Obtinerea de enzime mutante New mutant enzymes · Atasarea proteinelor pe unele situsuri de legare...

Obtinerea de enzime mutante

https://www.neb.com/

β-lactamaza

1982

New mutant enzymes

β-lactamase

1982

Mutații

punctiforme

Site-directed

mutagenesis


New mutant enzymes

Tirozil-transfer ARN sintaza Tyrosyl–Transfer RNA Synthetase

Rodger, K.J. And Shiozawa, N., 2008, IntJBioChemCellBiol, 40(8), 1452

Autoantibodies directed against the

aminoacyl transfer RNA (tRNA)

synthetases are associated with

myositis, arthritis, Raynaud’s

phenomenon, mechanic’s hands,

fever, and interstitial lung disease,

clinically referred to as

anti–synthetase syndrome

Anticorpii pentru sintetaza aminoacil

ARNt sunt asociați cu miozită, artrită,

fenomen Raynaud, mâini mecanice,

febră și boală pulmonară interstițială

denumire clinică sindrom anti-

sintetază

Mutageneza Proprietatile enzimelor

stabilitate

Modificarea

specificitatiiEficienta catalitica

kcat/Km


New mutant enzymes

Mutagenesis Key properties of enzymes

stability

enhancing

Altering

substrate

specificity

Improving overall

catalitic activity

kcat/Km

Stabilizarea enzimelor

Utilizate în aplicații industriale

Enzyme Stability

Wide range of industrial applications

Îmbunătațirea performanțelor

în condițiile de lucru

To improve enzyme performance

under process conditions

Condiții extreme

temperatura

presiune

pH

alți parametri

Extreme conditions

temperature

pressure

pH

other conditions

Enzime hiperstabile

(condiții extreme de mediu)

Hyperstable enzymes

(extreme environmental

conditions)

2 strategii de stabilizare

prezenta unor suprafețe

mari cu interacții electrostatice

tendință de multimerizare

2 stabilizing strategies

large surface networks of

electrostatic interaction

tendency to be multimeric


O serie de strategii-mutații

Enzyme Stability

Several approaches-mutations

o Reducerea entropiei

proteinelor despachetate

punți S-S

↑nr. Pro (bucle mobile)

o To reduce the enthropy

of unfolded proteins

disulfide bridges

↑no Pro (mobile loops)

o Cresterea nr de -helixuri

Gly→Ala

impachetare

centru hidrofob

o To increase the number of -helices

Gly→Ala

well-packed

hydropobic cores

o Inginerie de punți de sare o Engineering of salt bridges

o Analiza filogenetica

proiectare logică

AA preferati in

organsimele termofile

o Engineering of salt bridges

Rational design

favoring common residues

(thermophiles)

Stabilizarea enzimelor Enzyme Stability

Strategii de stabilizare utilizate in proiectarea logica a mutatiilor dintr-o enzima

Stabilizing stratgies used in rational enzyme design

Ridoldi et al., APLBioeng, 2, 011501 (2018)

Termolizina (proteaza)

B.Strearothermophilus

Mutatii specifice: A4T, T56A, G58A,

T63F, S65P, A69P

Introducere 2 cisteine: N60C si G8C

Van den Burg et al. PNAS 95 (1998)

8 mutatii specifice

temperatura optima 58 0C

37 0C (nativa)


Enzyme Stability

Thermolysin (Bacillus thermoproteolyticus)

Aspartame synthesis assisted

by thermolysin

Sheldon, R.A. et al.

Green Chemistry 2 (2002)


McGrath-Directory of therapeutic enzymes

CRC Press-2006

O noua xiloz-izomeraza

producerea siropului

de fructoza

Enzyme Stability

A new xylose-isomerase

high fructose-corn syrup

Cho et al. Str. Biol Comm, 69(10), 2013 1127–1130

Stabilizarea enzimelor Enzyme Stability

Quax, W. J. et al. Biotechnology, 9 (1991)

Stabila in solutie 60–70°C

Mai putin susceptibila la glicozilare

de către glucoză (T)

t1/2 (60°C) > 1550h

nativa 607 h.

Whiterhouse-Enzyme in food technology-CRC Press-2002

More stable in solution 60–70°C

Less susceptible to glycosylation (T)

half-time (60°C) > 1550h

wild-type 607 h.

