Lecture  2       Key  reactions  of  Biosynthesis        

1.0 ATP and Activation

Enzymes which use ATP to build bonds are called SynthETases.

Adenosine 5' triphosphate (ATP) stores chemical energy which is used to activate carboxylic acids.

HO

O

N

N

bicarbonate OH

N

O O O OH P P P HO O O O OH OH

NH2

N O

O

HO

N

N

O

ADP

NH2

N

N O O O OH P P P HO O O O OH OH

O

HO

NH2

N O

HN

N

N

Lys HO

Nu-H

OH

H N

O

+

P OH OH

HO

O

S

Acyl Adenylate

biotin

O

OH O HN

Nucleophile could be OH, SH, NH2 or C on a small molecule or attached to a protein

Nu

Lys

Examples:

N

O

S

O OH P O O

O

N

N

O

OH N

N

OH

HO

H

O

CoA

R

S

O

N

Malonyl CoA

A

O

T

SH

OH

O

ACP S

HO

O

CoA

NH2

N O

O

R

Acetyl CoA

CoA-SH

O OH P O O

H

H N

OH

CoA

Abbreviated chemical mechanism of acetyl CoA carboxylase S

HO

N

O

NH2

N

ACP-SH

C H

O

O

OH

O

N

H

AMP

O

P O OH

OH

OH N O OH P O O

O

ATP

OH

OH O

O

Acetyl ACP

NH2

O

ATP

NH2

Adenylation and Thiolation Chemistry from NRPS

T

A

 

2.0 CoA and ACP thiolesters are used to hold, transport and activate acyl groups CoA Thiolesters 'Chemical Handle' - recognised and held by proteins

thiol

N N O O H H P P N N N O O O O OH OH O O HO HO O OH P Coenzyme A (CoA-SH) O OH

HS

NH2

Mimics of CoA/ACP are often used in the study of biosynthetic proteins both in vivo and in vitro

H N

HS

N-acetyl Cysteamine (NAC) O

N H N

HS

OH

H N O

Pantetheine

OH O

Acyl Carrier Protein (ACP) - holds starter units, extenders and intermediates as thiolesters.

Acyl Carrier proteins (ACP) of FAS and PKS and the Thiolation (T) domains of NRPS are initially produced in their apo forms, i.e. lacking a Phosphopantetheine (PP) Prostehtic group. PP is added to a conserved serine

ACP

by a special enzyme called a PP-transferase to form holo-ACP.

Protein - ca 70-80 amino acids

SH malonate

These enzymes are sometimes called "holo-synthase" or PPT-ase.

H N

HS

O O P O O O OH P O O

H N

CoA

O

O

thiol sulfur

ATP Serine 42

O 2-O

3PO

N

N

The Actinorhodin ACP See Crump et al J. Mol.Biol. 2009, 389, 511-528.

HO N

NH2 N

PPTase

ACP

Mg2+

OH Phosphopantetheine

apo-CP

Serine 42 wobbly line indicates PP

ACP O O

O P

O

HO

ACP HS

H N

H N O

O

SH

O S

H N

OH

H N O

O O

OH O

holo-CP

O P O O OH

HO

O SACP

O

HN

N H

 

3.0 Carbon - Carbon Bond Forming Reactions Thiolase (Primary Metabolism) O

O

O

SCoA

SCoA

O

O

SCoA

SCoA

SCoA

H

S makes α-H more acidic

Acetoacetyl CoA

S is good leaving group

B

O

Hydroxy Methyl Glutaryl CoA Synthase (Primary Meatbolism and some PKS) A H O

O

O

O

O

SCoA

OH

O

SCoA

O

OH SCoA

SCoA

H SCoA

O

S is good leaving group

SH Cys111

SCoA

H2O B

O

O

S

Glu79

Cys111

O O

S

S

O

OH

Cys111

Cys111

SH Cys111

S makes α-H more acidic -keto acyl synthase (FAS and PKS)

C-methyl transferase (PKS)

OH R

S makes α-H more acidic

KR

CMeT

O

O R

SACP NH2 Me

O

S

Me

O SAM

Ado-Met is excellent leaving group

KR

DH SACP

OH

Ado

ER

KS

AT

S

OH

S

O

OH O

HO

DH

CO2 ACP TE

O

ER

KS

AT

SH

OH

ACP TE S O

S makes α-H more acidic

KS and ACP catalyse chain extension

OH

O

Acyl group resides on ACP for subsequent processing

 

