Fluorescence-Activated Cell Sorting of Human l-asparaginase Mutant

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Article

Fluorescence-activated cell sorting of human Lasparaginase mutant libraries for detecting enzyme variants with enhanced activity for the physiological substrate Christos S. Karamitros, and Manfred Konrad ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00283 • Publication Date (Web): 21 Jul 2016 Downloaded from http://pubs.acs.org on July 25, 2016

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ACS Chemical Biology

Fluorescence-Activated Cell Sorting of Human L-asparaginase Mutant Libraries for Detecting Enzyme Variants with Enhanced Activity Christos S. Karamitros1 & Manfred Konrad Enzyme Biochemistry Group, Max-Planck Institute for Biophysical Chemistry, Göttingen, D-37077, Germany KEYWORDS. Human L-asparaginase-3, glycine-dependent activation, enzyme engineering, high throughput screening, acute lymphoblastic leukemia. ABSTRACT. Immunogenicity is one of the most common complications occurring during therapy making use of protein drugs of non-human origin. A notable example of such a case is bacterial L-asparaginases (L-ASNases) used for the treatment of acute lymphoblastic leukemia (ALL). The replacement of the bacterial enzymes by human ones is thought to set the basis for a major improvement of antileukemic therapy. Recently, we solved the crystal structure of a human enzyme possessing L-ASNase activity, designated hASNase-3. This enzyme is expressed as an inactive precursor protein, and post-translationally undergoes intramolecular processing leading to the generation of two subunits which remain non-covalently, yet tightly associated and constitute the catalytically active form of the enzyme. We discovered that this intramolecular processing can be drastically and selectively accelerated by the free amino acid glycine. In the present study we report on the molecular engineering of hASNase-3 aiming at the improvement of its catalytic properties. We created a fluorescence-activated cell sorting (FACS)-based high-throughput screening system for the characterization of rationally designed mutant libraries, capitalizing on the finding that free glycine promotes autoproteolytic cleavage which activates the mutant proteins expressed in an E. coli strain devoid of aspartate biosynthesis. Successive screening rounds led to the isolation of catalytically improved variants showing up to 6-fold better catalytic efficiency as compared to the wild-type enzyme. Our work establishes a powerful strategy for further exploitation of the human asparaginase sequence space to facilitate the identification of in vitro-evolved enzyme species that will lay the basis for improved ALL therapy. 

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Introduction

cell epitopes requires extensive mutagenesis at

Cancer is considered as one of the most difficult

sites of the protein that may have a negative im-

diseases to cure, and its treatment is often limited

pact on the catalytic activity and stability of the

because of acute toxicities toward normal cells

molecule.11

caused by chemotherapeutics.1,2 A noteworthy

The alternative approach focuses on the devel-

complication of cancer treatment is immuno-

opment of enzyme drugs derived from proteins of

genicity3 when proteins or enzymes of non-hu-

human origin. Human enzymes in most cases are

man origin are used in anti-neoplastic regimens.4

characterized by immune tolerance, and therefore

To circumvent side effects associated with im-

it is generally believed that their use for disease

munogenicity, two experimental strategies have

treatment can be advantageous in comparison to

predominantly been put forward : i) combinatori-

their non-human homologues.12 However, a main

al deimmunization of non-human proteins based

drawback inherent to this approach is that hu-

on epitope analyses5,6,

and ii) molecular engi-

mans often do not encode enzymes displaying the

neering of weakly active human enzymes aiming

desirable catalytic and pharmacological features

at improving catalytic efficiency to replace the

adequate for therapeutic applications. Therefore,

non-human molecules in therapeutic settings.7 In

the development of such drugs largely depends

the first case, the goal is the mutational disruption

on successful protein engineering strategies. 13,14,15

of amino acid sequences of the target molecule which could be recognized by the adaptive im-

A prominent example of bacterial enzymes which

mune system either as B- or T-cell epitopes. This

have been used for more than fifty years for ther-

necessitates the identification of such sequences

apeutic application in humans are L-asparaginas-

which can be achieved by in silico, in vitro, and

es (L-ASNases) which catalyze the hydrolysis of

in vivo methods.8 However, the reliable predic-

L-asparagine (L-Asn) to L-aspartate (L-Asp) and

tion and the removal of B-cell epitopes is excep-

ammonia.16 The clinically relevant enzymes, ex-

tionally difficult due to their conformational

clusively originating from the two bacterial

complexity, as well as our limited awareness of

species Escherichia coli and Erwinia chrysan-

how the antibody repertoire varies among distinct

themi, have been used at the frontline for the

human populations.9 On the other side, it has

treatment of acute lymphoblastic leukemia

been evidenced by a number of different reports

(ALL), an aggressive cancer of the white blood

that the disruption of T-cell epitopes can lead to

cells.17 The principle of this therapy is based on

reduced immunogenicity and, consequently, low-

the observation that the metabolic enzyme as-

er antibody responses.10 Yet, the removal of T-

paragine synthetase of ALL cells is downregulat-


2

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ed, such that the levels of intracellularly synthe-

L-ASNase-3 (hASNase-3), an enzyme which cat-

sized L-Asn cannot satisfy the high metabolic

alyzes primarily the hydrolysis of β-aspartyl-

demands of the malignant cells.18 As a conse-

dipeptides (end products of degraded proteins),

quence, the survival of the cancerous cells is

and secondarily L-Asn, and belongs to the N-

solely dependent on the exogenous supply of L-

terminal nucleophile (Ntn) super-family of en-

Asn from the blood stream.19 Intramuscular or

zymes.23,24 The main biochemical feature of this

intravenous administration of L-ASNase prepara-

family of enzymes is that they are expressed as

tions deplete the available L-Asn from the pa-

inactive single polypeptide chain-precursors and

tients’ blood, and this results in total shortage of

post-translationally undergo an intramolecular

this amino acid for the cancerous lymphoblasts,

autoproteolytic cleavage step at a scissile peptide

ultimately leading to blockage of protein synthe-

bond generating two subunits (termed protomers

sis and apoptosis.20

α and β), which remain non-covalently, yet tight-

Numerous nocuous side effects such as immuno-

ly associated and represent the active form of the

genicity and hypersensitivity reactions have been

enzyme (Figure S1).25 A second human protein

well documented over the years for ALL treat-

identified as enzyme displaying L-ASNase activi-

ment using L-ASNases, and these side effects

ty is the 60-kDa lysophospholipase.26 This two-

have been mainly attributed to the bacterial origin

domain protein consists of an N-terminal L-AS-

of the enzymes.21 Polyethylene glycol (PEG)-

Nase domain which resembles the cytoplasmic

modified versions of the bacterial L-ASNases,

type-I bacterial L-ASNases, and a C-terminal

that were shown to have a longer serum half-life

ankyrin repeat domain.27 Recently, we showed

and to be less immunogenic, have been clinically

that the N-terminal domain of the 60-kDa human

approved to overcome certain drawbacks of the

lysophospholipase (designated hASNase-1) can

native enzymes.22 However, it appears reasonable

be expressed as truncated form (369 amino acids)

to believe that the replacement of the current bac-

and possesses L-ASNase activity. A remarkable

terial enzymes by related ones of human origin

feature of hASNase-1 is its pronounced sig-

would substantially improve ALL treatment.

moidal steady-state kinetic profile, which is simi-

However, the human genome does not encode

lar to the one reported for its E. coli homologue.28

homologues of catalytically efficient bacterial L-

The present work focuses on molecular engineer-

ASNases. Instead, human enzymes that exhibit

ing of hASNase-3 aiming at the improvement of

L-ASNase activity are catalytically very poor

its catalytic properties for the physiological sub-

and, therefore, lack therapeutic potency.

