The cysteine dioxygenase homologue from Pseudomonas aeruginosa is a 3-mercaptopropionate dioxygenase

JBC Papers in Press. Published on August 13, 2015 as Manuscript M114.635672

The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M114.635672

Thiol Dioxygenase from Pseudomonas aeruginosa

The Cysteine Dioxygenase Homologue from Pseudomonas aeruginosa is a 3-Mercaptopropionate

Dioxygenase*

Egor P. Tchesnokov^a,‡, Matthias Fellner^a,‡, Eleni Siakkou^a, Torsten Kleffmann^b, Lois W. Martin^b,

Sekotilani Aloi^a, Iain L. Lamont^b, Sigurd M. Wilbanks^b, Guy N. L. Jameson^a

^aDepartment of Chemistry and ^bDepartment of Biochemistry, University of Otago, PO Box 56, Dunedin

9054, New Zealand.

*Running Title: Thiol Dioxygenase from Pseudomonas aeruginosa

To whom correspondence should be addressed: Guy N. L. Jameson, Department of Chemistry, University

of Otago, PO Box 56, Dunedin 9054, New Zealand. Tel: +64

guy.jameson@otago.ac.nz

3

479 8028; Email:

Keywords: dioxygenase; Pseudomonas aeruginosa; enzyme kinetics; Mössbauer spectroscopy; substrate

specificity; crystal structure

Background: Thiol dioxygenation is catalyzed by

enzymes specific for each substrate.

Results: Kinetic, structural and spectroscopic data

describes an enzyme from P. aeruginosa that is a

3-mercaptopropionate dioxygenase with secondary

cysteine dioxygenase activity.

Conclusion: An arginine to glutamine switch and

absence of a cis-peptide bond correlates with

substrate preference.

cysteine and 3-mercaptopropionic acid (3-

MPA) in vitro, with a marked preference for 3-

MPA. We therefore describe this enzyme as a 3-

mercaptopropionate dioxygenase. Mössbauer

spectroscopy suggests that substrate binding to

the ferrous iron is through the thiol but

indicates each substrate could adopt different

coordination geometries.

Crystallographic

cysteine

comparison with mammalian

Significance: Characterization of this enzyme

deepens our understanding of substrate specificity

in thiol dioxygenases.

dioxygenase shows that the overall active site

geometry is conserved but suggests that the

different substrate specificity can be related to

replacement of an arginine by a glutamine in

the active site.

ABSTRACT

Thiol dioxygenation is the initial oxidation

step that commits a thiol to important catabolic

or biosynthetic pathways. The reaction is

catalyzed by a family of specific non-heme

mononuclear-iron proteins each of which is

reported to react efficiently with only one

substrate. This family of enzymes includes

cysteine dioxygenase, cysteamine dioxygenase,

The

global

sulfur

cycle

involves

interconversion of sulfur through a wide range of

oxidation states. The most abundant oxidation

states of sulfur are -2, as thiol/ate (eg. cysteine or

hydrogen sulfide), 0 as elemental sulfur, and +6 as

sulfate. Within the sulfur cycle, microbiological

transformation dominates the conversion of

inorganic sulfur and to some extent organic

sulfur.(1) While bacteria are very flexible in terms

of their sulfur source, animals require dietary

intake of sulfur-containing amino-acids such as

mercaptosuccinate

mercaptopropionate

dioxygenase

dioxygenase.

and

3-

Using

sequence

alignment

to

infer

cysteine

dioxygenase activity, a cysteine dioxygenase

homologue from Pseudomonas aeruginosa

methionine, which serves as

a biosynthetic

(p3MDO)

has

been

identified.

