Biochemistry CHEM-C2330

Lecture 11, Oct. 8th 2024

Biocatalysis

Feedback tool presemo: Some slides: Modified from Prof. Jan Deska

http://presemo.aalto.fi/c2330
Prof. Silvan Scheller
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 Learning Outcomes for L11: Be Able to…
• explain advantages and disadvantages of biocatalysis over classical
catalysis
• describe strategies for cofactor recycling
• explain the differences between isolated enzyme and whole-cell
biocatalysis
• name examples for state-of-the-art biocatalysis

2
Transaminases use Pyridoxal phosphate (PLP)
pyridoxal 5'-phosphate (PLP)
What happens?
vitamin B6 • C=O bond → C-N bond
• C-N bond → C=O bond

ω-Tra ns ami na se

PLP Sitagliptin

→ How is the equilibrium pushed to the right side?

See L4: Chemical Reactions at Equilibrium
• Consider the general reaction:

aA + bB cC + dD

• The equilibrium constant, K, can be written as:

 [C]c [D]d 
K = a b 
 [A] [B] eq

 [C ]c [ D]d 
G = G + RT ln  a b 
 [ A] [ B] 
Transaminases use Pyridoxal phosphate (PLP)
ω-Tra ns ami na se

PLP Sitagliptin

→ How is the equilibrium pushed to the right side?

• Isopropylamine is deaminated to acetone

• Acetone is less soluble in water, thus can be removed by slight vacuum,
elevated temperature or N2-purge

– Requires relatively high concentrations of isopropylamine

 𝞈 Transaminases
How to shift equilibrium to the product side?

unfavourable equilibrium!

→ How can we remove pyruvate?

http://presemo.aalto.fi/c2330
 𝞈 Transaminases
Enzyme-coupled pyruvate removal!

• Lactate dehydrogenase (Ldh)

removes pyruvate
Problem:
– NADH is expensive
– → How to solve this issue?
 𝞈 Transaminases
Enzyme-coupled pyruvate removal!

• Lactate dehydrogenase (Ldh)

removes pyruvate
• Requires cofactor recycling systems
such as Gdh (glucose dehydrogenase)
or Fdh to provide sufficient NADH
 𝞈 Transaminases
Extra Slide: Even more advanced: Enzyme-coupled alanine recycling

• Alanine dehydrogenase and ammonia recover the

used alanine amine donor
 𝞈 Transaminases
Extra Slide: Even more advanced: Enzyme-coupled alanine recycling

• Alanine dehydrogenase and ammonia recover the

used alanine amine donor
 𝞈 Transaminases
Application of this method
biocatalytic reductive amination

Fuchs, Koszelewski, Tauber, Kroutil, Faber, Chem. Commun. 2010, 5500

 What we can Learn from Sitagliptin Story
Tailored-made optimized biocatalysts for pharma production (see L10)
• Nitrogen-functionalities widespread in modern pharmaceuticals
• Over the past decade: new and powerful amination procedures evolved
from initially very limited basic enzymatic functions
• Today: transaminases, imine reductases, monoamine oxidases and amine
dehydrogenases are state-of-the-art tools for synthesis of bioactive amines

✓ late-stage bio-amination as alternative to

typical heavy metal-based process
✓ 2017, approval to replace existing production
route for Merck's Januvia/Janumet API
 Biocatalysis on an Industrial Scale
Advantages / Disadvantages?
 Biocatalysis on an Industrial Scale
Catalysis in general
▪ Acceleration of a reaction by providing a path with lower activation energy
▪ In many cases catalysis makes reactions happen that are not happening at
all under uncatalyzed conditions

Biocatalysis
▪ Enzymes are used to facilitate chemical processes

Major advantages over chemical catalysis

▪ very high selectivities (chemo-, regio-, and stereoselectivity)
▪ particularly mild conditions (important pillar of the Green Chemistry idea)
Isolated Enzymes vs. Whole Cell Biocatalysis

Advantages: Advantages:

Disadvantages: Disadvantages:
Isolated Enzymes vs. Whole Cell Biocatalysis

Advantages: Advantages:
• Highly controlled conditions • Optimal conditions
• Simple reaction equipment • Low enzyme degradation
• High catalyst concentrations • No cofactor recycling needed
• Nonaqueous environment • Metabolic engineering possible
possible (many steps in a row)