strate L-Asn, ultimately resulting in the selection

Recently, we reported on the structure of human

of a human enzyme that could serve for replace-


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ment of the bacterial L-ASNases as anti-leukemic

stronger fluorescence. Importantly, we relied on

agents. We chose to center our work on hAS-

our recent discovery that the free amino acid

Nase-3. rather than hASNase-1, for the following

glycine drastically accelerates the autoproteolytic

reasons: i) hASNase-1 displays strong allosteric

activation of hASNase-3.24 Therefore, the cells

regulation by its substrate L-asparagine, which

were grown in the presence of glycine for the in-

complicates the engineering scheme, and ii) the

duction of the enzyme’s self-processing. In the

crystal structure of hASNase-3 in complex with

absence of glycine, the intramolecular activation

its product L-aspartate is known, providing de-

of hASNase-3 is extremely slow, not allowing the

tailed structural information on the functional

monitoring of detectable levels of enzymatic ac-

role of sequence regions to be targeted for direct-

tivity.

ed evolution of the enzyme molecule. We em-

We generated several site-saturation-mutant

ployed a FACS-based high-throughput screening

(SSM) libraries by randomizing specific residues

(HTS) to select for catalytically improved vari-

chosen by inspecting the available structure of

ants of human L-ASNase-3. This HTS (Scheme

hASNase-3. The major challenge in this particu-

1) is based on the use of a 5-gene-deletion strain

lar engineering scheme is the generation of muta-

of E.coli (devoid of all genes which contribute to

tions which do not negatively affect the intramol-

the biosynthesis of L-Asp) whose survival is

ecular processing, yet may have a positive impact

solely dependent on the availability of L-Asp

on the catalytic activity. Subsequent mutant se-

from the growth medium.29,30 Genetic comple-

lection rounds resulted in the isolation of variants

mentation by hASNase-3 mutants growing in L-

catalytically improved in comparison to the wild-

Asp-free minimal medium rescues these E.coli

type enzyme with respect to both kinetic con-

cells through the supply of L-Asp generated by

stants, KM and kcat, and whose activation equally

enzyme-catalyzed L-Asn hydrolysis, the growth

relies on the availability of glycine. Beneficial

and proliferation of the bacterial cells being pro-

mutations then were combined in order to inves-

portional to the activity of the hASNase-3 mu-

tigate possible positive epistatic effects. Our data

tants. The co-expression of the enhanced green

suggest that the combination of such mutations

fluorescent protein (eGFP) provided an additional

resulted in improved conformational stability

quantification level of L-Asp availability, corre-

rather than catalytic efficiency as evidenced by

lating intracellular eGFP fluorescence intensity

differential scanning fluorimetry (DSF) analyses

with the mutants’ L-ASNase activity. Thus, high-

of the recombinantly produced purified mutant

er availability of L-Asp results in higher expres-

enzymes.

sion levels of eGFP which, in turn, results in ACS Paragon Plus Environment

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George Georgiou’s lab (University of Texas at Austin, Department of Chemical Engineering and Institute for Cellular and Molecular Biology). pBAD33 is a medium copy number plasmid (~ 25 copies per cell) harboring the chloramphenicol antibiotic resistance marker cmR coding for chloramphenicol acetyltransferase (CAT). The E. coli five-gene deletion strain, designated JC1(DE3), was also a kind gift from Dr. George Georgiou’s Scheme 1. Representation of the high-throughput screening approach for the identification of catalytically improved hASNase-3 variants.

lab. The deleted genes of this strain are the following ones: The L-ASNase genes ansA (encoding L-ASNase-1), ansB (encoding L-ASNase-2), iaaA (encoding L-ASNase-3), the aspartate

EXPERIMENTAL SECTION

aminotransferase gene aspC, and the tyrosine Plasmids, E.coli strains, cDNA, and chemicals.

aminotransferase gene tyrB. The parental strain

Plasmid pET-14b (purchased from Novagen) is a

used for the chromosomal deletions was E. coli

medium copy number plasmid (~30 copies per

MC1061. All restriction and DNA-modifying en-

cell); it carries the pBR322 origin of replication

zymes were from New England Biolabs. All

and the ampicillin antibiotic resistance marker

oligonucleotides used in this study were obtained

ampR coding for the enzyme beta-lactamase

from IBA (Goettingen, Germany). Yeast extract,

(BLA). A modified version of this plasmid, des-

peptone, agar, ampicillin, chloramphenicol, iso-

ignated pET14b-SUMO,31 includes the SUMO

propyl-beta-D-thiogalactoside (IPTG), arabinose

tag (101 residues, derived from the Saccha-

and glucose were purchased from Applichem

romyces cerevisiae smt3 gene) positioned be-

(Gatersleben, Germany). L-asparagine, flavine

tween the N-terminal His6-tag and the multiple

adenine dinucleotide (FAD), nicotinamide ade-

cloning site (MCS). The His6-SUMO-tag was

nine dinucleotide (NADH), horseradish peroxi-

removed by cleavage with SUMO protease as

dase (HRP type X, 386 U/mg, Mr ~ 44 kDa), glu-

described previously.31 The E.coli host strain C41

tamate dehydrogenase from bovine liver (GDH,

(DE3) was obtained from Lucigen/BioCat. The

44 U/mg, Mr ~ 56 kDa), α-ketoglutarate, L-aspar-

XL1-Blue and DH5α strains were purchased

tate, all other amino acids used for M9 minimal

from Stratagene and Invitrogen, respectively.

medium, and the SYPRO Orange dye were ob-

Plasmid eGFP-pBAD33 was a kind gift from Dr.

tained from Sigma Aldrich (St. Louis MO, USA).


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Page 6 of 29

Amplex Red (AR) was purchased from the Cay-

zyme served as control for the FACS HTS analy-

man Chemical Company (Michigan, USA). The

sis. EcASNase-2 displays a ~ 2,000-fold higher

gel filtration chromatography column Superdex

catalytic efficiency as compared to hASNase-3

200 was from Pharmacia/GE Health Care Life

(KM and kcat of EcASNase-2 for L-Asn: 20 µM and

Sciences (Uppsala Sweden). Coomassie Brilliant

11 s-1 at 25 ̊ C, respectively, as determined in the

Blue G-250 (Bradford reagent) for protein assays

present study). FACS analysis of the five-gene-

was from Roth (Karlsruhe, Germany). All PCR

deletion E.coli strain JC1(DE3), genetically com-

reactions were performed with KAPA HiFi poly-

plemented by wildtype hASNase-3 and EcAS-

merase purchased from PeqLab (Erlangen, Ger-

Nase2 provided information about the sensitivity

many). Gel extraction and PCR purification kits,

of the screen (Figure S4). L-AspOx is an essen-

and nickel resin for affinity purification of poly-

tial helper enzyme for the three-step fluorescent

histidine-tagged proteins was from Macherey

assay we developed for monitoring L-ASNase

Nagel (Düren, Germany), plasmid purification

activity in 96-well plate formats.32 This assay was

kits from Fermentas (Thermo Fisher Scientific,

employed for the activity analysis of single

Germany), kits for genomic DNA preparation

clones following the final sorting round of the

from Qiagen (Hilden, Germany). The hASNase-3

mutant libraries.

open reading frame was amplified using as template cDNA from a human skin and meninges

Assays for L-asparaginase activity determination and kinetic characterization of the enzyme.

library (Source Bioscience, UK). All DNA constructs involving PCR amplifications were con-

i) The NADH-dependent assay is a continuous

firmed by sequencing (SeqLab, Goettingen,

spectrophotometric coupled-enzyme assay which

Germany) according to the company’s instruc-

monitors the conversion of α-ketoglutarate plus

tions.