Mass

precursor of cysteine.(2) In organisms, sulfur is

present in proteins, nucleic acids, sulfate esters of

polysaccharides, steroids, phenols, and sulfur-

containing coenzymes.(3,4) Therefore, sulfur-

dependent enzymology represents an important

means of biological sulfur utilization. In particular,

spectrometry of P. aeruginosa under standard

growth conditions showed p3MDO is expressed

in low levels suggesting that this metabolic

pathway is available to the organism. Purified

recombinant p3MDO is able to oxidize both

1

Thiol Dioxygenase from Pseudomonas aeruginosa

two thiol-containing compounds, cysteine and 3-

mercaptopropionic acid, are of interest as they are

both abundant and are irreversibly removed during

acid, resulting in deformation, seizures or death of

rats.(38-40)

The

gram-negative

proteobacterium

their

respective

metabolic

cycle

by

Pseudomonas aeruginosa is an opportunistic

pathogen, which can infect plants and animals,

including humans. This bacterium has gained

importance as a nosocomial pathogen that infects

immune-compromised patients and is highly

resistant to antibiotic treatment.(41) Due to its

ability to utilize a wide range of organic matter

and cope with different environmental conditions

this organism can colonize many natural

environments including soil, water and skin. By

implication, P. aeruginosa metabolic enzymology

illustrates various means of environmental

adaptation. Considering that P. aeruginosa

colonizes human hosts it is not surprising that it

can utilize cysteine or methionine as a sulfur

source.(42) Although it has been established that

cysteine can be fully reduced to H₂S in the course

of P. aeruginosa metabolism,(43) oxidation of

cysteine to CSA through CDO or an equivalent

enzyme has not been reported. Similarly, little is

known about bacterial enzymes involved in 3-

MPA metabolism. Strains from the Pseudomonas

family are capable of 3-MPA utilization.(28)

Conversion of 3-SPA to 3SP-CoA in

Proteobacteria has been attributed to succinyl-

CoA synthetase,(44,45) however enzymatic

oxidation of 3-MPA to 3-SPA remains to be

identified. CDO homologues from bacteria

(Ralstonia eutropha, PDB accession 4QMA

(2GM6), Bacillus subtilis, 3EQE) are structurally

very similar to mammalian CDO, for which

several X-ray crystal structures are available, but

the substitution of glutamine in bacterial enzymes

for arginine at position 60 of the mammalian

active site suggests altered substrate specificity.

In this study we report identification,

classification, structural, kinetic and spectroscopic

dioxygenation.(2,5-7)

In mammals, cysteine levels are maintained

directly from dietary sources and/or through

biosynthesis from methionine.(2,7) High cysteine

levels have been implicated in a number of

neurodegenerative diseases due to its exitotoxic

effect in the nervous system.(8-12) Accumulation

of cysteine has been attributed to impaired

function of cysteine dioxygenase (CDO)^†,(9,13-

15) an enzyme catalyzing dioxygenation of

cysteine thiolate to sulfinate (CSA).(16-21)

Functional CDO occurs widely and has been

identified in mammals, lower eukaryotes and in

bacteria.(21-23) CDO belongs to a family of non-

heme mononuclear-iron dioxygenases, but stands

out due to a number of unique characteristics. The

iron atom has His₃coordination and, in

mammalian

modification involving

crosslink is present.(24,25)

forms,

a

post-translational

a

cysteine-to-tyrosine

3-mercaptopropionic acid (3-MPA) is

a

structural analogue of cysteine (Fig 1), lacking

only the amine group. In spite of the fact that it is

one of the most abundant thiols in wetlands(26)

and fresh water lakes(27) most bacteria cannot use

3-MPA as a sole carbon source.(28-30) Instead,

bacteria are capable of growing on various 3-MPA

precursors, which are catabolized to 3-MPA inside

the cell.(28-31) In bacteria, 3-MPA is a central

metabolite in both catabolic and assimilatory

sulfur metabolism (5) and while bacteria can form

3-MPA as

homocysteine

a

metabolite of methionine,

(32) or

dimethylsulfoniopropionate,(31-33) little is known

about the dissimilation of 3-MPA. In one

bacterium, Variovarax paradoxus, 3-MPA

utilization was shown to involve dioxygenation of

3-MPA to 3-sulfinopropionic acid (3-SPA),

characterization

of

P.

aeruginosa

thiol

dioxygenase (p3MDO). This enzyme is a 3-MPA

dioxygenase with secondary cysteine dioxygenase

activity. This is the first report of enzymology of

cysteine and 3-MPA dissimilation in P.