Disadvantages: Disadvantages:
• Protein stability • A lot biomass as waste
• Cofactor stability / recycling • Substrate needs to enter cell
• Substrate might be toxic to cell
 From in vitro to in vivo:
There are procedure “in between”, depending on the application:
• Purified, recombinant enzymes (e.g. His6-tag)
• Overexpressed enzymes, whole cell lysate is used.
o If heterologous thermostable enzyme: native enzymes might
be denatured at higher temperature
• Whole cells are used for one enzyme
o dead or alive (or something in between)
o Substrate goes into the cell, gets converted, product leaves
• Enzyme cascade inside cells. Substrate taken up, product leaves
• Products derived from the substrate of the cells (e.g. glucose)
= “Metabolic Engineering”
• Cells produce the desired product as their primary metabolism
(e.g. ethanol from glucose in yeast)
Strategies to Improve Whole-Cell Biocatalysis

Lin and Tao Microb Cell Fact 2017, 106

Identification and relief of bottlenecks, pathway balancing to maximize flux

towards the product, blocking competing pathways, improving the precursor
supply, engineering cofactor or co-substrate balance and chassis optimization
Engineering Cofactor or Co-Substrate Balance

NAD(P)H regeneration
systems formed via coupling
with a regeneration reaction

Alcohols → amines using alcohol

dehydrogenase (ADH) and amine
dehydrogenase (AmDH)

Lin and Tao Microb Cell Fact 2017, 106

Some Examples of Industrial Applications
• Transaminations (see sitagliptin before) → amines (e.g. enantioselective)
• Eneoat reaductases → reductions of C=C (e.g. enantioselective)
• Arene dioxygenases → 2 stereogenic centers introduced simultaneously
• Biocatalytic Cascades → Many steps in the same pot (all in water)
• Biocatalysis by Microbial Consortium → Mixture if different microbes
Enoate reductases Enoate Reductases
Replacement of carbonyl by polarized olefin as substrate

R R *
EWG EWG
enoate
R' reductase R'
or or
NAD(P)H
R' R'
R R
EWG * EWG
EWG = ester, ketone, aldehyde, nitro,...

"Old Yellow Enzymes" (found in various yeasts and bacteria)

§ physiological role not entirely clear

§ flavoproteins (tightly bound FMN)
§ no metal cofactor
§ NAD(P)H-dependent
Enoate reductases Enoate Reductases
Applications
old yellow enzyme
(S. cerevisiae)
H NADH H
GDH recycling system
O O O O
aq. buffer, tBuOMe
O O 97% ee

Helional
(fragrance)

enoate reductase YqjM

(B. subtilis)
O NAD+ O
GDH recycling system
HO OMe aq. buffer HO OMe
99% ee

"Roche ester"
used in synthesis of vitamins,
fragrances, antibiotics etc

Winkler, Tasnádi, Clay, Hall, Faber, J. Biotechnol. 2012, 162, 381-389.

Arene diox y genas es Arene Dioxygenases
certain strains from Pseudomonas putida accumulate cis-dihydroxycyclohexadienes

benzene catechol
dioxygenase OH dihydrogenase OH

O2, NADH OH NAD+ OH

cis-dihydroxycyclohexadiene catechol

P. putida 39/Dcatechol
and F1 mutants grow on
toluene as2,3-dioxygenase
carbon source but suffer from a
somewhat sluggishO2 dehydrogenase

§ biosynthesis of complex organic building blocks OH

acetate, pyruvate,...
CO2H
P. putida F1 diastereoselectivity: pure cis
Cl OH O
Cl grown on enantioselectivity: pure (+)
toluene OH

Gibson, Koch, Schuld, Kallio, Biochem. 1968, 7, 3795-3802.

 Synthetic use of Arene Dioxygenases
Synthetic us e of arene diox y genas es
excellent for
fermentative production of basic synthetic building blocks follow-up chemistry

Br Br
Escherichia coli JM109
OH 99% ee
99% cis
OH

Br
Br
Pseudomonas putida 39/D I
I 99% ee
99% cis
HO
OH

CO2H HO2C OH
Alcaligenes eutrophus B9
OH occasionally also
1,2-selectivity
 Synthetic use of Arene Dioxygenases
Synthetic us e of arene diox y genas es

Synthesis of antitumor agents (pancratistatin family), Hudlicky 1996

I
Ph NTs Br
Br Br O
E. coli JM109 Cu(acac)2
O
then protection MeCN O
O N
Ts

Bu3SnH
[Cu] AIBN
O
O O O
deprotection CONEt2 O
O lactamization O OMOM
O O
NHTs O
O NH O N
CONEt2
MOMO O OMOM Ts