ammonia to glutamate in a glutamate dehydrogenase (GDH)-coupled reaction (Figure S2).34 The

Cloning, expression and purification of hASNase-3, E.coli L-asparaginase-2 and E.coli Laspartate oxidase.

disappearance of NADH was monitored continuously at 340 nm and was directly proportional to

The cloning in pET14b-SUMO, recombinant ex-

the L-ASNase activity. For these measurements,

pression, purification, and glycine-induced acti-

a Jasco UV/VIS V-650 spectrophotometer was

vation of hASNase-3, E.coli ASNase-2 (EcAS-

used. All enzymes used for the kinetic experi-

Nase-2; the current anti-leukemic drug) and

ments were free of the protease-cleaved His6-

E.coli Aspartate Oxidase (L-AspOx) have been

SUMO tag which was removed in the size exclu-

described elsewhere.32,33 The EcASNase-2 en-

sion chromatography step. For steady-state kinet-


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ic analysis of all hASNase-3 species, L-Asn con-

Asn concentration was chosen in order to select

centrations were varied in the range of 0-5KM (0-

mutants for improved KM given the fact that wild-

15 mM) in a final volume of 1 mL of 50 mM

type hASNase-3 exhibits a KM value ~ 3 mM.23

Tris-Cl, 100 mM NaCl, pH 8. The final enzyme

The reaction kinetics was monitored continuously

concentration was typically in the range of 1-1.5

for 20 min using a fluorescent plate reader (Mol-

µM (~ 30-50 µg in 1 mL). The obtained V/E (ve-

ecular Devices, SpectraMax Paradigm, and filter

locity/total enzyme concentration) values were

settings Ex. 532, Em. 592).

plotted against the respective substrate concentra-

The assay conditions for the steady-state kinetic

tions. The kinetic constants KM and kcat were calcu-

characterization of the enzymes using L-Asp-β-

lated by non-linear regression using the

methyl ester as substrate were the following: 1

Michaelis-Menten model (equation 1) and ana-

mL of 50 mM Tris-Cl, 100 mM NaCl, pH 8, final

lyzed by the SoftZymics software (Igor Pro,

enzyme concentration typically in the range of ~

Wavemetrics):

40 nM (~ 1 µg in 1 mL). The tested substrate concentrations covered the range 0-5KM (0-3

$

mM). The assay was performed at room temperature using similarly to the NADH assay a Jasco

(1)

UV/VIS V-650 spectrophotometer adjusted at 570 nm that is the maximum absorbance of Re-

ii) The Amplex Red-dependent fluorescent as-

sorufin, the final product of the three-step assay

say was used for the final screening step in 96-

(Figure S3). The kinetic constants KM and kcat

well plates of the selected mutants following the

were calculated as described above.

last FACS sorting round, as well as for the kinetic characterization of the hASNase-3 mutants using

Generation of hASNase-3 mutant libraries.

as substrate the dipeptide L-Asp-β-methyl ester.23

Site-Saturation-Mutagenesis libraries (SSM-Lib)

One of the products of this reaction is L-Asp, and

were generated using the overlap extension PCR

this can be detected using the Amplex Red fluo-

methodology (Figure S5) and screened for cat-

rescent assay. The assay solution using 96-well

alytically improved hASNase-3 variants. The li-

plates during the last screening step for the iden-

braries were designed using as template the crys-

tification of active mutants contained: 5 µM

tal structure model of hASNase-3 (PDB entry:

(~300 µg/mL) L-Aspartate Oxidase (L-AspOx),

4OSY) and based on a massive amino acid se-

10 µM FAD, 100 nM (0.1 U/mL) HRP, 50 µM

quence alignment of more than 1000 homologous

Amplex Red, and 1 mM L-Asn in a final volume

enzymes from different organisms using the pro-

of 50 µL per well. The use of 1 mM as final L-


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Page 8 of 29

gram Geneious.35 Figures S6A and S6B show

initiated at 95 ̊ C for 3 min, followed by 30 cycles

the results of this alignment for the randomized

of denaturation at 98 ̊ C for 20 s, primer annealing

regions of SSM-Lib1 and SSM-Lib2. Additional-

at 63 ̊ C for 30 s, and extension at 72 ̊ C for 40 s.

ly, Figure S7 shows the first ten consensus

The amplification reactions were terminated after

residues of this alignment (NPVIAIHGG), high-

a 5-min polishing step at 72 ̊ C. The PCR products

lighting in a red frame the highly conserved triad

were gel-purified (1.2% agarose, kit from

His8-Gly9-Gly10 (counting starts from Met1)

Macherey Nagel) and then mixed in equal molar

which is critical for intramolecular processing

amounts for the final overlap-extension PCR us-

and catalytic activity. The alignment served as

ing the external FWhASNase3wt and RVhASNase3wt

tool to determine highly conserved residues in the

primers (Table S1). The reaction parameters were

hASNase-3 sequence, thus avoiding mutating

identical to the ones described above, except for

them since they could play a pivotal role for the

the annealing step which was done at 65 ̊ C, and

enzyme’s activity and stability. Codons were ran-

the total number of cycles was 25. The final PCR

domized using degenerate oligonucleotides (Ta-

product was gel-purified, digested with NdeI and

ble S1) following the NNS scheme (N: A, T, G,

BamHI H.F., purified with PCR clean-up kit, and

C; S: G, C). For randomization, the NNS-type of

then ligated overnight at 16 ̊ C into the pET14b-

codons was chosen in order to minimize the

SUMO vector using T4 DNA ligase (molar ratio

probability of generating stop-codons. This com-

1:3; vector:insert). The ligation mixture was used

bination of nucleotides allows all the twenty pos-

to transform electro-competent DH5α E.coli cells

sible amino acids to be encoded, but only one

which were streaked onto ampicillin-containing

stop-codon (UAG). In case of SSM-Lib1 and

2xYT plates and incubated at 30 ̊ C overnight. In-

SSM-Lib2, where two residues were randomized,

sert-containing clones were identified by restric-

the theoretical diversity on the DNA level was

tion digestion with NdeI and BamHI H.F., and

~103, and on the amino acid level 4x102.

finally by sequencing of the cloned DNA insert to

The PCR reactions for the construction of the

verify proper randomization of the codons at the

SSM-Libs described above were run in a final

desired sites. In cases where the background liga-

volume of 50 µL and included the following

tion reaction of the negative control (without in-

components: 10 ng of plasmid DNA template

sert) accounted for >5% of the positive control,

(pET14b-SUMO-hASNase-3), 100 pmoles of

the plates were discarded, and the process was

each primer, 1X KAPA HiFi buffer, 0.5 mM

reinitiated from the cloning level. In case of suc-

dNTPs, and 1 Unit KAPA-HiFi DNA poly-

cessful library construction, the cells were

merase. In the first two PCRs, the reactions were

scraped from the original plates and resuspended


8

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in 2xYT medium supplemented with 200 µg/mL

trifuged for 5 min at 8,000g at 4 ̊ C and washed

ampicillin. A fraction of this suspension was used

3X with ice-cold 0.9% NaCl. Subsequently, the

to culture 0.5 L of 2xYT medium, and the rest of

cells were resuspended in M9 minimal medium

the cells were pooled and stored at -80 ̊ C as glyc-

containing 1% glycerol, 3.5 µg/mL thiamine, 1

erol stocks. Following overnight growth at 30 ̊ C,

mM MgSO4, 0.1 mM CaCl2, 160 µg/mL of L-Tyr,

the 0.5 L culture was centrifuged, and the plas-

5 mM L-Asn, and 80 µg/mL of the remaining 18

mid DNA was extracted using a MIDI-PREP kit

amino acids except L-Asp, 200 µg/mL ampicillin,

(Macherey Nagel). Approximately 100 ng of the

35 µg/mL chloramphenicol, 1 mM IPTG, 2%

plasmid mutant library (pET14b-SUMO-hAS-

arabinose and 50 mM glycine. The cultures were

Nase-3) were used to transform the electro-com-

incubated at 37 ̊ C by shaking at 250 rpm for 2 h.