aeruginosa metabolism and the first example of a

CDO homologue which utilizes a second substrate

with near stoichiometric coupling to dioxygen

consumption.

catalyzed

by

a

CDO

homologue,

3-

mercaptopropionate dioxygenase (3MDO) which

showed high specificity for 3-MPA but not for

cysteine.(6) Interestingly, 3-MPA acts as an

inhibitor of metallo-β-lactamase, an enzyme

involved in antibiotic resistance in bacteria.(34,35)

In mammals 3-MPA is toxic by inhibiting the

oxidation of fatty acid(36,37) and the synthesis of

the inhibitory neurotransmitter γ-aminobutyric

Experimental Procedures

2

Thiol Dioxygenase from Pseudomonas aeruginosa

Protein Sequence Identification, Expression

and Purification. The NCBI reference sequence

(NC_002516.2) for P. aeruginosa (strain

absence of a posttranslational modification present

in mammalian CDO(53-56) (Fig 3). Protein

preparations were found to be at least 97.5% pure

and contained ~15% endogenously bound iron as

judged by colorimetric assay using ferrozine in a

96-well format as previously described.(57)

PAO1)(46)

contains

a

region

gi|110645304:2945264-2945869 coding for

a

hypothetical protein PA2602 (NP_251292.1).(47)

BLAST search(48) of putative bacterial CDO

sequence (YP_299237.1) from R. eutropha (strain

JMP134) was performed using the SIB-BLAST

network service and identified PA2602 as a CDO

homologue (Fig 2). The experimental design for

the PCR-amplification of the PA2602 coding

sequence was optimized using Primer D'Signer

software (IBA). DNA sequence coding for CDO

was PCR-amplified using Pfu DNA polymerase

(Stratagene) and inserted into pPR-IBA1, a Strep-

tag affinity-based expression plasmid (IBA)

digested with BsaI (Fermentas), according to

standard procedures to yield an open reading

frame encoding the full PA2602 protein with a C-

terminal extension of SAWSHPQFEK. Both

strands of the coding region of the P. aeruginosa

Protein

concentrations

were

determined

spectrophotometrically at 280 nm using an

extinction coefficient of 29700 M^-1cm^-1estimated

from a bovine serum albumin standard curve

quantified by SDS-PAGE densitometry. For

anaerobic

studies

protein

preparations,

corresponding buffers and solvents were made

anaerobic by alternating cycles of vacuum and

purging with argon gas on a Schlenk line kept on

ice slurry. All anaerobic manipulations were

performed in a nitrogen atmosphere in a glovebox

(Belle Technology, <1 ppm oxygen).

Assessment of PA2602 protein expression in

Pseudomonas aeruginosa. P. aeruginosa strain

PAO1 was grown in L-broth to late logarithmic

phase (OD600 ~3.3) and the cells collected by

centrifugation. Approximately 0.6 g cell pellet was

re-suspended in 2.3 mL 100 mM phosphate buffer

pH 7.5 in the presence of protease inhibitors. Cells

were sonicated (SONICS Vibra cell) on ice for 5

min (9 s on, 9 s off, amplitude 38%). The lysed

cells were spun down at 30,000 g at 4 °C for 30

min. 100 µL of the supernatant were mixed with

500 µL ice-cold acetone and stored at -20 °C for

45 min to precipitate the proteins. After spinning

the solution for 30 min at 16,000 g at room

temperature the supernatant was discarded and the

pellet air-dried on the bench overnight. The dried

pellet was dissolved in SDS-buffer and proteins

were separated by SDS-gel electrophoresis.

