Abstract
Formylglycinamide ribonucleotide amidotransferase (FGAR-AT) is a 140 kDa bi-functional enzyme involved in a coupled reaction, where the glutaminase active site produces ammonia that is subsequently utilized to convert FGAR to its corresponding amidine in an ATP assisted fashion. The structure of FGAR-AT has been previously determined in an inactive state and the mechanism of activation remains largely unknown. In the current study, hydrophobic cavities were used as markers to identify regions involved in domain movements that facilitate catalytic coupling and subsequent activation of the enzyme. Three internal hydrophobic cavities were located by xenon trapping experiments on FGAR-AT crystals and further, these cavities were perturbed via site-directed mutagenesis. Biophysical characterization of the mutants demonstrated that two of these three voids are crucial for stability and function of the protein, although being ∼20 Å from the active centers. Interestingly, correlation analysis corroborated the experimental findings, and revealed that amino acids lining the functionally important cavities form correlated sets (co-evolving residues) that connect these regions to the amidotransferase active center. It was further proposed that the first cavity is transient and allows for breathing motion to occur and thereby serves as an allosteric hotspot. In contrast, the third cavity which lacks correlated residues was found to be highly plastic and accommodated steric congestion by local adjustment of the structure without affecting either stability or activity.
Highlights
An efficient strategy employed by nature to sequester unstable intermediates along various biosynthetic pathways is by evolving separate enzymes with coupled reactions that are only active in consort as multiprotein complexes [1,2]
Solving the structure of StPurL protein crystals (StPurL-Xenon complex) which were pressurized for 4 min did not show any increase in the number of xenon atoms bound as compared to the crystals derivatized for 1 min 1 min pressurization of xenon was used for further studies
Xenon trapping was used as a tool to identify these cavities and subsequently these cavities were perturbed via mutations
Summary
An efficient strategy employed by nature to sequester unstable intermediates along various biosynthetic pathways is by evolving separate enzymes with coupled reactions that are only active in consort as multiprotein complexes [1,2]. In some instances analogous systems have emerged that retain these activities together via synthesis of the enzymes as a single polypeptide chain [3,4]. Conjoining various domains with multiple activities leads to complex folding and unfolding profiles, and results in creation of interfaces that play a critical role in coordinating function of spatially distant active centers. An analysis of genomes and present protein sequence databases suggests that 40–60% of proteins exist in multidomain format highlighting the importance of studying such systems [6,7]. Efforts to understand and mimic such systems have been pursued for optimizing the production of molecules important from the perspective of industry and medicine [8,9]
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