petent JC1(DE3) five-gene-deletion strain which

Next, the cells were centrifuged 5 min at 8,000g

already harbors the pBAD33-eGFP plasmid

at 4 ̊ C and washed twice with ice-cold PBS. Ul-

(chloramphenicol resistance). The transformed

timately they were resuspended in PBS solution

cells were cultured in 0.5 L 2xYT medium sup-

at a final OD600 of 0.05 for subsequent FACS

plemented with 200 µg/mL ampicillin and 35 µg/

analysis.

mL chloramphenicol. When OD600 reached ~ 2,

Flow cytometric analyses were performed with a

0.5 mL aliquots were pooled and stored at -80 ̊ C

BD Biosciences Influx FACS instrument using a

as glycerol stocks. For subsequent screening, a

488-nm solid-state laser for excitation and a 495-

0.5 mL aliquot was used to inoculate a 50 mL

525 bandpass filter for detection. The cells corre-

culture of 2xYT medium as described in the next

sponding to the 5% of the most highly fluores-

section where the screening process is discussed.

cent cells of the parental population were sorted in a throughput of ~ 4-5,000 cells per second in

FACS-based screening of hASNase-3 mutant

the single-cell mode. The sorted cells were col-

libraries. Frozen aliquots of JC1(DE3) cells co-

lected in tubes containing 2xYT medium supple-

transformed with an SSM library and pBAD-

mented with 200 µg/mL ampicillin and 35 µg/mL

eGFP plasmids were used to inoculate 50 mL

chloramphenicol, and were finally plated onto

2xYT cultures supplemented with 0.4% glucose,

2xYT plates with the respective antibiotics. Fol-

200 µg/mL ampicillin, 35 µg/mL chlorampheni-

lowing overnight growth, the clones were pooled

col, and 50 mM glycine (which induces the in-

and stored at -80 ̊ C in aliquots for the next sorting

tramolecular activation of hASNase-3); the start-

round.

ing OD600 was ~ 0.1. The cells were grown at 37 ̊ C, and when OD600 reached 1, the cells were cen-

Identification of catalytically improved hAS-


9


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Page 10 of 29

Nase-3 mutants. After the last FACS-sorting

15C swinging bucket plate-centrifuge. The su-

round of the mutant libraries, the pooled clones

pernatant culture medium was discarded using a

were cultured, and their plasmid DNA was ex-

12-channel Eppendorf pipette; the pelleted cells

tracted (plasmid DNA contains both pET14-

were resuspended in 50 mM Na2HPO4, 300 mM

SUMO and pBAD33 plasmids). The extracted

NaCl, pH 8, supplemented with 1X BugBuster

plasmid DNA was used as template for the ampli-

(Novagen) cell lysis reagent and 5 KU/mL re-

fication of the coding region of hASNase-3 mu-

combinant lysozyme (rLysozyme, Novagen). The

tants using the primers FWhASNase3 wt and RVhASNase3

plates were incubated at RT for 30 min under

wt. The amplified sequences were gel-purified,

mild shaking (50 rpm), and were then centrifuged

digested with NdeI and BamHI H.F., and finally

for 20 min at 3,000g. The supernatants were

ligated into pET14b-SUMO overnight at 16 ̊ C

transferred to Ni-NTA-coated HisSorb 96-well

using T4 DNA ligase. The overnight ligation

plates (Qiagen) and were left overnight at 4 ̊ C

mixture was made salt-free using a PCR-clean-up

under mild shaking. Ultimately, the supernatants

kit, and subsequently electroporated into

were discarded, and the plates were rinsed twice

C41(DE3) electrocompetent cells. The cells were

using 50 mM Na2HPO4, 300 mM NaCl, 20 mM

resuspended in 2 mL SOC medium and then in-

imidazole, pH 8, before washing them twice with

cubated for 1 h at 37 ̊ C for recovery. Finally, they

PBS, which was the final assay buffer. Enzymatic

were streaked onto 2xYT plates supplemented

activities were determined applying the Amplex

with 200 µg/mL ampicillin, and placed at 30 ̊ C.

Red-dependent fluorescent assay directly in the

Single clones were selected from the plates and

HisSorb plates containing 300 µg/mL (5 µM) L-

used to inoculate wells of a sterile 96-well plate

Aspartate Oxidase (L-AspOx), 12 µM FAD, 100

containing 120 µL 2xYT medium per well, with

nM HRP (0.1 U/mL), 50 µM Amplex Red, and 1

200 µg/mL ampicillin, 0.4% glucose and 50 mM

mM L-Asn in a final volume of 50 µL per well,

glycine. The plates were placed at 37 ̊ C under

using a fluorescent plate reader (Molecular De-

vigorous shaking at 300 rpm for 3 h. Next, 100

vices, SpectraMax Paradigm, Ex. 532, Em. 592).

µL were transferred to a second 96-well platecontaining 200 µg/mL ampicillin, 0.4% glucose, 1

Biochemical characterization of catalytically

mM IPTG, and 50 mM glycine, while the rest of

improved mutants. The most highly active mu-

the cells from the first plate (~ 20 µL) were tem-

tants, which were identified after the final activi-

porally stored at 4 ̊ C. The second plate was incu-

ty measurements using the fluorescent plate read-

bated at 37 ̊ C for further 3 h, followed by cen-

er, were traced back to the original 96-well plates

trifugation for 20 min at 3,000g using a Sigma 4-

where they had grown; they were re-cultured in


10

Page 11 of 29

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50 mL for plasmid extraction, and aliquots were

(Microseal B adhesive Sealer) to prevent evapo-

stored at -80 ̊ C. The mutations were identified by

ration. The protein-melting experiments were

sequencing, and plasmid DNA was used to trans-

performed using a CFX96 RT-PCR machine

form C41(DE3) E.coli cells for recombinant ex-

(Bio-Rad) with the following settings: 2 min pre-

pression and purification of selected mutant en-

warming step at 30 ̊ C, and subsequent tempera-

zymes. Subsequent kinetic characterization was

ture gradient between 31-95 ̊ C with 1 ̊ C/min in-

done by applying the NADH-dependent continu-

crements. SYPRO Orange fluorescence was

ous spectrophotometric assay and the Amplex

monitored using FAMex (492 nm) and ROXem

Red-dependent assay for the substrates L-Asn

(610 nm) filters. Data were exported as Excel-

and L-Asp-β-methyl ester, respectively.

based worksheet and further analyzed by Igor-Pro (Wavemetrics). Melting temperatures (Tm) were

Thermal stability determination of wildtype and mutant hASNase-3 enzymes by differential scanning fluorimetry.

obtained by plotting the first derivative d(RFU)/ dT of the raw data as a function of temperature increase.

