Download solving the structure and mechanism of novo

Enzymes for sustainable C-C bond formation:
Doing it de NovO way
Joanna Sadler1,2, Dr. Luke Humphreys 2, Dr. Glenn Burley1, Dr. Chun-wa Chung2
1University
of Strathclyde, Cathedral Street, Glasgow, G1 1QX; 2GlaxoSmithKline R&D, Gunnels Wood Road, Stevenage, SG1 2NY
Enzymes are Nature’s catalysts
Enzymes are a tool for sustainable chemistry
Enzymes are protein molecules which speed up a chemical reaction. They are ubiquitous
in Nature:
For example, the enzyme amylase breaks down starch into maltose in our digestive system.
Metal Catalysed
Enzyme Catalysed
Organic Solvents
Aqueous Solvent
Extremes in pH and temperature
Mild pH and temperature
Toxic Metal Waste
Biodegradable Waste
Can Lack Selectivity
Highly Selective
Wide substrate scope
Narrow substrate scope
NovO: Nature’s Answer to an Old Problem
Project Aim:
Develop a toolbox of
enzymes for sustainable
alkylation reactions
Alkyl groups on aromatic rings are common in industrially important molecules. For
example, methyl groups (CH3, shown in red) are found in many top-selling pharmaceuticals:
Celebrex (anti-inflammatory)
Nexium (proton pump inhibitor)
Gleevec (anti-cancer)
The Friedel –Crafts alkylation is a very important and widely used reaction to make such
compounds, however it is not environmentally sustainable and can give low yields:
S-adenosyl methionine (SAM)
Novobiocin
Streptomyces sp.
James Crafts
(1839-1917)
Novobiocin is an antibiotic made by Stretomyces spheroides. The enzyme responsible for
alkylation of the coumarin core is called NovO.
Charles Friedel
(1832-1899)
NovO: Structure
NovO: Mechanism
>1000
crystallisation
conditions
screened
Crystal structure of NovO solved to 1.9 Å resolution
100
Computationally modelled
substrate into active site
90
NovO: Scope and Evolution
Using these results, we can design new enzymes for
sustainable chemical synthesis.
% Conversion
80
70
60
50
40
30
Tested model with a
series of single point
mutants of NovO in
proposed active site
Detailed understanding of
catalytic mechanism
Tested need
for OH next
to
methylation
position
20
10
0
Mutation
In situ cofactor synthesis: an efficient process
The very high cost (~£900/ g) and instability of the cofactor (SAM) limits the use of NovO on a large scale.
To address this, we coupled the enzyme SalL, from Salinospora tropica to NovO in one pot, thereby
employing the cheap and readily available amino acid methionine (~35 p/ g) as the terminal methyl
source.
Directed
Enzyme
Evolution
Salinospora tropica
In Summary… NovO: Structure → Mechanism → Scope →
Platform for design of new biocatalysts
for sustainable organic synthesis