Enzyme design and evolution

Hovedområde:Protein chemistry
Målgruppe:Biochemistry, Biology
Niveau:Bachelor, Masters

We work on elucidating the mechanism of catalysis and regulation for enzymes involved in complex polysaccharide degradation (amylase, mutanase and alpha-mannosidase), for proteases (HIV protease), antibiotic resistance conferring enzymes like beta-lactamase and small molecule converting enzymes mainly from nucleotide metabolism.

Enzyme design, enzyme regulation and specificity, and drug resistance
We combine enzymology and enzyme design with the development of genetic selection systems based on the growth of Escherichia coli and Lactococcus lactis to evolve new enzyme activities directed towards e.g. new peptide recognition sequences (HIV protease), deglycosylation of glyco-proteins and the mechanisms that guide the specificty excerted by binding modules and -sites in reactions catalysed by maybe otherwise promiscous endo- or exoglycosidases. These projects in part serve to make us wiser with respect to the rules governing the design of enzymes that can lead to a whole new tool-box addressing the problems faced with green chemistry, industrial waste etc.

We also try to gain insight into e.g. the complex rules that govern substrate specificity or development of resistance to anti-HIV drugs in HIV protease. Likewise, we also work on the M2 proton transport channel of the influenza virus to elucidate the mechanisms by which resistance to drugs by mutation also takes place for this channel. Hopefully, we may be able to predict the efficiency of drugs from the combination of data and computational chemistry. These projects take place in a close collaboration with Assoc. Prof. Kresten Lindorff-Larsen and Prof. Jakob R. Winther.

Many of the projects also involve basic characterisation of enzymatic properties. An example could be alpha-amylase I from barley, where we study the complex kinetics of amylopectin degradation. Enzymes from the nucleotide metabolism that we study involve the orotate phosphoribosyltransferase where we study in great detail the catalytic mechanism to an almost nerdy degree of details and also the bifunctional dCTP deaminase:dUTPase from M. tuberculosis is an example of an enzyme for which the subtle and intriguing mechanism of regulation has produced several projects over the years.

A third area of interest is to figure out good methods of generating mutant libraries with as many different mutations represented as possible. We have designed a system in which we perform error-prone PCR on the beta-lactamase gene (the enzyme that confers ampicillin resistance) to generate large libraries from which to select for mutations that promote the ability of the mutant enzyme to break down other antibiotics of the ampicillin family. Mutations and associated enzyme activities that are also known from multi-resistant pathogenic bacteria. At the same time, because beta-lactamase represents a good selection system, we work to unravel by multifactorial analysis the mechanisms guiding the outcome of mutations from varying the reaction conditions for error-prone PCR. This is done in order to generate a general description and effective protocol for error-prone PCR, regardless of template properties.

Projects where we contribute
Recently, we have initiated a collaboration with collegues in our the section and headed by Prof. Karen Skriver on intrinsically disordered proteins, IDP's, that are involved in transcriptional regulation in plants. The project addresses the interesting question of how a protein can transform into a highly specific signal mediator through interaction with other protein partners, despite a seeming lack of structure. We contribute to the collaboration mainly via our expertise in microcalorimetry, where we investigate and assist in interpretating the thermodynamic parameters determined for the IDP's and their interaction with binding partners. Projects within this subject will typically be co-supervised with Karen Skriver who is the expert on the particular proteins and their related biological context.

About the lab
In the lab we routinely use all common molecular biology and protein chemistry techniques such as: PCR, cloning, mutagenesis, DNA sequence analysis (sequencing performed by outside supplier), plasmid and chromosomal DNA purification, design of DNA primers and synthetic genes, restriction enzyme analysis, protein purification using salting out, column chromatography a.o. methods, protein analysis by SDS-PAGE and biophysical methods.

In addition we do: enzyme kinetics using spectrophotometry or isothermal titration calorimetry (ITC), ligand binding and protein-protein interaction using ITC and differential scanning calorimetry (DSC), analysis of protein stability using (DSC), bacterial growth and genetic analysis, bacterial strain construction, design of selection systems for enzyme development and much more.

If you apply and are assigned to a project you will be introduced to the subject and the relevant methods by working as a team with an experienced lab. member. After a few months depending on how comfortable you are with working in the lab. you will start working more independently on your own project as a part of our research programme.

Quite a number of bachelor and master students apply for projects each year within the Protein Biology Group at Section for Biomolecular Sciences, so unfortunately space is limited and we tend to offer projects on a first come, first served basis. You are very welcome to contact us about the current status for available projects within the above areas. These change all the time with the development in the ongoing research in the laboratory. Also, you may want to contact the master students already in the lab and hear aboout how it is to work with us and what they do.

Anvendte metoder:See text
Keywords:Generating selections sytems, Enzyme mechanism, Microcalorimetry, Proteases and glycosidases, Drug resistance
Vejleder(e): Martin Willemoës