The hASNase-3 mutants selected for enhanced asparaginase activity were further analyzed by differential scanning

fluorimetry36

RESULTS AND DISCUSSION

(DSF) in order

to investigate the potential impact of mutations

zyme samples from the -20 ̊ C glycerol stocks

Identification of the catalytically most improved mutant hASNase-3 enzymes and evaluation of their intramolecular processing state.

were dialyzed (Pierce, Slide-A-Lyzer, 10,000

We generated a number of different site satura-

MWCO) against 50 mM Tris-Cl, 0.1 NaCl, pH 8,

tion mutagenesis (SSM) libraries where we ran-

to remove glycerol, and were mixed with SYPRO

domized four residues located in proximity to the

Orange (Sigma-Aldrich) in a final volume of 20

catalytic centre. In these cases, Ile4Val5Val6Val7

µL. The final concentrations of the enzyme and

(SSM-Lib3) and Met193Val194Cys202Leu203

the dye were 1 µM and 10 % v/v, respectively;

(SSM-Lib4) were randomized based on the NNS

the DMSO stock solution of the dye (5,000X)

scheme (Oligonucleotides are shown in Table

was pre-diluted in H2O giving a 100X solution,

S1). In addition, two epPCR libraries (PCR con

from which aliquots were used according to the

ditions: 0.2mM dATP, 0.2 mM dGTP, 1 mM

experimental needs. The samples were mixed in a

dCTP, 1 mM dTTP, 7 mM MgCl2, 0.5 mM Mn-

96-well plate suitable for real-time (RT)-PCR

Cl2, 5 µg/mL BSA, 50 nM template DNA, 0.03

measurements, centrifuged at 500 rpm for 30 s,

nmol/µL oligonucleotides, 1X Taq buffer and 2.5

on the structural stability of the enzymes. En-

and finally sealed with heat-resistant membranes


11


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Page 12 of 29

Units NEB Taq polymerase in 100 µL final volume) with an average error rate of 0.5% and size of 108 mutants were screened for five successive rounds. The error rate was estimated based on the sequencing of 10 randomly selected variants and represents the genetic error rate. The error rate on the protein level is expected to be lower given the fact that in epPCR many neutral mutations are generated that do not result in amino acid substitutions.37 Moreover, misincorporation of nu-

and Val190 are not highly conserved (see Figure

cleotides preferentially exchanges A and T, rather

S6). On the other hand, Arg143 and Arg147 point

than C and G (base pairing via two and three hy-

away from the active site and are part of an α-he-

drogen bonds, respectively), and even less likely

lix of the enzyme’s α-subunit.

are transversions between purine and pyrimidine nucleotides, thus yielding incomplete libraries. The screening of all these libraries did not show any fluorescence signal increase which would

Figure 1. Cartoon representations of the overall hASNas-3 dimeric structure and the randomized hASNase-3 structural regions. (A) hASNase-3 dimer. The black circle indicates the active site of the first monomer. Black-labeled is the product of the enzymatic reaction, L-Asp. Red-labeled are the residues which constitute the catalytic centre of the enzyme and are directly involved in substrate binding. These are: Asn62, Thr186, Arg196, Asp199, Thr219, Gly220, and the catalytic threonine Thr168* which plays the critical role of the catalytic nucleophile.23,24 (B) Randomized residues of SSM-Lib1. With cyan is colored the α subunit, and with magenta the β subunit. The amino acids which were randomized (Ile189, Val190) are green-labeled and are indicated by a black arrow. (C) Randomized residues of the SSM-Lib2. Arg143 and Arg147 which were mutated are green-labeled and are indicated by a black arrow similar to Figure 1B. Figures were generated by PyMol.38

indicate enrichment with catalytically improved variants (data not shown). Therefore, we created two smaller SSM libraries where only two residues were mutated. In the case of SSM-Lib1, Ile189 and Val190, which are located very close to the catalytic site, were randomized (Figure 1B), while SSM-Lib2 is the result of the randomization of Arg143 and Arg149, which are located on the surface of the enzyme (Figure 1C). More specifically, the side-chain of Ile189 points towards the substrate binding pocket and is in space very near to Thr186 and Arg196 which contribute to the binding of the substrate L-Asn. Despite the fact that they are located very close to

After two selection rounds for each library, we

the active site being part of a flexible loop, Ile189

observed fluorescence enrichment (as evidenced


12

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by the increase of the arithmetic fluorescence

Arg147Lys (Quadruple Mutant 2; QDM 2) were

mean, µ) as shown in Figures 2A and 2B. This is

generated.

a first indication that the sorted cells of the final rounds harbor catalytically improved hASNase-3 variants. After the final sorting rounds of both libraries, we further analyzed the isolated cells in 96-well plates where the activity of hASNase-3 variants was monitored by applying the very sensitive fluorescent assay for L-ASNases.32 Enzymatic activity measurements revealed that a few clones were more active than the wildtype which served as control. Those clones were sequenced, and we identified predominantly two different variants from the SSM-Lib1, and one from the SSM-Lib2. The mutants isolated from SSM-Lib1 carried the following mutations: Ile189Thr-

Figure 2. Fluorescence enrichments of the cell populations of the two screened hASNase-3 libraries. (A) Fluorescence profiles of the two sorting rounds of the hASNase-3 SSM-Lib1. The histograms show the fluorescence distribution of 10,000 cells of each sorting round (S0: starting population; S1 and S2: first and second sorting round, respectively). From each round, ~ 5% of the most highly fluorescent cells from the parental population were sorted (see Supporting Information). With µ is denoted the arithmetic fluorescence mean. (B) Fluorescence profiles of the two sorting rounds of the SSM-Lib2. Similar to Figure 2A, the histograms show the fluorescence distribution of 10,000 cells.

Val190Ile (Double Mutant 1; DM1) and Ile189Val-Val190Ile (Double Mutant 2; DM2), while the mutant from SSM-Lib2 was Arg143GluArg147Lys (Double Mutant 3; DM3). The discovery of these three isolated mutants inspired us to generate by site-directed mutagenesis two additional variants which combined the identified mutations in one single mutant. This would allow us to investigate possible epistatic effects39,40 of the combined mutations on the catalytic and conformational characteristics of the enzyme, given

At this point, we should like to emphasize that

the fact that the two sets of mutations are located

the primary concern about the five isolated mu-

in distant sites of the enzyme. Therefore, the

tants was their potential failure to undergo the

quadruple mutants Ile189Thr-Val190Ile-

autoproteolytic activation step which is directly

Arg143Glu-Arg147Lys (Quadruple Mutant 1;

related to their catalytic activity, and whether this

QDM 1) and Ile189Val-Val190Ile-Arg143Glu-

activation could be accelerated by glycine as in


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Page 14 of 29

wildtype hASNase-3. This is because mutants

recombinantly expressed and purified in larger

showing distinct degrees of autocleavage would

amounts (> 2 L culture medium yielding ~ 15-20

be variably active, and this could be misleading

mg of pure protein). Upon their in-vitro activa-

in their overall activity evaluation. Although the

tion with glycine, and removal of glycine by

three main mutants (DM1, DM2 & DM3) were

dialysis, they were kinetically characterized for

sorted from a library growing in medium sup-

their ability to hydrolyze L-Asn and the dipeptide

plemented with 50 mM glycine, in principle, their

L-Asp β-methyl ester.

intramolecular processing state (autoproteolytic cleavage) was unknown up to the level of the single-clone activity assay in 96-well plates and subsequent SDS-PAGE analysis of the purified enzyme. Hence, we had to confirm that the observed improved activity of the mutants was not due to different auto-activation rates, but indeed

