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McKinnie Lab
Lukas Varga
Abstract
Enzymes have become increasingly prevalent as a complement to chemical synthesis in order to simplify chemical reactions. However, many chemical systems involve temperature or solvent conditions that drastically reduce the capability of an enzyme to generate the desired compounds, preventing their employment.
One emerging technique for obtaining more stable enzymes is ancestral sequence reconstruction (ASR), which uses a bioinformatics approach. In our lab we study vanadium dependent haloperoxidases (VHPOs), which are a family of enzymes that can perform halogenation reactions in selective manners.
In my research, I sought to use this technique in order to generate new forms of VHPO enzymes and investigate whether these ancestral versions had more flexible chemistry.
Biocatalysis
Biocatalysis
Biocatalysis is the emerging effort to use enzymes to simplify current reaction pathways to create industrially, medically, or chemically relevant compounds . Properly implemented enzymes can often reduce multi-step reactions to single-steps, reducing environmental impact of generating compounds.
Targeted application of enzymes in production systems can solve multiple inefficiencies such as reaction conditions, unwanted side products, and simplifying chemical reactions to potentially eliminate harsh reagents. While incredibly powerful, biocatalysis is hindered by low stability and tolerance to temperature and solvent conditions different from the native system of the enzyme
Ancestral Sequence Reconstruction
Ancestral Sequence Reconstruction
Ancestral sequence reconstruction (ASR) is the process of generating an evolutionary tree by using modern forms of an enzyme class as roots . The methodology involves using sequence alignment to identify mutations that separate the modern forms of an enzyme family. By comparing their sequences and organizing them into phylogenies, theoretical common ancestors can be inferred.
ASR provides valuable data, as ancestral forms of enzymes are often more resistant to temperature and solvent changes. They can also show an increased promiscuity for substrates, which is highly relevant for creating an enzyme for biocatalytic purposes.
(Figure adapted from Prakinee et al.)
Vanadium dependent Haloperoxidases
Vanadium dependent Haloperoxidases
Vanadium dependent haloperoxidases (VHPOs) are a diverse class of enzymes found in marine bacteria, fungi, and algae that install halogen atoms into organic substrates . They are characterized by the presence of a vanadate prosthetic group and the use of hydrogen peroxide to selectively oxidize halides to produce hypohalous acid (HOX). The proposed reaction cycle is seen on the left.
Some VHPOs release the halide as freely diffusible HOX, while some (known as selective) hold onto the HOX by creating a putative haloamine linkage to a conserved lysine residue, and perform halogenation on specific organic substrates. One unique characteristic of the selective VHPOs is their ability to oxidize and release less electronegative halides than the halide they are selective for, an example being a chloroperoxidase holding onto chloride upone oxidation, but releasing hypobromous acid upon bromide oxidation.
We are interested in these selective forms since they perform highly controlled halogenation chemistry, an ability highly relevant to biocatalysis. The mechanisms of their regioselective halogenation are not fully understood, so we are aiming to use this ASR technique in order to generate ancestral VHPOs that display more promiscuity and compare them to more selective, extant forms.
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