McGrath-Directory of therapeutic enzymes-CRC Press-2006

Stabilizarea enzimelor Increasing of the enzyme stability

Inițial – mutantii termostabili

reducere actv catalitica

Improving enzyme thermostability

reduction catalytic activity

Balanță rigiditate - flexibilitate

(stabilitate) (activitate)

Balance rigidity - flexibility

(stability) (activity)

Structurile enzimelor pot fi

stabilizate prin reticulare

intramoleculara

Enzyme structures can be stabilized

by forming intracellular crosslinks

Stabilizarea enzimelor Increasing of the enzyme stability

glutaraldehida glutaraldehyde

Cristale enzimatice reticulate

cu glutaraldehidaCrosslinked enzyme crystals

Glucoamilaza, lipaza

Subtilizina, termolizina

Glucoamylase, lipases

subtilizin, thermolysin

Atasarea proteinelor pe unele

situsuri de legare ale polimerilor

Enzyme attachment to multiple sites

of a suitable polymers

Invertaza / Chitosan sau pectina65 grade 5 min → 5h, 5 min → 2 zile

Invertase / Chitosan and pectin65 degree 5 min → 5h, 5 min → 2 days

DAAO instabila > 55 grade

atasare pe dextranstabila pana la 75 grade

DAAO unstable > 55 degree

DAAO/dextranstability up to 75 degree

Glucoz oxidaza- biosenzori glucozaInd. alimentara, fermentatie

GO / bile de sticla

stabila pana la 75 grade

crestere cu 180% activitate

Glucose Oxidase – glucose biosensorsfood and fermentation processes

GO / Glass beads

stability up to 75 degree

180% increase of relative activity

Frances H. Arnold

Evoluție enzimatică directă Enzyme directed evolution

noi catalizatori

Enzimele posedă

abilități neexploatate

Enzyme posses

unexplored abilities

fla

tha

tne

ws.c

om

Subtilizina E

10 mutații

60%DMF

Subtilisin E / casein (substrate)

3td generation mutant worked

256 X better in DMF


Metodologia se bazează pe A methodology relying on

1) Identificarea unei enzime

adecvate pentru cerința

1) Identification of a suitable starting

enzymes for the chosen task

2) Construcția unui set de

secvențe ADN ce acoperă

subsetul ales din secvență

2) DNA-sequence library construction

to cover well-chosen subset

of sequence space

3) Identificarea

unor criterii de selectie pentru

îmbunătățiri/ funcții noi

metodele de selectie a

versiunilor de mutanti

optimizate

3) Identification

selection criteria that will lead

to enhanced/ new function

methods for selection of optimized

enzyme variants

4) Rediversificarea genelor pentru

a crea noi librarii de ADN pe baza

secvențelor din prima selectie

4) Re-diversification of the genes

to create new DNA-sequence libraries

arround the sequences from

the first selection

5) Configurarea criteriilor de selecție

pentru stabilirea numarului de

runde necesare pentru a atinge

nivelul țintă al performanței enzimelor

5) Setup of selection criteria with

increased stringency, for as many rounds

as needed to reach the target level

of enzyme performance

Oxidare anti-Markovnikov Anti-Markovnikov oxidation

Hammer et al., Science, 358 (6360), 215-218, 2017

DOI: 10.1126/science.aao1482

Modificarea a 12 AA din P450

Modifying of 12 AA residue of P450

Sinteza 4-Cianotriptofanului în soluție apoasă

asistată de mTrpB din Thermotoga maritime

Synthesis of 4-cyanotryptophan in aqueous solution

Assisted by a Trp variant of Thermotoga maritime

Boville et al., JOC., 83 (14), 7447–7452, 2018

DOI: 10.1021/acs.joc.8b00517

η = 78%


Formarea de leg C-Si utilizând un mutant al citocromului c din Rhodothermus marinus

Mutant design of cytochrome c from Rhodothermus marinus - formation of C-Si bonds

Kan et al., Science, 354, 1048, 2016, DOI: 10.1126/science.aah6219


Noi legaturi chimice New chemical bonds

Genă a CytP450 BM-3 din Bacillus megaterium folosită în Ecoli conversia alcanilor în alcooli

A gene of CytP450 BM-3 from Bacillus megaterium cloned in Ecoli and used in alcohol biosynthesis

Beilen and Funhoff, Curr. Opin. Biotechnol, 16, 308-314, 2005

DOI:10.1016/j.copbio.2005.04.005


Producerea de biocombustili Biofuel production

Avantaje

alternativa verde

biotehnologii blânde

consum scăzut de solventi

↓produsi secundari, deseuri

Advantagesgreen alternative

milder biotehnology

low consumption organic

solvents

↓side products, waste

Herbabdez, K. E. et al., ACS Catal., 6 (11), 7810–7813, 2016

DOI: 10.1021/acscatal.6b02550 Inhibitor al agregării

plachentare

(prevenirea atacului

vascular/cerebral)