Figure 3. SDS-PAGE profiles of pre- and postglycine treatment of the isolated variants. (A) 15% SDS-PAGE analysis of hASNase-3 wildtype and five mutants without glycine treatment. After a short NTA-affinity purification step, the soluble proteins were analyzed in order to evaluate their auto-proteolytic activation. Lane 1: wildtype; Lane 2: DM1; Lane 3: DM2; Lane 4: DM3; Lane 5: QDM1; Lane 6: QDM2. The upper intense band corresponds to the full-length hASNase-3 inactive precursor (calculated Mr: 33 kDa), and the lower ones shows the α and β protomers (calculated Mr: 18 and 15 kDa, respectively). The SUMO-tag was removed by gel filtration chromatography. (B) SDS-PAGE analysis of wildtype and mutant hASNase-3 enzymes. The figure shows the purity and the activation status of wildtype and the five hASNase-3 mutants after treatment with glycine. Lane 1: wildtype; Lane 2: DM1; Lane 3: DM2; Lane 4: DM3; Lane 5: QDM1; Lane 6: QDM2. The two generated subunits α and β are the major protein bands. The electrophoretic profiles of the enzymes reflect the final purity obtained after the gel filtration step which removed the His6-SUMO tag. Auto-proteolytic processing of the enzyme precursors (weak bands seen at about 40 kDa) was induced by including 200 mM glycine in the bacterial growth

because of improved catalytic efficiency. To this end, all five variants, and the wildtype enzyme, were initially produced in C41(DE3) E.coli cells grown in 2xYT medium without glycine at low temperature (at 16 ̊ C overnight), in order to drastically slow down their activation process. After a rapid small-scale (expression in ~ 100 mL culture medium) NTA-affinity purification step at 4 ̊ C and incubation with SUMO-protease (for 4 h at 4 ̊ C; 1:100 molar ratio of protease:enzyme) to cleave the His6-SUMO tag, the protein samples were analyzed by SDS-PAGE to evaluate their cleavage state. As shown in Figure 3A, all mutants had an intramolecular activation profile similar to the wildtype enzyme indicating that the mutations had no effect on this posttranslational processing step which is absolutely essential for catalytic activity. Subsequently, the mutants were


14

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medium, as well as upon further treatment with 500 mM glycine immediately after the first affinity purification step.

sus 3 mM for the wildtype). The DM3 variant, which was isolated from the SSM-Lib2 and which was the only mutant to be identified as

Kinetic characterization of wildtype and mutant hASNase-3 enzymes using L-Asn and LAsp β-methyl ester as substrates.

catalytically improved from this library, showed

The ultimate goal of the present work was to

is similar to that of wildtype, unlike the KM value

identify catalytically improved hASNase-3 vari-

which is improved ~ 4-fold (0.77 mM).

ants for hydrolysis of the physiological substrate

steady-state kinetic plots for the wildtype, DM1,

L-Asn. Wildtype hASNase-3 hydrolyzes L-Asn

DM2, and DM3 enzymes are shown in Figure 4.

approximately 4-fold amelioration of the kcat/KM ratio. As with DM2, the turnover number of DM3

The

with poor catalytic properties as shown in our

The rationally designed quadruple mutants

previous studies24,41 and in those from others.23

QDM1 and QDM2, which combine the improved

Yet, this human isoform appears to be a more at-

characteristics of the double mutants, showed

tractive candidate for molecular engineering than

some further improvement, but overall they did

the second human L-ASNase (designated hAS-

not exceed the highest level of advancement

Nase-1)28, envisioning possible substitution of

achieved by the DM1 variant (6-fold). The

the currently used bacterial enzymes.

QDM1 enzyme, which is a combination of the

All five hASNase-3 variants (DM1, DM2, DM3,

DM1 and DM3 variants, displayed almost the

QDM1 and QDM2) are characterized by im-

same catalytic constant as DM3 (0.7 s-1) and a

proved kinetic parameters kcat and KM in compari-

similar KM value as DM1 (1.45 mM), resulting in

son to the wildtype enzyme. More specifically,

a 2-fold kcat/KM overall improvement compared to

the DM1 variant displayed a ~ 6-fold improve-

the wildtype. The second quadruple mutant,

ment regarding the overall catalytic efficiency

QDM2, showed the highest kcat value (~ 2.2 s-1 )

(kcat/KM). Interestingly, both kinetic constants

among all the mutants. However, it also had the

were improved; the turnover increased 2.5-fold,

highest KM (2.3 mM) among all the variants

and the KM value decreased 2.3-fold. In contrast,

which resembles the one from the wildtype en-

the second mutant which was isolated from the

zyme (3 mM). These kinetic parameters of

same library, DM2, showed a 2-fold improve-

QDM2 rank this variant second to DM3, having

ment of kcat/KM. The kcat of this variant is similar

kcat/KM values of ~ 960 M-1 s-1 and 1000 M-1 s-1 for

to that of the wildtype enzyme (~ 0.8 s-1), but the

DM3, respectively. The Michaelis-Menten plots

binding affinity for L-Asn was 2-fold enhanced

for QDM1 and QDM2 are depicted in Figure 5,

as evidenced by the lower KM value (1.6 mM ver-

and Table 1 summarizes the steady-state kinetic


15


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constants for all variants including the wildtype

Page 16 of 29

the substrate concentration. Data points are represented as means ± SD of duplicate sample measurements. Plots were prepared and analyzed by the SoftZymics software (Igor Pro, Wavemetrics) by non-linear regression using the MichaelisMenten equation.

enzyme for L-Asn as substrate. The rate (velocity) at which the enzyme hydrolyzes L-Asn is proportional to the ratio kcat/ KM multiplied by the free L-Asn concentration ([L-Asn]) if [L-Asn] < KM. When comparing the efficiencies of wildtype and mutant L-ASNases catalyzing L-Asn hydrolysis it is important to consider the actual substrate concentration at which they operate.42,43 In blood serum of hu-

Figure 5. Steady-state kinetic plots for quadruple-mutants QDM1 and QDM2 of hASNase-3 for the L-Asn as substrate. V/E versus [L-Asn] plot for (A) QDM1 and (B) QDM2. Activity measurements were performed and plots were generated as detailed in Fig. 4 legend.

mans and mice, [L-Asn] has reliably been reported to be close to 50 µM, falling below 5 µM upon L-ASNase treatment (respective values for L-Asp: 10 µM and 50 µM).44,45

Table 1. Steady-state kinetic constants for wildtype and mutant hASNase-3 enzymes for L-Asn hydrolysis.

Figure 4. Steady-state kinetic plots for wildtype and double-mutant hASNase-3 enzymes for LAsn as substrate. V/E versus [L-Asn] plot for (A) wildtype hASNase-3, (B) DM1, (C) DM2, and (D) DM3. Activities were measured in 1 mL 50 mM Tris-Cl, 100 mM NaCl, pH 8, at 25 ̊ C, using a final enzyme concentration of ~ 1 µM (~ 30 µg in 1 mL; hASNase-3 Mr: 33 kDa). Steady-state turnover rates (s-1) are expressed as a function of

Enzyme

kcat (s-1)

KM (mM)

kcat/KM (M-1 s-1)

wildtype hASNase-3

0.78 ± 0.02

3 ± 0.16

0.26 (±0.02) x 103

DM1 (Ile189ThrVal190Ile)

1.89 ± 0.07

1.3 ± 0.16

1.45 (±0.23) x 103

DM2 (Ile189ValVal190Ile)

0.83 ± 0.01

1.58 ± 0.06

0.53 (±0.03) x 103

DM3 (Arg143GluArg147Lys)

0.78 ± 0.03

0.78 ± 0.10

1 (±0.17) x 103

QDM1 (Ile189Thr-Val190IleArg143Glu-Arg147Lys)

0.7 ± 0.01

1.46 ± 0.03

0.48 (±0.02) x 103

QDM2 (Ile189Val-Val190IleArg143Glu-Arg147Lys)

2.2 ± 0.11

2.3 ± 0.33

0.96 (±0.20) x 103


16

Page 17 of 29

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would have to be tested in order to get a better picture of the structure-function relationships Given the fact that hASNase-3 is primarily a β-

prevailing in hASNase-3 mutants The DM1 and

aspartyl-dipeptidase, we assessed the impact of

DM2 variants were isolated from a library with

the upper mutations on this activity. We tested as

two randomized amino acids (Ile189 and Val190)

substrate the L-Asp-β-methyl ester (see above)

located very close (~ 5 Å) to the binding pocket.