Comparison to the retention of purified p3MDO

on a separate gel and with the use of molecular

weight markers a region of interest of about 1 cm

between 21.5 and 31 kDa was excised from the gel

to be measured by mass spectrometry. The

proteins were digested ‘in gel’ with trypsin using a

robotic workstation for automated protein

digestion (DigestPro Msi, Intavis AG, Cologne,

Germany) essentially following the protocol as

described by Shevchenko et al.(58)

PA2602

IBA1/PA2602/P.

expression

construct

were

(pPR-

fully

aeruginosa)

sequenced at the Genetic Analysis Services at the

University of Otago to confirm the absence of

adventitious mutations. A molecular weight of

23746.5 Da was calculated based on the amino

acid sequence of the expressed protein.(49,50)

PA2602

expression

was

induced

in

BL21(DE3)pLysS cells (Novagen) and purified

using Strep-tag affinity chromatography according

to the manufacturer’s protocol (IBA).(51,52) We

employed the following modifications to the

standard

protein

expression/purification

procedures: (i) cells were incubated overnight at

18 °C during protein expression, (ii) cell lysis

buffer contained 100 mM TRIS (pH=8.0), 1.15 M

NaCl, 1mM EDTA, 5 mM Tween 20 (1.5% v/v)

and a Roche complete protease inhibitor, and (iii)

cells were lysed using a French pressure cell

(model FA-078, SLM Aminco). The purified

protein preparations were dialyzed extensively

(>10⁹dilution factor) against buffer compatible

with the subsequent experiment. Protein was

concentrated

on

Vivaspin

centrifugation

concentrators (GE Healthscience) to the

appropriate concentrations for the subsequent

experiments. As expected, P. aeruginosa CDO

homologue preparations resolved into a single

band when subjected to SDS-PAGE revealing the

Parallel reaction monitoring (PRM) assays

were performed by nanoflow uHPLC-coupled

TripleTOF 5600+ mass spectrometry (AB Sciex,

Framingham, MA). The m/z values of doubly and

triply charged precursor ions and their 10 strongest

3

Thiol Dioxygenase from Pseudomonas aeruginosa

singly charged transitions (m/z of fragment ions)

were predicted for seven proteotypic peptides of

peptides with a maximum of 3 missed cleavage

sites and the variable modifications of

p3MDO

using

the

skyline

software

carboxyamidomethylated

cysteine,

oxidized

(https://skyline.gs.washington.edu).(59) An aliquot

of the sample was first analyzed by an

unscheduled PRM assay using the target peptides

given in Table 1.

methionine and deamidated asparagine/glutamine.

Precursor and fragment ion accuracy were 25 ppm

and 0.1 Da respectively.

Crystallization and Structure Determination.

Crystals were grown using protein that had been

reconstituted with iron (see below) by both the

hanging-drop and the sitting-drop vapor diffusion

methods. To prepare hanging-drops, 300 nL of

protein at 11 mg/mL in water was mixed with 150

nL of 100 mM HEPES, 100 mM NaCl, 1.6 M

(NH₄)₂SO₄, pH=7.5. Crystals grew as extended

rhombohedra of ~100 µm at 16 °C in less than 14

days. Crystals were transferred to a 15% (v/v)

solution of glycerol in the well solution and

mounted in a loop at -180 °C. Sitting-drop crystals

were produced in 70 µL reservoirs by mixing 2 µL

protein (11 mg/mL) with 1 µL mother liquor

containing 200 mM sodium acetate, 8% (w/v)

PEG 4000, pH=5.7. Crystals grew quickly as

extended rhombohedra of ~500 µm within days at

18 °C and these were transferred to 25% (v/v)

ethylene glycol in well solution and mounted in a

loop at -180 °C.

LC separation of peptides was performed on

an Ekspert 400 LC-system (Eksigent, AB Sciex) at

a flow rate of 500 nL/min using a homemade

emitter tip column (75 µm ID PicoTip fused silca

tubing (New Objectives, Woburn, MA) packed

with 3 µm C-18 beads on a length of 12 cm). The

gradient was developed from 2% acetonitrile,

0.2% formic acid in water to 25% acetonitrile,

0.2% formic acid in water over 45 min followed

by an increase to 90% acetonitrile, 0.2% formic

acid in water over 15 min. The mass spectrometer

was operated in precursor mode with a precursor

isolation window of 0.7 units (unit-resolution) and

an ion accumulation time of 100 ms to allow for

the acquisition of both charge states of all 7

precursors (14 ions per cycle) within a 2 second

cycle time. For precursor fragmentation rolling

collision energy was selected and all transitions

were monitored in parallel by the TOF analyzer in

a high resolution fragment-ion spectrum. Raw data

of the unscheduled PRM were imported into the

skyline software for compound detection and

determination of retention times. Skyline was then

used to build a scheduled PRM method of only the

strongest detected charge state of each precursor

within a retention time window of 2 min to be

used by the instrument software (Analyst software,

AB Sciex). For the scheduled PRM method the

same LC-gradient and MS-settings were used as

for the unscheduled PRM with the exception of an

increased ion accumulation time of 150 ms for

each precursor and a maximum of 4 precursors per

cycle resulting in a cycle time of 0.9 sec.

