Engineering allosteric control to an unregulated enzyme by transfer of a regulatory domain Penelope J. Crossa, Timothy M. Allisona, Renwick C. J. Dobsonb,c, Geoffrey B. Jamesond, and Emily J. Parkera,1 aBiomolecular Interaction Centre and Department of Chemistry, University of Canterbury, Christchurch 8140, New Zealand; bBiomolecular Interaction Centre and School of Biological Sciences, University of Canterbury, Christchurch 8140, New Zealand; cBio21 Molecular Science and Biotechnology Institute, Department of Biochemistry and Molecular Biology, University of Melbourne, Parkville, Victoria 3010, Australia; and dInstitute of Fundamental Sciences, Massey University, Palmerston North 4442, New Zealand Edited by George H. Lorimer, University of Maryland, College Park, MD, and approved December 26, 2012 (received for review October 14, 2012) Allosteric regulation of protein function is a critical component of domain, of ∼80 amino acids in length, was first identified as a metabolic control. Its importance is underpinned by the diversity ligand-binding regulatory module in a number of proteins of di- of mechanisms and its presence in all three domains of life. The verse function, leading to its description as a “conserved evolu- first enzyme of the aromatic amino acid biosynthesis, 3-deoxy-D- tionarily mobile ligand-binding regulatory module” (17). Most arabino-heptulosonate 7-phosphate synthase, shows remarkable proteins in which the ACT domain has been recognized catalyze variation in allosteric response and machinery, and both contem- early steps in amino acid and purine metabolic pathways, and the porary regulated and unregulated orthologs have been described. ACT domain serves as a binding site for pathway end products. The To examine the molecular events by which allostery can evolve, ligand-binding sites for the allosteric effector(s) are typically asso- we have generated a chimeric protein by joining the catalytic ciated with interfaces between two or more ACT domains. The domain of an unregulated 3-deoxy-D-arabino-heptulosonate 7- underlying details of the allosteric mechanisms associated with the phosphate synthase with the regulatory domain of a regulated ACT domain are now starting to be revealed: generally, ligand enzyme. We demonstrate that this simple gene fusion event on binding in these sites elicits structural changes that alter the its own is sufficient to confer functional allostery to the unreg- catalytic function of the remote active site (13, 15, 16, 18, 19). ulated enzyme. The fusion protein shares structural similarities 3-Deoxy-D-arabino-heptulosonate 7-phosphate synthase with its regulated parent protein and undergoes an analogous (DAH7PS) is the first enzyme of the shikimate pathway, which BIOCHEMISTRY major conformational change in response to the binding of allo- is responsible for the biosynthesis of the aromatic amino acids steric effector tyrosine to the regulatory domain. These findings Phe, Tyr, and Trp (Fig. 1A). Although all DAH7PS enzymes help delineate a remarkably facile mechanism for the evolution share a core catalytic (β/α)8 barrel, this barrel is variably deco- of modular allostery by domain recruitment. rated with additional structural elements, creating a remarkable array of diverse examples of modular allostery (Fig. S1). The ACT domain | shikimate most intricate system discovered to date is demonstrated by the DAH7PS from Mycobacterium tuberculosis, where multiple rotein allostery, where ligand binding is coupled to a func- subdomains and ligand-binding sites grafted onto the (β/α)8 barrel Ptional change at a remote site, is critical for the control of are associated with a complex synergistic allostery by Phe and metabolism. For enzymes of key metabolic pathways, allostery Trp, largely mediated by changes in protein flexibility (20). The allows precise control of catalysis in response to cellular demand. DAH7PS enzymes from Escherichia coli and Saccharomyces Although this important phenomenon was first described many cerevisiae have N-terminal barrel extensions providing a single years ago, it is only more recently that the molecular details that aromatic amino acid binding site (21, 22). Other DAH7PS govern this precise control of enzyme function have been ex- enzymes are fused to a functional chorismate mutase domain plored for a diverse range of protein systems (1–3). Functional for the purposes of allosteric control (23, 24). The simplest change results from changes in the protein energy landscape contemporary DAH7PS enzymes show no extra barrel exten- elicited by ligand binding (4). This change in energy landscape sions and hence are unregulated. This latter group includes the may lead to conformational change and/or may be more subtly structurally characterized enzymes from Pyrococcus furiosus expressed as a change in protein molecular dynamics (5–12). (25) and Aeropyrum pernix (26). We recently demonstrated that The importance of protein allostery is underpinned by the the DAH7PS from Thermotoga maritima, structurally composed observation that it is ubiquitous. As more proteins are studied in of an