which has been shown to be converted best of a

In the DM1 variant, Ile189 is substituted by Thr,

series of β-aspartyl-dipeptides.23 Figures S8 and

and Val190 by Ile, while the DM2 variant (~ 2-

S9 show the Michaelis-Menten kinetic plots for

fold kcat/KM improvement as compared to the wild-

all five variants and the wildtype for the hydroly-

type) had the wildtype residues mutated in a re-

sis of L-Asp-β-methyl ester, and Table S2 sum-

versed manner, i.e. Ile189 was substituted by Val,

marizes their steady-state kinetic constants. Strik-

and Val190 by Ile. In the crystal structure of wild-

ingly, in contrast to L-Asn as substrate, the KM of

type hASNase3, the side chain of Ile189 points

all mutants remained almost unaffected as com-

towards the active site, thus providing a space-

pared to the wildtype, but the kcat was slightly

filling hydrophobic group in the substrate binding

improved in all cases, the highest value being ~

pocket. The replacement of Ile by Thr offers

12.5 s-1 for both QDM1 and QDM2. Taken to-

more space for substrate binding, though space

gether, the kinetic data suggest that the mutations

alone does not necessarily lead to higher activity.

selected by FACS sorting differentially affected

An additional hint to the possible rationalization

the two catalytic functions of hASNase-3, i.e. L-

of the effect of these mutations can be obtained

asparaginase and β-aspartyl-dipeptide hydrolase

by inspection of DM2, which has a Val residue at

activity, respectively.

this position: the two variants, DM1 and DM2,

The DM1 variant, in which Ile189 is replaced by

carry the same mutation at position 190 (Ile), but

Thr, a less bulky and more polar amino acid, was

DM1 has a Thr, and DM2 has a Val at position

only 1.6-fold improved for the substrate L-Asp β-

189. The two mutants have very similar KM val-

methyl ester as opposed to 5.6-fold for L-Asn. In

ues (1.3 mM for DM1, and 1.58 mM for DM2),

contrast, the DM2 variant, which has an Ala at

but different kcat numbers, with the one of DM1

position 189, displayed 2-fold improvement re-

being ~ 2.5-fold higher. These observations sug-

garding both hydrolytic activities. This finding

gest that the presence of a polar residue at posi-

points to the hypothesis that residues with more

tion 189 particularly favors a chemical step of L-

polar character may favor the catalysis of L-Asn

Asn hydrolysis rather than binding of this sub-

over the dipeptide conversion. More dipeptides

strate; both sets of mutations similarly lowered (~


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Page 18 of 29

50 %) the KM value for L-Asn, while it remained

displayed similar improvement for both catalytic

almost unaffected for the dipeptidase activity.

functions, was the only variant among all five

The DM3 variant, which contains two mutations

identified species whose peptidase activity was

on the surface of the enzyme molecule, exhibited

improved more than its L-ASNase activity. Table

a 2-fold improvement for L-Asp-β-methyl ester

2 shows the improvement of catalytic efficiencies

hydrolysis, and a 4-fold for L-Asn hydrolysis.

of hASNase-3 variants for both hydrolytic func-

Mutations which are located far away from the

tions in comparison to the wildtype enzyme.

active site and yet affect catalysis like those of the DM3 variant (~ 17 Å from Arg143, and 14 Å

Table 2. Factors of improvement of catalytic efficiency (kcat/KM) of hASNase-3 variants for both catalytic functions.

from Arg147), are difficult to interpret.46,47 Often, such distant mutations affect the transition

Enzyme

L-Asn hydrolysis

L-Asp methylester hydrolysis

variant, the positively charged Arg143 is replaced

DM1 (Ile189Thr-Val190Ile)

5.6

1.6

by a negatively charged Glu residue, and Arg147

DM2 (Ile189Val-Val190Ile)

2

1.8

by Lys, either residue being positively charged.

DM3 (Arg143Glu-Arg147Lys)

4

2

QDM1 (Ile189Thr-Val190IleArg143Glu-Arg147Lys)

1.85

4.2

QDM2 (Ile189Val-Val190IleArg143Glu-Arg147Lys)

3.7

3

state of catalysis by altering the dynamics of the whole enzyme

molecule.48

Interestingly, in this

This variant showed the lowest KM value for LAsn hydrolysis as compared to the other isolated mutants, being ~ 4-fold lower than that of the wildtype enzyme, while kcat values are similar. The side chains of both Arg143 and Arg147 point outward from the enzyme’s active site, while the distance between them (~ 5 Å) is somewhat larg-

The mutations showed distinct effects on the conformational stability of the hASNase-3 enzyme. In addition to studying kinetic features of the

er than the upper allowable limit for van der Waals interactions (side chains interact via van der Waals interactions when they have less than 4

identified catalytically improved enzyme vari-

Å distance).49 Yet, this does not exclude the pos-

ants, we addressed the question of how the muta-

sible interaction of the oppositely charged side

tions impact on the stability of hASNase-3 using

chains of Glu143 and Lys147, thereby stabilizing

differential scanning fluorimetry (DSF) as an an-

the α-helix which accommodates them.

alytical technique to monitor thermal shifts of the

Finally, concerning the rationally-designed

melting temperature (Tm) associated with the un-

quadruple mutants, QDM1, unlike QDM2 which


18

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folding of the protein.36,50 The results of our DSF

imity to it (DM1 and DM2). The combination of

analysis implied that the different mutations had

the mutations occurring in QDM1 and QDM2

distinct influence on the stability of hASNase-3.

resulted in an increase of their Tm values which

As shown in Figure 6, the melting curves for all

are higher than their individual ones, indicating a

hASNase-3 enzymes were monophasic, with

dominating stabilizing effect of DM3’s muta-

some of them indicating stabilizing and others

tions.

destabilizing effects of mutations. The Tm values for all the variants including the wild type are

Table 3. Tm values for the variants and the wildtype enzyme of hASNase-3 obtained from DSF experiments. The conditions of the experiments are described in detail in the main text.

summarized in Table 3. More specifically, among the double mutants, the only one which exhibited a higher Tm in comparison to the wildtype is

Enzyme

Tm (°C)

wild type

62.5

DM1 (Ile189Thr-Val190Ile)

61

unfolding is decreased.51 Both DM1 and DM2,

DM2 (Ile189Val-Val190Ile)

61.8

which were isolated from SSM-Lib1, showed

DM3 (Arg143Glu-Arg147Lys)

66.5

lower Tm values, 61 and 61.8 ̊ C, respectively.

QDM1 (Ile189Thr-Val190Ile-Arg143GluArg147Lys)

68

QDM2 (Ile189Val-Val190Ile-Arg143GluArg147Lys)

68

DM3 (~ 66.5 C versus 62.5 ̊ C). It is generally believed that mutations at specific surface locations can considerably increase stability, possibly because the propensity of these regions to initiate

Strikingly, the quadruple mutants displayed the highest Tm values which were considerably improved in comparison to the wildtype enzyme: 68 ̊ C for both of them. These data strongly suggest that the variant with the best catalytic efficiency

We did not study reversibility of thermal unfold-

(DM1) is characterized by the lowest melting

ing aiming to apply equilibrium thermodynamics

temperature (61 ̊ C) and, consequently, lowest

analysis, which would allow us to reliably ex-

stability; this also holds true for the second vari-

trapolate stability parameters to lower tempera-

ant isolated from the same library (DM2, with Tm

tures, such as at physiological (37°C) or storage

of 61.8°C), though being catalytically 3-fold less

(4°C) temperatures. The Tm values, that were all

improved. These results purport the idea that

determined at 1 µM protein, may vary with the

there is a sort of positive epistasis associated with

protein concentration, yet they are assumed to

mutations which are located at far distance from

adequately reflect relative thermal stability of

the active site (DM3) and those located in prox-

these hASNase-3 species. We did not observe


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Page 20 of 29

aggregation or precipitation of the protein under

of the enzyme has not yet been elucidated, given

these conditions which would indicate kinetically

its main activity as a dipeptidase it is believed to

controlled irreversible protein unfolding depen-

play a key role in the cellular detoxification of L-

dent on the heating rate.