4.0 Carbon Heteroatom Reactions 4.1 Acyl Transferases (e.g. During PKS and FAS) KR ER

KR ER

KR ER

KR ER

KR ER

DH

DH

DH

DH

DH

KS AT ACP TE

KS AT ACP TE

KS AT ACP TE

KS AT ACP TE

KS AT ACP TE

SH OH SH OH

SH O

S

S

S

AT loads starter unit S

SH OH

AT loads Extender

O

O

HO

AT transfers starter to KS

CoA

OH SH OH

S O

O

O

T

S

C

Ac

O

T

S

NH2 HO

H2N H2N

Aa

SH

O

T

SH

C

O

HO

O

OH S

O O

AT transfers extender to ACP

OH O

HO

O

O

4.3 Ester Formation/Hydrolysis (e.g. Thiolesterase)

Ac

O N H

T

KR ER

KR ER

KR ER

DH

DH

DH

S

SH

KS AT ACP TE

KS AT ACP TE

KS AT ACP TE

SH OH S

SH OH SH O

SH OH SH OH

OH O

O

SH OH

O

CoA

4.2 Amide Formation (e.g. During Nonribosomal Peptide Synthesis)

Aa

O

OH R

OR'

O

R'HO

O

R R

 

 

4.4 Aminotransferase Reactions

Also Decarboxylation of Amino Acids O

O

O P HO O OH

OH N

NH2 +

R

R'

NH2

O P HO O OH

Me

N

pyridoxal phosphate (PLP)

R

O

OH

+

R

OH N

O P HO O OH

R'

Me

OH

pyridoxamine phosphate (PMP)

N H

Me

O

Notes R''

PLP / PMP effectively transfer amino groups by interconverting carbonyls (usually ketones) and secondary amines.

R'''

Also racemases O

O

O P HO O OH

OH

NH2

+ R''

N

OH

OH

NH2

R'''

NH2

Me Alanine Racemase

pyridoxal phosphate (PLP)

4.5 Lyases

O

PLP

4.6 non-PLP Racemases (Epimerases) NH3 O

O OH

NH2 Phenylalanine Ammonia Lyase (PAL) No Cofactor required, but see: Proc. Natl. Acad. Sci. USA, 92, 8433-8437, 1995 for mechanism

O OH

O

HO

O OH

NH2

NH2

O

HO

OH NH2

e.g. Diaminopimelate epimerase (DAP epimerase)

NH2

 

4.7 Glycosyl Transferases

O O NDP

Retaining Glycosyl Transferase

O

O

Inverting Glycosyl Transferase

XR

RXH is termed the Acceptor

Donor

SN2

OH O HO

The sugar can be almost anything.... X = O or N (very infrequently C)

H

HO HO

Donor NDP = nucleotidyl diphosphate Uridine, Guanosine, Cytidine

XR

RXH

H

H

OH

OH

HO OH H OH OH O O O O P P O O

Uridine diphosphate glucose Inverting Mechanism

H N

O NH

O

O

HO HO HO

SN1

OH

H

O

OH

O

HO HO HO

HO OH H OH OH O O O O P P O O

Uridine diphosphate glucose

H N

O

HO HO

H

HO

O

OH

NH O

Retaining Mechanism OH HO HO

O HO

O

H

 

5.0 Redox Reactions 5.1 NAD(P)H mediated reactiuns

H

N

N N

NH2

OH O P OH O O P O

NH2

N

O O

nicotinamide - 'the business end'

O

O

Mechanism H+

This is the oxidised form - it can be reduced by hydride.

N

R H H

HO

Reduction

Oxidation

N N

NH2

O

N

O O

O

HO

N

This is the reduced form - it is a reducing agent - equivalent to NaBH4 in organic chemistry.

OH

Note that HproR and HproS are diastereotopic - i.e. they are distinguishable.

NADH HO

OH

NH2

N

N

R

R

NAD(P)H

NAD(P)+

Notes reduced nicotinamide - 'the business end'

HR HS O

N

X

O

NH2

make NADP+. This does not alter the chemistry. Some enzymes use NAD+, others use NADP+.

NH2

H

O

H

OH

OH This hydroxyl can be phophorylated to

OH O P OH O O P

R

X

NAD+ HO

EH

E

The reaction tends to run in this direction because NAD(P)+ is aromatic, but is reversible so can be driven backwards under certain circumstances. The electrophilic species is usually a carbonyl examples are known of ketones, aldehydes, thiolesters, αβ-unsaturated carbonyls and iminium ions.

 

5.2 Monooxygenases 5.2A Cytochrome P450 monooxygenases

In Monooxygenation One atom from O2 ends up in the substrate - the other usually ends up as H2O

Cytochrome P450 enzymes are versatile oxidases, using molecular oxygen as the oxidant.

Top view - heme is usually deeply bound within the protein. The iron atom is usually also bound by a cysteinyl sulfur on one face.

NH O

N HN

S

N Fe

N

N

Side view - cysteine can be omitted for clarity bold lines represents heme side view.

