Structural and functional analysis of the AAA ATPase Bl-ARC from Bifidobacterium longum
Abstract
Analysis of the Bifidobacterium longum subsp. infantis (ATCC15697) genome revealed a hypothetical ORF with 72% sequence identity against the AAA ATPase family of proteins. Phylogenetic analysis using CLANS tool showed it belongs to the ARC (AAA ATPase forming Ring-shaped Complexes) branch of the AAA family, thus we termed it Bifidobacterium longum ARC (Bl-ARC). The gene was expressed in E. coli and the recombinant protein was functionally assessed for activity. The gene encodes a protein with a molecular mass of 56 kDa. The purified protein had ATPase activity and formed a complex of approximately 336 kDa that corresponds to an apparent hexameric ring. The specific enzyme activity with ATP was 47 nmol/mg/min, and the kinetic parameters were: Km 0.21 mM and Vmax 51.28 nmol/mg/min. The maximum activity was reached at 37ºC and pH 4.5, however it remained stable in a pH range from 1.5 to 5.0, thus resistant to the human gastrointestinal tract environment. We assume that Bl-ARC is the orthologue of the previously characterized Rhodococcus erythropolis ARC.
References
This study was supported by CONACYT Fondos Sectoriales SEP grant no. 101568.
Sela DA, Chapman J, Adeuya A, Kim JH, Chen F, Whitehead TR, Lapidus A, Rokhsar DS, Lebrilla CB, German JB, Price NP, Richardson PM, Mills DA. The genome sequence of Bifidobacterium longum subsp. infantis reveals adaptations for milk utilization within the infant microbiome. Proc Natl Acad Sci U S A. 2008. 105(48):18964-9.
Frickey T, Lupas A. CLANS: a Java application for visualizing protein families based on pairwise similarity. Bioinformatics. 2004. 20(18):3702-4.
Wolf S, Nagy I, Lupas A, Pfeifer G, Cejka Z, Müller SA, Engel A, De Mot R, Baumeister W. Characterization of ARC, a divergent member of the AAA ATPase family from Rhodococcus erythropolis. J Mol Biol. 1998. 20;277(1):13-25.
