Microbial transglutaminase (mTG) forms trans-peptide bonds between glutamines and suitable amine donors such as lysines. In this study we use this concept to covalently graft proteins in a programmable manner by employing lysine and glutamine rich tags on N- and C- termini and by controlling their access to each other and mTG. In this way, we can theoretically create poly enzyme assemblies that preserve function and enhance or modulate flux. Moreover, the method is amenable to applications in or on devices. To demonstrate this concept we use two quorum sensing enabling enzymes LuxS and Pfs, which act in a cascade to produce Autoinducer-2 (AI-2), a quorum sensing signaling molecule and attach these enzymes together using mTG. Such a cascade should enable substrate channeling, or enhanced flux, by reducing delays due to molecular diffusion or transport. In this study, we show that LuxS and Pfs can be attached through mTG and can result in functional enzyme cascade leading to production of AI-2. Western blots verify the molecular weights of the LuxS-Pfs heterodimers. Their enzymic activity is measured through biochemical assays measuring AI-2 concentrations. We also show that not just two subunits but multiple subunits can be attached in a controlled fashion and demonstrate by assembly of a trimer, dimer of one enzyme coupled with a monomeric second enzyme. Building trimer with two enzymes could be of use when mismatched rates of enzymes are encountered for optimizing flux. In our case, first enzyme Pfs is ~2 orders of magnitude faster than LuxS and we therefore explored whether two LuxS molecules coupled with Pfs leads to increased overall generation rate of AI-2. We suggest this methodology may be of some benefit in certain applications where multiple enzymatic steps are needed for enabling product synthesis and the presentation of enzymes on various surfaces afford local control and synthesis of small molecule products.

Microbial transglutaminase mediated two step enzyme cascade fabrication leading to increased product flux

247th National Meeting (2014)

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