Science - 06.12.2019

SCIENCE sciencemag.org

GRAPHIC: A. KITTERMAN/

SC

IENCE

By Elaine O’Reilly and James Ryan

N

atural biosynthesis assembles a

vast array of complex natural prod-

ucts starting from a limited set of

building blocks, under physiologi-

cal conditions, and in the presence

of numerous other biomolecules.

Organisms rely on the extraordinary se-

lectivity of enzymes and their ability to

operate under similar reaction conditions,

meaning that these catalysts are perfectly

adapted to mediate cascade reactions. In

these multistep processes, the product of

one biocatalytic step becomes the sub-

strate for the next transformation ( 1 – 3 ).

On page 1255 of this issue, Huffman et al.

( 4 ) report the development of an impres-

sive nine-enzyme biocatalytic cascade for

the synthesis of the investigational drug

islatravir for the treatment of human HIV.

This study represents a partnership be-

tween scientists from Merck and Codexis.

These two companies have a history of

successfully collaborating to develop bio-

catalysts for the synthesis of important

pharmaceuticals. Almost a decade ago,

they developed a chemoenzymatic route

for the synthesis of the type 2 diabetes

drug sitagliptin (Januvia), relying on a key

enzyme-catalyzed transamination with

a highly engineered (R)-selective trans-

aminase ( 5 ). The work was considered a

landmark example of directed evolution

and functioned to highlight the potential

application of biocatalysis to revolutionize

industrial chemical processes.

The cascade for synthesizing islatravir

was inspired by the bacterial nucleoside

salvage pathway, which recycles precious

nucleosides by using three key enzymes:

a purine nucleoside phosphorylase (PNP),

a phosphopentomutase (PPM), and a de-

oxyribose-5-phosphate aldolase (DERA)

(see the figure). However, to achieve the

synthesis of the target molecule, Huffman

et al. required the natural nucleoside deg-

radative cascade to run in reverse. The

reversible nature of enzymes is central to

the design of this cascade and is one of

the important features that sets biocata-

lysts apart from the majority of traditional

chemical catalysts.

The success of the cascade developed by

the team also relied on all three enzymes

accepting non-natural substrates bear-

ing a fully substituted carbon at the C-4

position of the 2-deoxyribose ring. The

authors reconstructed the reverse nucleo-

side salvage pathway from a PNP and PPM

found in Escherichia coli and a DERA from

Shewanella halifaxensis. The native E. coli

enzymes required engineering to improve

their activity. The DERA displayed existing

high activity and stereoselectivity for the

formation of the desired sugar phosphate

enantiomer, but it required engineering to

improve its ability to operate at high sub-

strate concentration.

One of the many advantages of per-

forming biocatalytic cascade reactions is

the effective displacement of unfavorable

reaction equilibria that can be achieved

through product removal. However, de-

spite performing the PNP and PPM steps

in tandem, the reaction proceeded with

poor conversion, and the inorganic phos-

phate by-product inhibits the enzymes. An

elegant solution to these issues was the in-

clusion of an auxiliary sucrose phosphory-

lase, along with its sugar substrate, which

removed free phosphate and effectively

displaced the reaction equilibrium toward

product formation.

Having assembled enzymes for the

three key steps in the cascade, Huffman et

al. sought to develop a biocatalytic route

for the synthesis of the DERA substrate

2-ethynylglyceraldehyde 3-phosphate.

Extensive screening of a broad range of

kinases resulted in the identification of

pantothenate kinase (PanK) from E. coli,

which displayed low levels of activity (~1%

conversion) toward the (R)-enantiomer of

the target aldehyde. Despite the modest

initial activity, directed evolution was suc-

cessfully used to substantially improve the

productivity and stability of this enzyme.

Finally, after 12 rounds of evolution, the

authors reversed the enantioselectivity

and improved the activity, stability, and ex-

pression of a galactose oxidase variant for

the desymmetrization of the starting sub-

strate, 2-ethynylglycerol.

Advancements in protein engineering,

BIOCATALYSIS

Biocatalytic cascades go viral

An investigational drug targeting the HIV virus

is synthesized with nine enzymes

School of Chemistry, University College Dublin, Belfield,

Dublin 4, Ireland. Email: [email protected]

GOase

Catalase

By-product

removal

Maintains metal

oxidation state

Cofactor

HRP regeneration

PanK

DERAPPMPNP

AcK

HO O 2 (air)

HO OH

Immobilized enzyme; Me, methyl

HO

HO O

HO

HO

HO

• HO
  3 PO
  O

O

Me OPO 3 H–

O

Me H

NH 2

NH (^2) H
2 PO 4

• 
  

Sucrose

Islatravir

Reverse nucleoside

salvage pathway

By-product

removal

Fructose

Glucose 1-

phosphate

NH N O SP

N

N

N

NN

F

N

F

+

+

PPM Bacterial

DERA Bacterial

PanK Bacterial

GOase Fungal

Panthothenate kinase

Galactose oxidase

Deoxyribose-5-phosphate aldolase

Phosphopentomutase

EVOLVED ENZYME ABBREVIATION SOURCE OF ENZYME

SP Bacterial

AcK Bacterial

HRP Plant

• Mammalian
  Horseradish peroxidase

Catalase

Acetate kinase

Sucrose phosphorylase

AUXILIARY ENZYME ABBREVIATION SOURCE OF ENZYME

Purine nucleoside phosphorylase PNP Bacterial

Engineering a biocatalytic cascade

The synthetic pathway for the production of the investigational drug islatravir (MK-8591) was inspired by the natural bacterial nucleoside salvage pathway. The cascade

created by Huffman et al. uses a total of nine enzymes, five of which were evolved to optimize their properties and four additional auxiliary enzymes.

6 DECEMBER 2019 • VOL 366 ISSUE 6470 1199

Published by AAAS

on December 12, 2019^

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