OH OH
OH HO OH

O OH or O OH
O NH O NH

HO O narciclasine HO O pancratistatin

Hudlicky, Tian, Königsberger, Maurya, Rouden, Fan, J. Am. Chem. Soc. 1996, 118, 10752-10765.
 Synthetic use of Arene Dioxygenases
Activity on non- arene substrates

Chemical asymmetric cis-dihydroxylation (Sharpless dihydroxylation)

K2OsO2(OH)4 OH N N N N
(DHQD)2PHAL O O
OH
MeO OMe
K3Fe(CN)6
N N
(DHQD)2PHAL

Benzene dioxygenase also accepts e.g. indene

N

OH Ph
E. coli JM109 N OH
H OH
OH N N

O
O NHtBu

indinavir (anti-HIV)

Gally, Nestl, Hauer, Angew. Chem. Int. Ed. 2015, 54, 12952-12956.
 Biocatalytic Cascades

Merck & Co developed a total enzymatic synthesis of the HIV drug islatravir built on five key enzymatic steps.

AcK, acetate kinase; PanK, pantothenate kinase;

DERA, deoxyribose phosphate aldolase; PPM, phosphopentamutase;
GOase, galactose oxidase; PNP, purine nucleoside phosphorylase;
HRP, horseradish peroxidase; SP, sucrose phosphorylase;

Bell et al. Nature Reviews “Methods Primers” 2021, Article citation ID: (2021)1:46
 Application of Whole Cells
Surplus Electricity to Gas
Hydrogenotrophic methanogens*
CO2 + 4 H2 CH4 + 2 H2O

* Product (methane) is formed via the

energy metabolism of the cells

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 Outlook: New Project at Aalto
Surplus Electricity to Propane?
Microbes engineered
to produce propane?*
3 CO2 + 10 H2 C3H8 + 6 H2O
Engineered Microbes

Propane?

* Product (propane) should be formed

via the energy metabolism of the cells

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Whole Cells: 3 CO + 6 H2 → Isopropanol (IPA)
Utilization of waste-gasses from steal industry
or biomass pyrolysis

3 CO
 Summary
• Enzymatic reactions happen often close to the thermodynamic equilibrium
and require (by)product removal
• Redox-cofactors need to be regenerated
• Transamination is important for biocatalysis, since many drugs are chiral and
have amine functionality
• Different ways: purified enzymes or whole-cell Biocatalysis. Or even
“metabolic engineering”.
• Few cases: wanted product = main waste product of the cell = very high yield
• Advantage of biocatalysis:
• Higher selectivity (very important for enantioselectivity)
• Milder conditions (“green chemistry”)
• Disadvantage of biocatalysis:
• Development takes a long time (large investments)
• Difficult for late-stage processes (useful for the beginning, Sitagliptin is an
exception that was quite challenging)
• Restricted in the types of chemistries enzymes are doing 31
 Tasks:
• Revise lecture 11
• Study concepts for cofactor recycling
• Prepare for exam (→ check ILOs!)

• Participate in the poster presentation

• Submit Self-evaluation for posters (after presenting; latest this Friday noon)

• No lecture on Friday. (I will come to the room to answer questions, if needed)

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 C2330 ”Enzyme Poster” Presentation
Poster presentation procedures:
- Start: 1015 (or 1515 for Tuesday / Thursday sessions). Find your group. Tape
papers together, if not already done before. Hang up poster with the lowest
EC number
- 1020: Start presenting the first poster. (7-10 minutes). Followed by 4-7
minutes questions. Everybody should ask at least one question. 1 min time
for changing.
- 1035: Poster 2 (with 2nd lowest EC number, same procedures)
- 1050: Poster 3
- 1105: Break
- 1120: Poster 4
- 1135: Poster 5
- Keep poster hanging until the end, so that other groups also can see them
- At the end: please make sure to remove all tape/glue from the walls
- Fill self-evaluation
 Poster Rooms (see next slide):
Tuesday 8. Oct. 1515: Jeti lecture room:
Groups 11-27

Wednesday 9. Oct. 1015: Undergraduate center U2:

Groups 31-52

Thursday 10. Oct. 1515: Jeti lecture room:

Groups 61-76

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8/10. Oct. : Jeti lecture room: 9. Oct. : U2 lecture room:

