Principles of Biochemistry 8 |Enzyme Design| Class Notes |HarvardX

The enzymes were used in different areas to fit different purposes. But there are so many restrictions before applying them in utility. As a result, enzyme modification is a huge leap for the enzyme industry.

Tree enzyme design approaches:

Rational Design

Using existing structural information of the enzyme, and using a rational approach to replace specific amino acids with others.
It is based on the structural information and another ligand for the enzyme.

Drawbacks:

• Full structural information of the enzyme with and without substrate is required.
• Time-consuming
• Money
• Complexity

Directed by Evolution

Evolution on a very small scale in a controlled environment.

White type protein;
introducing a variety of mutations;
Screen and select the result;
back to the first step and mutated it again.

Drawbacks:

• Not all enzymes are amenable to high-throughput screens.

Semi-rational Design

Iterative Saturation Mutagenesis (ISM)

• the selected sites are saturated with mutations.
• saturation mutagenesis is repeated to obtain an optimum variant

Saturation Mutagenesis

• Mutation improves activity or modifies the specificity of enzymes located at, or near, the active site.
• The mutation improves the stability of the enzyme tend to away from the activity site.

Example: lipase A

T50: the temperature at which 50% of the enzyme remains properly folded and active after a given time interval.

1. B-factor which could measure the flexibility of amino acid was applied, and the top 10 highest B-factor AA at 8 sites in wild-type structure were selected.

2. Hypothesis: the mutation of these sites could increase the rigidity of the protein and wouldn’t affect the activity of the enzyme.

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Result:
Condition: pre-incubation at $75^ \circ C$ for $60 min$

Protein	Residual Activity
WT	0%
Variant X	60%
Variant XI	90%

ISM applications

Iterative Saturation Mutagenesis:

1. Crystal structure -> choose AA
2. ‘Smart’ mutant library
3. Assay enzyme activity
4. Repeat

Universal Blood

Back ground:

Universal blood is the blood, which could be used for any of the patients.

AB blood type system: the different sugar groups found on glycolipids on the surface of the red blood cell.

They have a glycoside hydrolase that cleaves the antigenic trisaccharides found in type A and B red blood cells.

Goals: improve the activity of glycoside hydrolase

1. Figure out the crystal structure of the enzyme
2. Generate mutant library by degenerate-code PCR
3. Screen for enzyme activity to cleave fluorescent glyco-substrate.

Result: By repeating screen and selection, scientists finally replaced 6 AAs and increased 170-fold of activity compared to the wild type.

Halohydrin dehydrogenase

manufacture of pharmaceutical compounds.

Background:
Challenge: The production of the drug is often a mixture of stereoisomers.
epoxide group: a common precursor in chemical synthesis which is very active.
Normal circumstance:
Halohydrin dehydrogenase + R-2-chloro-1-phenylethanol -> pure product.
S-2-chloro-1-phenylethanol: remains no change.

Solution: Design an enzyme that could tolerate the S form of the substrates.

1. Solving the crystal structure and determine the (7) amino acids in activity sites.
2. Generate mutant enzyme library by degenerate codon PCR
3. High throughput colorimetric screen for enzyme activity.

Result: As a result, the combination use of the wild and mutation type enzyme was used to convert both forms of the substrates to the desired product.

Diabetes sensors

Background:

Glucometers: convert glucose into a product and release electrons.

Naturally, oxygen would accept electrons and lead to underestimating the level of glucose.

Goals: Increasing the accuracy of the Glucometers.

1. Target glucose oxidase amino acid sites were chosen based on functional information.

2. build a mutagenesis lib:

   • alanine 173 & 332 increases both mediator and oxygen activities.
   • phenylalanine 414, increased mediator activity, and decreased oxygen activity.
   • valine 560 dramatically decreased oxygen activity.

3. Muti-site saturation mutagenesis to find optimal oxygen-insensitive enzymes.

Result: Variant 7

Wilde	Position	Mutate
Adenine	173	valine
Alanine	332	serine
Phenylalanine	414	isoleucine
Valine	560	threonine

Alter the use of the cofactor

Target: Increased the by-path method of reaction.

1. The crystal structure was resolved and the residuals near the active site were determined.
2. Site-saturation mutagenesis via PCR with NNK degenerate primers at the 54 positions.
3. High throughput screens identified mutations at 12 sites that boosted NAD(H) usage.
4. The best variant, Q350N was used as the template for ISM for the remaining 11 positions.
5. An additional variant, Q350N/H171A, was discovered with further increased NAD(H) usage.