4 Creation of switchable enzymes using the Split Protein approach
5.1 Guidelines for the choice of components
5.1.1 Regulatory Domains
We defined following 4 criteria for the choice of regulatory domains in Domain Insertion:
First, a crucial characteristic of regulatory domains is their existence as self-contained proteins or domains. In many enzymes, the allosteric binding site is not formed by an isolated domain. Instead, amino acids at different positions within the primary structure of the protein are needed for effector binding and signal transduction, as for instance described for LeuA 118. Such allosteric binding sites are difficult to isolate and transfer and therefore not suited for Domain Insertion. Second, regulatory domains are preferred that undergo a large conformational change upon binding of their allosteric regulator. The larger a conformational change is, the more likely it is that this change can be transmitted to the fused enzymatic domain and thereby, the more likely is it that the enzymatic activity can be modulated. Third, we preferred regulatory domains and proteins that act as monomers. The reason for this it that enzymatic domains inserted into oligomer forming regulatory domains could sterically impede their oligomerization and consequently their functionality. The fourth criterion is the allosteric effector. Whereas in later applications, this criterion will be one of the most important ones as the allosteric effector defines the function of the resulting enzyme – regulatory domain fusion, for a first test of concept, the identity of the allosteric effector is less important.
Chapter 5 - Creation of switchable enzymes using the Domain Insertion approach
insertion of proteins into parts required for the oligomerization. And third, similar to other periplasmic sugar binding proteins, the protein consists of two separate domains with a groove between them which contains the sugar binding site. It has been shown previously that MBP undergoes a large conformational change when binding maltose 119,120 (Figure 22). Moreover, MBP has already been used as regulatory domain in other applications of Domain Insertion 73,78,79.
Figure 22: Conformational change of MBP upon binding of maltose.
Alignment of structures of MBP in the unbound, open form (blue, PDB 1JW4, 121) and when bound to maltose (green) in a closed form (red, PDB 1ANF, 122). Upon binding of maltose, the clamp shaped structure closes. The hinge angle increases as a result of this motion by 35° 119.
5.1.2 Enzymatic domains
For enzymatic domains in parts similar guidelines apply as for regulatory domains: First, monomeric enzymes are preferred to enzymes that have to oligomerize in order to form catalytically functional enzymes. The reason for this is that an insertion into a regulatory domain might sterically hinder the enzymes’ ability to form oligomers. Second, in later applications, enzymes will be mainly chosen to control certain reactions. For our first attempts to create metabolic enzymes with synthetic regulations this criterion is less important. In addition to these criteria, two other rules apply for enzymatic domains: One is that N- and C-terminus of the enzymes are ideally in as closest proximity as possible. The idea behind that is that the insertion of the enzymatic domain separates the two fragments of the regulatory domain with the same distance as the distance between the N- and the C-termini of the enzymatic domain. The longer this distance is, the less realistic is a successful folding as well as a functional reassembly of both fragments of the regulatory domain. The second additional rule is that the enzymatic activity has to result in a measurable phenotype, e.g. fluorescence 72, antibiotic resistance 73 or changes in growth rates.
Based on these criteria, for our first attempts to create metabolic enzymes with synthetic allosteric regulation we selected murine dihydrofolate reductase mDHFR (folate biosynthesis)
Chapter 5 - Creation of switchable enzymes using the Domain Insertion approach
and 2-isopropylmalate synthase LeuA (leucine biosynthesis) as our enzymes. Furthermore, the usage of ArgA (arginine biosynthesis) and HisG (histidine biosynthesis) has been evaluated. mDHFR had been chosen because of its relatively simple structure. It acts as a monomer and possesses termini that are in very close proximity (murine DHFR: 14.8 Å, see Figure 23),
allowing the insertion of DHFR in regulatory domains with relatively small linkers. In addition, mDHFR has already been used in Domain Insertion although with a more targeted approach and with DHFR as acceptor domain 77. For us, DHFR has in addition the advantage that we also used it as Split Protein (Chapter 4).
Figure 23: Structure of mDHFR.
PDB 3d80 123. Marked is the distance between both termini.
LeuA has been chosen for its higher relevance for biotechnology 124,125 but has also the advantage of having a relatively simple structure consisting of an N-terminal catalytic domain and a C-terminal regulatory domain, connected through a subdivided linker structure 118 (Figure 24). LeuA forms dimers and although both N-terminal domains of a dimer form independent catalytic sites, the C-terminal domain is required for catalytic activity, presumably by ensuring protein dynamics 126. We therefore decided to work with the full length LeuA and assume chimera with LeuA as enzymatic domain will form oligomeric complexes.
Chapter 5 - Creation of switchable enzymes using the Domain Insertion approach
Figure 24: Domain Structure of LeuA
126.
LeuA consists of 3 domains, a N-terminal catalytic domain (green), a C-terminal regulatory domain (purple) and a linker structure in between which consists of two subdomains (blue and red). LeuA forms homodimers with two catalytic sites and one leucine binding site.