Producerea de intermediari

In industria medicamentelor

Production of intermediates used

in pharmaceutical industry

Patel, T. V. et al., Ann Trop Med Public Health, 6 (1), 14, 2013

Ticagrelor binds reversibly

and directly to the

ADP P2Y12 receptors on

the platelets change the

conformation

of these receptors

inhibits platelet activation

and eventual aggregation

Designul enzimelor terapeutice Engineering therapeutic enzymes

Proteina C activate (PCA) Redesigned activated protein C (APC)

Serin proteazaantitrombotic

antiinflamator

rol in homeostazia vasculara

atenueaza coagularea sangelui

Serine protease

antithrombotic

antiinflammatory

a role in vascular homeostasis

attenuate blood coagulation

Serpinele (serin proteaze)inhibitori reversibili ai PCA

Serpins (serine protease)

irreversible inhybitors of APC

Preston, R.J.S. Biochemical Society Transactions, 2015, 43 (4) 691-695.

L194S PCA mutant↑6x stabilitate plasma

↑5x descreste inactivarea / serpine

L194S APC mutant

↑6x increase plasma half-life

↑5x decrease the inactivation

by serpines

Designul enzimelor terapeutice Engineering therapeutic enzymes

Butirilcolinesteraza Butyrylcholinesterase (BChE)

Tratamentul toxicitatii

cu cocainadetoxificare - hidroliza

The treatment of acute

cocaine toxicity

hydrolyze cocaine

Un mutant dublu (AME-359)hidrolizeaza 40 x mai repede

A designed double-mutant

hydrolyze cocaine with 40 x improved kcat

Xie, W. wt al., Molecular farmacology, 1999, 55, 83-91.

4. Caracterizarea enzimei

4.6. Pachete de vizualizare 3D - Swiss PDB viewer

File Open 1HEW (baza de date)

Ferestre

de lucru Fereastră grafică

Control Panel Catenele laterale

Catena polipeptidică

Etichetări atomi

Reprezentare schematică

îngroşatăBara de control

Bara de aliniere

(Alignment)Ferestre

specifice

Wind/Alignment

Structura primara

Swiss PDB viewer

Selectare secvente scurte

Resturi marcate cu rosu

in Control PanelMutarea cursorului

mouse-ului

Localizare aminoacid individual

In structura tridimensionala

Graficul RamachandranFerestre

specifice

Wind / Ramachandran Plot

Swiss PDB viewer

Restrictiile conformaţionale

ale resturilor din proteină

Fiecare punct

Unghiurile φ şi θ

(fiecare rest din proteina)

DisplayInteriorul

proteinei

Slab

Swiss PDB viewer

Vizualizarea

atomilor

C-alb

S-galben

O-rosu

P-portocaliu

N-albastru

H-cian

Vizualizarea

3D

faţă Alt+5

sus Alt+8

bază Alt+2

stanga Alt+ 4

dreapta Alt+6

initial Alt+ 7

Swiss PDB viewer

Vizualizarea atomilor de O

din legaturile peptidice

Display / Show backbone

Catena laterala

aminoacid

Control Panel / side

Selectare aminoacid

Vizualizarea pozitiei

unui aminoacid

Control Panel / Label

Vizualizarea densitatii

electronice a unui

aminoacid

Control Panel / ::v

Motivele structurale

4. Caracterizarea enzimei4.4. Determinarea structurii secundare/supersecundare

Scop

http://scop2.mrc-lmb.cam.ac.uk/0.html

Relatiile dintre proteine

Clase structurale

Tipuri de proteine

Elemente de evolutie structurala

Clase structurale

Impachetarea proteinelor in

functie de continutul

structurii secundare



Scop

Protein Relationships

Other Relationships

Motif

CGxCxxCx(n)C motiv legare ion metalic

N - terminal ADH

Motivul Rosmann element de conexiune în buclă

GX[GS]XX[GSTA]

GS - contact cu pirofosfat (nucleotidă)