ACT domain fused to the N terminus of a catalytic (β/α)8 detail, one of the striking revelations is the variety of allosteric barrel, undergoes a remarkable conformational change associated mechanisms that are used to control protein function. These with ligand binding (27). The associated domain reorganization mechanisms can be broadly divided into three groups that reflect controls enzyme functionality by physically gating substrate ac- the evolutionary path taken to acquire the allosteric functionality cess to the active site upon binding of Tyr (Fig. 1B). (5). In the first group, modification of existing structural features How readily can this modular allostery demonstrated by of a protein creates an allosteric site. Second, the formation of Tyr-regulated T. maritima DAH7PS (TmaDAH7PS) be acquired homo- and heterooligomeric assemblies may lead to the de- byasimplegenefusionevent?TheDAH7PSfamily,with velopment of allosteric sites at the interface of subunits. Third, allostery may be endowed by domain fusion to create a modular protein with a distinct domain responsible for binding of the Author contributions: P.J.C. and E.J.P. designed research; P.J.C., T.M.A., and R.C.J.D. per- allosteric effector. A detailed understanding of this modular al- formed research; P.J.C., T.M.A., R.C.J.D., G.B.J., and E.J.P. analyzed data; and P.J.C., G.B.J., lostery, in which the allosteric effector binding site is associated and E.J.P. wrote the paper. with a discrete protein domain, offers the potential to generate The authors declare no conflict of interest. engineered proteins with tailored functionality. This article is a PNAS Direct Submission. fi The ACT domain has been identi ed as a modular regulatory Data deposition: The atomic coordinates and structure factors have been deposited in the unit associated with the control of a variety of metabolic pro- Protein Data Bank, www.pdb.org (PDB ID code 4GRS). cesses (13–16). The name of this domain originates from the 1To whom correspondence should be addressed. E-mail: [email protected]. fi fi three proteins in which it was rst identi ed: aspartokinase, This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. chorismate mutase, and prephenate dehydrogenase (17). This 1073/pnas.1217923110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1217923110 PNAS Early Edition | 1of6 Downloaded by guest on September 24, 2021 Fig. 1. (A) DAH7PS catalyzes the first step of the shikimate pathway, the condensation of PEP and E4P to give 3-deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P), for the biosynthesis of the aromatic amino acids. (B) A superposition of the barrels of the open Tyr-free (PDB ID code 1RZM, green) and closed Tyr- bound (PDB ID code 3PG9, purple) conformations of TmaDAH7PS showing the rearrangement of the ACT domains in the presence of Tyr. contemporary examples of both unregulated enzymes and those identical to that inferred from the TmaACT-PfuDAH7PS ORF. containing a discrete ACT regulatory domain controlling catalysis Circular dichroism spectrophotometry indicated the enzyme was by physical gating of the active site, is an ideal family to examine correctly folded (Fig. S4). the evolution of this important enzyme property. In this work, Tma Pfu we describe a regulated DAH7PS chimera (TmaACT-PfuDAH7PS), ACT- DAH7PS Is Catalytically Active and Inhibited by Tyr. Tma Pfu constructed by combining the ACT domain from TmaDAH7PS ACT- DAH7PS is catalytically active with kinetic parame- β α ters similar to those of WT PfuDAH7PS, the enzyme from which with the unregulated catalytic ( / )8 barrel domain from P. furiosus Tma Pfu DAH7PS (PfuDAH7PS). This article demonstrates the transfer the catalytic barrel of ACT- DAH7PS was derived (Table of functional allostery from one contemporary protein to the 1). The chimeric enzyme displays limited cooperativity with re- fi spect to substrate concentrations and negligible change in the ortholog from another genetically distinct species. These ndings fi demonstrate that the acquisition of allosteric properties by gene Hill coef cient is observed in the absence and presence of Tyr fusion is surprisingly facile. (Fig. S5). TmaDAH7PS is strongly inhibited by Tyr and to a lesser ex- Results tent inhibited by Phe (27). These two amino acids were tested as Tma Pfu Design Considerations: Construction of Fusion Protein TmaACT-PfuDAH7PS. inhibitors of ACT- DAH7PS (Fig. 2). Both Tyr and Phe Unregulated PfuDAH7PS, whose subunit comprises the basic had an inhibitory effect on the enzyme, with Tyr being a more effective inhibitor. The inhibition by Tyr was less pronounced for (β/α) barrel housing the active site, was chosen as the scaffold 8 TmaACT-PfuDAH7PS than for TmaDAH7PS. for fusion to the regulatory ACT domain of TmaDAH7PS. Kinetic parameters were determined for TmaACT-PfuDAH7PS Both PfuDAH7PS and TmaDAH7PS crystallize with a similar in the presence of Tyr (Table 1). The apparent Km for phospho- homotetrameric architecture, which has been shown to be criti- enolpyruvate (PEP) remained relatively unchanged by the pres- cal for the allosteric mechanism of TmaDAH7PS (25, 28).