title: 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).
authors.affiliation: Leiden University, Institute of Molecular Plant Sciences, Clusius Laboratory, The Netherlands.
authors.forename: T
authors.initials: T
authors.lastname: Ritsema
authors.forename: A M
authors.initials: AM
authors.lastname: Gehring
authors.forename: A R
authors.initials: AR
authors.lastname: Stuitje
authors.forename: K M
authors.initials: KM
authors.lastname: van der Drift
authors.forename: I
authors.initials: I
authors.lastname: Dandal
authors.forename: R H
authors.initials: RH
authors.lastname: Lambalot
authors.forename: C T
authors.initials: CT
authors.lastname: Walsh
authors.forename: J E
authors.initials: JE
authors.lastname: Thomas-Oates
authors.forename: B J
authors.initials: BJ
authors.lastname: Lugtenberg
authors.forename: H P
authors.initials: HP
authors.lastname: Spaink
mesh_terms: D000213:Acyl Carrier Protein / Q000235:genetics*; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000235:genetics / Q000302:isolation & purification / Q000502:physiology*; D004926:Escherichia coli / Q000378:metabolism*; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism*; D012231:Rhizobium / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
chemical_list: C101898:AcpP protein, Streptomyces; D000213:Acyl Carrier Protein; D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; C068605:nodF protein, Rhizobium