MOTIF Search

http://www.genome.jp/tools/motif/


MOTIF Search http://www.genome.jp/tools/motif/

pepsinogen A

MKWLLLLGLVALSECIMYKVPLIRKKSFRRTLSERGLLKDFLKKHNLNPARKY

FPQWKAPTLVDEQPLEN YLDMEYFGTIGIGTPAQDFTVLFDTGSSNLWVPSV

YCSSLACTNHNRFNPEDSSTYQSTSETVSITYGTG SMTGILGYDTVQVGGISD

TNQIFGLSETEPGSFLYYAPFDGILGLAYPSISSSGATPVFDNIWNQGLVSQ DL

FSVYLSADDQSGSVVIFGGIDSSYYTGSLNWVPVTVEGYWQITVDSITMNGEA

IACAEGCQAIVDTGT SLLTGPTSPIANIQSDIGASENSDGDMVVSCSAISSLPDI

VFTINGVQYPVPPSAYILQSEGSCISGFQG MNLPTESGELWILGDVFIRQYFTV

FERANNQVGLAPVA


MOTIF Search

HUMAN Tyrosine-protein phosphatase non-receptor type 6 Homo sapiens MVRWFHRDLSGLDAETLLKGRGVHGSFLARPSRKNQGDFSLSVRVGDQVTHIRIQNSGDF

YDLYGGEKFATLTELVEYYTQQQGVLQDRDGTIIHLKYPLNCSDPTSERWYHGHMSGGQA

ETLLQAKGEPWTFLVRESLSQPGDFVLSVLSDQPKAGPGSPLRVTHIKVMCEGGRYTVGG

LETFDSLTDLVEHFKKTGIEEASGAFVYLRQPYYATRVNAADIENRVLELNKKQESEDTA

KAGFWEEFESLQKQEVKNLHQRLEGQRPENKGKNRYKNILPFDHSRVILQGRDSNIPGSD

YINANYIKNQLLGPDENAKTYIASQGCLEATVNDFWQMAWQENSRVIVMTTREVEKGRNK

CVPYWPEVGMQRAYGPYSVTNCGEHDTTEYKLRTLQVSPLDNGDLIREIWHYQYLSWPDH

GVPSEPGGVLSFLDQINQRQESLPHAGPIIVHCSAGIGRTGTIIVIDMLMENISTKGLDC

DIDIQKTIQMVRAQRSGMVQTEAQYKFIYVAIAQFIETTKKKLEVLQSQKGQESEYGNIT

YPPAMKNAHAKASRTSSKHKEDVYENLHTKNKREEKVKKQRSADKEKSKGSLKR

Motivele structurale-domenii/situsuri


InterPro

http://www.ebi.ac.uk/interpro/

IPR011895

Domeniul catalitic

Motiv de legare al

Fosfatului de tiamina

Domeniul EKR (bacterii,

eucariote inferioare)



https://www.cellsignal.com/


4. Caracterizarea enzimei4. 4. Determinarea structurii secundare/supersecundare

CATH

http://www.cathdb.info/

95 milioane de domenii proteice

6,199 superfamilii

Superfamilia

3.30.70.330Domeniul


4. Caracterizarea enzimei4. 4. Determinarea structurii secundare/supersecundare

CATH

Structuri 3D determinate experimental

https://www.rcsb.org/

4. Caracterizarea enzimei4.5. Baze de date cu structura tridimensionala a proteinelor

Structuri 3D determinate experimental

RCSB PDB


http://www.expasy.org/proteomics

Expert Protein Analysis System ExPASy


https://www.proteinmodelportal.org/

http://www.uniprot.org/ (pir.georgetown.edu)

5 milioane proteine – conexiune cu alte str 3D

UniProt


http://www.uniprot.org/

http://www.nextprot.org/

Proteine umane

NextProt

4. Caracterizarea enzimei4.5. Baze de date cu proteine

http://www.nextprot.org/

STRING http://string-db.org/

Interactiuni dintre proteine

4. Caracterizarea enzimei4.5. Baze de date cu interacțiuni între proteine

Comparari structuri

3D proteine

Dali

http://ekhidna.biocenter.

helsinki.fi/dali/

4. Caracterizarea

enzimei

Entrez Databases

http://www.ncbi.nlm.nih.gov/Class/MLACourse/Original8Hour/Entrez/

4. Caracterizarea enzimei

4.5. Baze de date cu structura tridimensionala a

proteinelor

Baze de date cu nomenclatura enzimelor

UniProtKB/Swiss-Prot http://enzyme.expasy.org/

Obtinerea de enzime mutante New mutant enzymes · Atasarea proteinelor pe unele situsuri de legare...

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