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 C2330 Overall Learning Outcomes:
Chemistry and Proteins (part of it is revision / making sure all are up to date):
• Be able to calculate pH values, making buffers etc. (chemistry in water)
• Be comfortable in drawing chemical reaction mechanisms relevant for
biochemistry (“shuffling electrons around”)
• Be able to describe the structure of proteins at different levels

Enzymes and Catalysis:

• Understand the foundation of enzyme catalysis
• Be able to explain Michaelis-Menten Kinetics
• Name different methods for protein engineering and evaluate their potential
• Describe how selected cofactors work and why they are needed

Applications and social skills:

• Be able to find state-of-the-art science
• Getting used to present scientific topics orally

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 L2: Learning Outcomes: Be able to..
• draw molecules and resonance structures
• explain what a resonance structure is
• select correct arrows for resonance structures and for equilibria
• draw the reaction mechanism of an aldol condensation
• draw the reaction mechanism of acid-catalyzed acetal hydrolysis
• explain how enzymes can carry out acid and base catalysis

• describe what Lysozyme is and what it’s chemical reaction is

• name different classes of enzymes

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 L3: Learning Outcomes: Be able to..
Ch 5: Amino acids and primary structure of proteins
• name different categories of amino acids (aa) and their R group
• recognize all aa and name them with name, 3 letter and 1 letter abbreviation
(for grade 5, and for students going to MSc program biotechnology or similar)
• explain why a peptide bond is planar and difficult to rotate

Ch 6: 3D-structure of proteins:
• define 1°, 2°, 3° and 4° structure of proteins
• name the non-covalent interactions that determine the 3D structure
• describe the structure of 𝛂 helices and 𝛃 sheets
• confidently participate in the computer exercise next Wednesday

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 Learning Outcomes for L4: Be able to..
• explain what the formula “∆G = ∆H – T∆S” means
• explain why proteins fold, even though becoming more ordered
• calculate equilibrium concentrations from ∆G values and vice versa
• explain why you should remember the numbers 5.7 kJ mol-1 and
-30 mV for estimating thermodynamics at given conditions
• define ∆G, ∆G°, ∆G°’ and ∆G’
• convert ∆E values to ∆G values

• draw a free energy diagram for a unimolecular reaction

• explain what a transition state is 39
 Learning Outcomes for L6: Be able to…
• explain the concept of transition state theory
• draw reaction vs. free energy diagrams (for chemical reactions)
and to interpret them
• explain the foundation of enzyme catalysis (evaluate different
models)
• predict the influence of stronger substrate binding on the rate of
the enzyme reaction
• explain mechanistically how a serine protease work

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 Learning Outcomes for L7: Be Able to..
• explain different strategies how enzymes can accelerate
reactions (continuation from L6)
• explain how the Michaelis-Menten equation is derived
• predict how the Michaelis-Menten equation behaves for very
low, and very high substrate concentrations
• determine kinetic parameters (KM, kcat, kcat/KM)
• describe reversible and irreversible enzyme inhibition
• interpret kinetic data, and predict which type of inhibition was
present

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 Learning Outcomes for L8: Be Able to…
• describe how stopped flow works
• explain how proteins can be overproduced in e-coli
• describe procedures to purify recombinant His6-tagged proteins
• name 3 different chromatography methods to purify proteins
and predict whether a given protein will elute early or late
• explain broadly the principle to sequence proteins via MS-MS
• broadly describe what proteomic is

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 Learning Outcomes for L9: Be Able to..

• explain why cofactors are needed, and give examples

• name different cofactors and describe their function
• draw the reaction mechanism of thiamine pyrophosphate (TPP)-
mediated decarboxylation of alpha-keto acids (umpolung), with
HCN as an analog of TPP
• broadly explain how pyridoxalphosphate chemistry works (e.g.
transamination)

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 Learning Outcomes for L10: Be Able to…
• name different methods for protein engineering
• differentiate between evolution and creation
• explain the concept of directed evolution
• describe how enzymes evolved for new function (enzyme
promiscuity)
• name example of how to use enzymes for sustainable
production of chemicals
• describe the challenges of protein design

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 Learning Outcomes for L11: Be Able to…
• explain advantages and disadvantages of biocatalysis over classical
catalysis
• describe strategies for cofactor recycling
• explain the differences between isolated enzyme and whole-cell
biocatalysis
• name examples for state-of-the-art biocatalysis

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