a substrate for the enzymes holo-ACP synthase, MCAT and acyl-ACP synthetase. Biological complementation studies show that the chimeric AcpP-NodF gene is able functionally to replace NodF in the root nodulation process in Vicia sativa. We therefore conclude that NodF is a specialized acyl carrier protein whose specific features are encoded in the C-terminal region of the protein. The ability to exchange domains between such distantly related proteins without affecting conformation opens exciting possibilities for further mapping of the functional domains of acyl carrier proteins (i. e., their recognition sites for many enzymes).

The application of computational methods to explore the diversity and structure of bacterial fatty acid synthase

“…This conclusion predicts that the protein• ACP interactions are achieved through a conserved set of electrostatic and/or hydrophobic contacts. This hypothesis is also supported by the fact that domains of ACPs from distantly related organisms ( E. coli and Rhizobium) can be interchanged without affecting the protein's conformation and function (14). Consistent with this, the primary sequence analysis of the ACPs identifies a motif that is conserved in ACP family members ( Fig.…”

“…The working hypothesis is that these ACPs have acquired additional novel structural features, outside the protein recognition site, to accommodate the different types of acyl chains they carry. For example, the C-terminal half of Rhizobial NodF may be specialized for sequestering polyunsaturated fatty acyl chains (14). The extended carboxy-terminus in M. tuberculosis AcpM is proposed to protect the extremely longchain fatty acyl chains in mycolic acid biosynthesis from the hydrophilic environment (10).…”

Expression and purification of four different rhizobial acyl carrier proteins The GenBank accession numbers for the nucleotide sequences reported in this paper are AF159244 and AF159243 for the acpP-containing regions of Sinorhizobium meliloti 1021 and of Rhizobium leguminosarum LPR5045, respectively.

“…3a, lanes 1 and 5). Purified holo-ACP synthase (AcpS) of E. coli (Lambalot & Walsh, 1995) is able to transfer in vitro 4h-phosphopantetheine to the specialized rhizobial ACPs NodF (Ritsema et al, 1998) and RkpF (Epple et al, 1998). To quantify the in vitro reaction between apo-ACPs and AcpS, we are presently determining the kinetic constants (K M , k cat ) for each one of the rhizobial ACPs as substrates.…”

“…However, the three-dimensional structure of the AcpP of E. coli has been determined (Kim & Prestegard, 1990) and an initial characterization of the secondary structure and the general tertiary fold of the NodF protein (Ghose et al, 1996) demonstrates that the overall structures of ACPs are surprisingly well conserved. In spite of the general structural similarity between NodF and AcpP of E. coli, AcpP cannot substitute for NodF in vivo in the synthesis of polyunsaturated fatty acids (Ritsema et al, 1998). Furthermore, these authors have shown that the NodF-specific functions are encoded in the C-terminal half of the protein.…”

SMb20651 is another acyl carrier protein from Sinorhizobium meliloti

“…It has been shown that NodF from Rhizobium leguminosarum bv. viciae can be malonylated in vitro using the enzyme FabD from E. coli (Ritsema et al, 1998), and therefore we used S. meliloti NodF as a positive control in this study. Although no conformational change was observed when NodF Sm was incubated with malonyl-CoA and FabD Sm (Fig.…”