Asp dipeptides occurring as products of protein degradation. We have been particularly spurred to engineer hASNase-3 aiming at the improvement of its L-ASNase activity. A great number of reports have shown that the use of bacterial L-ASNases for the treatment of the ALL-type of leukemia is associated with severe side effects.52 Therefore, we envision replacement of the bacterial enzymes by a better tolerated human L-AS-

$

Nase. However, there is no straightforward stratFigure 6. Differential scanning fluorimetry (DSF) melting curves recorded for wildtype and mutant hASNase-3 enzymes. The enzyme-melting experiments were performed using a CFX96 RT-PCR machine (Bio-Rad). SYPRO Orange fluorescence was monitored using FAMex (492 nm) and ROXem (610 nm) filters. Data were exported as Excel-based worksheet and further analyzed by Igor-Pro (Wavemetrics). Melting temperatures (Tm) were obtained by plotting the first derivative d(AFU)/dT of the raw data as a function of temperature increase. AFU, arbitrary fluorescence unit.

egy to accomplish this objective since humans lack a highly efficient L-ASNase homologue similar to the bacterial enzymes, and therefore engineering of hASNase-3 appears to be a promising route towards this challenge. We emphasize that structure-guided engineering, or in vitro-evolution, of hASNase-3 are particularly difficult and laborious processes because of the necessity for intramolecular activation of the enzyme by autoproteolytic cleavage exposing a specific catalyti-

CONCLUSION

cally essential threonine residue at the very

The present work focuses on directed evolution

amino-terminal position of the generated subunit

of hASNase-3, an enzyme which belongs to the

(protomer) of the enzyme.23,24,41 It could well be

N-terminal hydrolase super family of enzymes. 23,24,25

that certain mutations abolish the enzyme’s in-

Wildtype hASNase-3 exhibits dual catalyt-

herent property to activate itself, which, as a con-

ic activities converting primarily β-aspartyl-

sequence, would prevent detecting any enzymatic

dipeptides to L-Asp and the corresponding amino

activity. The big challenge here is to target

acid of the dipeptide moiety of the initial sub-

residues that will not impair the autoactivation

strate, and, secondarily, hydrolysis of L-Asn to L-

process, and simultaneously will have a positive

Asp and ammonia. Though the physiological role


20

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impact on the catalytic features. By employing a

quence regions to initiate a directed evolution

FACS HTS approach, we managed to isolate a

experiment is very challenging, since only few

number of mutants showing improved kinetic

pathways in Darwinian evolution lead to fitter

constants kcat and KM, the best one being about 6-

proteins.55 The generation of large libraries by

fold more active than the wild-type enzyme with

randomizing many amino acids (more than just

just two changes of amino acid residues. We un-

four residues that we have targeted here) might

derline that it is desirable to achieve the best cat-

be desirable for covering a larger sequence space,

alytic improvement with the least number of mu-

however, the likelihood of getting trapped in lo-

tations in order to lower the probability of mak-

cal minima (pathways of the fitness landscape

ing the enzyme immunogenic. The promising

which are dead-ends) is higher because non-func-

outcome of our site-saturation mutagenesis strat-

tional mutations accumulate resulting in non-vi-

egy is that the variants we have isolated showed

able species in cell-based selection schemes.56

simultaneously improved kinetic parameters KM

Analysis of published work on directed evolu-

and kcat towards the enzyme’s natural substrate (L-

tion suggests that in a number of cases successful

Asn), which has been rarely achieved in enzyme

engineering involved simple uphill walks on the

engineering. The obtained mutants can be sub-

fitness landscape, doing one mutational step at a

jected to more rounds of selection, iterated muta-

time.57,58 Quite often, ultimately, it turns out that

genesis, and kinetic characterization of identified

single amino acid mutations are responsible for

enzyme variants in order to further improve the

the functional change searched for, despite the

enzyme’s catalytic efficiency (Figure S10). In

fact that many mutations may have been intro-

such experimental enterprise, one needs to un-

duced.56 In case of hASNase-3, the four-codon

cover the most beneficial pathway among the

randomization libraries did not result in the isola-

vast number of pathways of the enzyme’s fitness

tion of catalytically improved variants. By con-

landscape.47 In the current study, the generation

trast, the ~ 103-fold smaller, two-codon random-

of small libraries (randomization of two residues)

ization libraries allowed us to unlock the en-

was more beneficial than the creation of larger

zyme’s regions which have a positive influence

libraries (targeting four residues). In this context,

on the catalytic properties upon mutagenesis. In

we note that according to results obtained on a

the case of the large libraries, the failure to detect

number of different proteins most of the muta-

improved variants might be due to the mutational

tional pathways led to dead-ends, and finally to

intolerance of the selected regions, rather than the

non-functional or less efficient enzyme variants.

number of randomized amino acids. In order to

53,54

substantiate this assumption, additional libraries

Therefore, the identification of suitable se-


21


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Page 22 of 29

1Present

need to be generated by mutating a smaller num-

Addresses: University of Texas at Austin, Department of Chemical Engineering, 2500 Speedway, Austin, Texas, TX 78712-1095.

ber of residues located around those regions. It is progressively becoming clearer through the different directed evolution studies that the best in-

Author Contributions

dicator for the evolvability of an enzyme is its

CSK designed the experiments and performed research. MK supervised the work. CSK and MK analyzed data and wrote the manuscript.

natural history.59 Enzymes from large families displaying diverse substrate activities are easier to evolve given the fact that the same natural evo-

Funding Sources CSK and MK thank the Max Planck Society for generous and continuous funding.

lutionary mechanisms govern the success or failure of obtaining new functions at the laboratory level as well. Transferring this belief to hAS 

Nase-3, we can be more optimistic about its

Notes 

evolvability since it satisfies the two aforementioned characteristics: It is a member of a very

ACKNOWLEDGMENT

large family of enzymes (Ntn-hydrolases), and it

We are thankful to S. Jakobs, Structure and Dynamics of Mitochondria research group at the MPIbpc, for allowing us to use their FACS machine, as well as to N. Jensen for his discussions on the analysis of our FACS data. We are also very grateful to G. Georgiou, Department of Chemical Engineering at the University of Texas at Austin, for the kind gift of their E. coli deletion strains.

accepts different types of substrates (primarily several derivatives of L-Asp dipeptides, and secondarily the free amino acid L-Asn). Finally, the recently developed strategy of structure-guided triple-code saturation mutagenesis will point the

ABBREVIATIONS

way to more effective directed evolution of this

ALL, Acute Lymphoblastic Leukemia; FACS, Fluorescence-Activated Cell Sorting; HTS, HighThroughput Screening; L-ASNase, L-asparaginase; hASNase-3, human asparaginase-3; hASNase-1, human asparaginase-1; DM1, Double Mutant 1; DM2, Double Mutant 2; DM3, Double Mutant 3, QDM1, Quadruple Mutant 1; QDM2, Quadruple Mutant 2;

human enzyme.60 ASSOCIATED CONTENT

Supporting Information REFERENCES

The Supporting Information is available free of charge on the ACS Publication website.

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AUTHOR INFORMATION * Corresponding author. Fax: +49 551 2011074 E-mail address: [email protected] (M.Konrad)


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Fluorescence-Activated Cell Sorting of Human l-asparaginase Mutant

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