Fe(II) Heme B

Initial coordination complex

O2 Fe(I I)

O

S

CO2H

HO2C

covalent peroxy species

O

O

Fe(II)

NADPH NADP+

peroxide

O

Fe(III)

Cys

O

e-

O

Fe(III)

Coupled reduction system e-

Oxidation of the organic substrate by insertion of O into CH bond

peroxide

H+

O

OH

H+

O

Fe(III)

R

H Fe(III)

Fe(V) H2O

R

OH

 

Details of the rebound mechanism for hydroxylation

Oxygen rebound mechanism

H

HO

O

O

OH

Fe(V)

Fe(IV)

Fe(IV)

Fe(III)

First step is hydrogen atom abstraction from the substrate, generating a carbon-centred radical. The radical recombines with the iron-OH species to complete the oxidation of the organic substrate and the reduction of the iron.

Details of the epoxidation mechanism

O O

O

Fe(V)

Fe(IV)

Details of the Baeyer-Villiger Mechanism O R O R' R R' O O O H+ O Fe(III)

Fe(III)

Compare to the use of mCPBA - here the mechanism is a one electron mechanism - i.e. a stepwise radical process.

Fe(III)

R

O

Compare to the use of mCPBA in the classic Baeyer-Villiger reaction

R' O OH Fe(III)

* Note - These are 'simplified' 'general' mechanisms - mechanisms may vary for individual cases.

 

Key Reactions in Natural Products Biosynthesis 1. Themes Example

- P450 in Human Health

Cytochrome P450 enzymes are versatile oxidases, using molecular oxygen as the oxidant. epoxidation

benzopyrene present in cigarette smoke

human cyp1A1 lung

Benzopyrene - DNA adduct blocking DNA polymerase. O

See L. S. Beese and Coworkers J. Biol. Chem., 2005, 280, p3764. H2O

binds covalently to deoxyguanine of DNA and blocks DNA replication

O N N

O

human cyp1A1 lung

HO OH

DNA 3'

O

O

HO

NH N

NH

HO HO

OH

O

5'

OH

 

5.2B FAD/FMN Dependent Monooxygenases O H H2N

N O

N

N

O P

O P

O O O HO HO HO

N OHOH

O

R

SCoA

SCoA H

OH OH N

Flavin Adenine Dinucleotide

O

O

N

O2

O

N

HO

N H

O O

OH

O

N

N

NH

N H HO

O

Electron rich Nucleophile

HO HO

O

O

N

N

NH O OH

HO FAD hydroperoxide

O

O P

HO O HO HO

FAD

O NH

N H2O

O

N

OH

N

N

O

HO

O NH

N H

OH N

O O

O

N

O O

NH

N

O NH

N H

O O

FADH2

N

NH

N H

NH

N

H N

N β-oxidation

N FAD

R

O

O Some enzymes use FMN - Flavin mononucleotide. The Chemistry is the same.

Also good for epoxidation

 

5.3 Non-heme Iron dioxygenases

In dioxygenases both oxygens end up bound to substrate - i.e no water formation

α-ketoglutarate Dependent Dioxygenases

H N

OH2 H2O H2O

(i) + α-keto glutarate (ii) + O2

His

II N

Fe

N

O Asp

O

NH

H H

O

O

Me

(iii) - CO2, H - succinate OH (iv) + substrate

His

H

O

H N

O H

His

IV N

Fe

NH

O

O O

H

His

O

O

OH

Asp

O H

H N

His

III N

Fe

N

O O

NH

H

O O

Me H

His

OH

O

O H

H N

His

II N

Fe

N

NH

O

His

O Asp

Asp

(iii)(iv) O

O

O

H+

H

O

Me

N

(i)(ii)

HO2CH2C

H H

H N

O

O His

III N

Fe

N

O O Asp

NH His

HO2CH2C

O O

O

H N

O IV N

Fe

N

O O

H N

O His

HO2CH2C

O

O

NH

O C O

His

Asp

IV N

Fe O

O Asp

Activation by O2 and α-ketoglutarate to form the active Fe(IV) oxo species.

N

His

NH

Me

O

Me O

His

OH O

Other oxidases There are many other types of oxidases, including Rieske Iron dioxygenases and copper dependent dioxygenases (also known as Laccases) The details of these mechanisms are beyond the scope of this course.

 

OH OH

OH

O

 

6.0 Why is all this important ? Because Types of Reactions are 'conserved' - similar reactions use similar cofactors for mechanistic reasons. Cofactors are regognised by Proteins using conserved structural domains. Conserved structural Domains are built from Conserved Sequence Motifs. These motifs can be found in new proteins (and thus genes). Concept of 'Genome Mining'

DEBS1 AT ACP KS AT KR ACP KS AT KR ACP

KR

Module 2

Module 1

KS AT ACP KR AT

ACP KS

AT ACP KS AT S

S O

KR ACP

S

O

O

HO

HO HO

S O O

H H

O NH2

N R NAD(P)H