Using a crystal A, grown from ammonium

sulfate, 180° of data were collected using 1°

oscillations at the MX1 and MX2 beamlines of the

Australian synchrotron with a wavelength of

0.9537

Å

and an Area Detector Systems

Corporation (ADSC) detector. Data were indexed

in space group P2₁2₁2₁, with unit cell dimensions

of 184.38, 63.02 and 35.49 Å, integrated in

Mosflm and scaled using Scala; 5% of reflections

were reserved for the calculation of the R_free

statistics (Table 2). Programs of the CCP4 suite

(The CCP4 suite, 1994) were used for structure

solution. Phases were solved by molecular

replacement using the R. eutropha structure

(2GM6, modified in Chainsaw) as a search model

in Phaser.(60) Two monomers were placed in the

asymmetric unit. Automated model refinement

was performed in Refmac 5,(61) with two TLS

groups defined in TLSMD (62) and medium NCS

restraints. Models were inspected in COOT(63)

and manual adjustment performed to match

chemical expectations, electron density maps and

anomalous electron density maps (this last for iron

atoms only). Electron density adjacent to the

active site iron was modeled as solvent molecules,

Raw data were analyzed using Skyline for

compound detection and the peak view software

(AB Sciex) for the extraction of high resolution

fragment-ion spectra and peak lists. Peak lists

were converted to a Mascot generic format for

software-assisted sequence assignment by the

database-dependent

search

engine

Mascot

(matrixscience.com). Spectra were searched

against the non redundant NCBI amino acid

sequence database (48,573,147 sequence entries)

allowing for the identification of semi-tryptic

4

Thiol Dioxygenase from Pseudomonas aeruginosa

including a sulfate ion, in agreement with other

CDO structures from crystals prepared in the

presence of sulfate and absence of substrate and

product. However, the resolution was not

sufficient to rule out other explanations such as

bound product (CSA) carried through the

purification. A final round of refinement in

Refmac5.5 used diffraction data to 2.7 Å but no

prior phase information (Table 2).

oxygen electrode (Rank Brothers) as previously

described.(57) Appropriate control experiments

were conducted to confirm that oxygen depletion

was not due to cysteine auto-oxidation under

reaction conditions.

Quantitative

Assessment

of

Cysteine

Dioxygenase activity of PA2602 Protein after Fe^II

Enrichment. PA2602 cysteine dioxygenase activity

after treatment of the protein with ferrous iron

sulfate followed by Chelex was measured using

HPLC-Evaporative light scattering detector

(ELSD) with a Luna 5 µ HILIC 200 Å column

(Phenomenex) as described previously (60, 61). 5-

13 µM PA2602 was incubated at 37 °C with 1-21

mM L-cysteine in the presence of 20-400 µM

bathocuproinedisulfonic acid (10 mM phosphate

buffer, 0-20 mM NaCl, pH 7.5). Reaction was

initiated by mixing 5 µL PA2602 with 845 µL

temperate buffered substrate. The reaction mixture

was incubated for 60 min with aliquots withdrawn

at regular time intervals. Both substrate and

product showed linear changes over 60 min and

the slopes were used to calculate the respective

reaction velocities. The velocities were plotted

versus initial cysteine concentrations and were

fitted to Michaelis-Menten equation with V_maxand

K_mconstants using Prism 5 (GraphPad) software.

V_maxwas normalized to the PA2602 active site iron

concentration to calculate k_cat. Alternative reaction

conditions of pH 6.5 were also tested, however the

rate of reaction was slower and therefore pH 7.5

was used for subsequent kinetic experiments.

A preliminary data set of 180° at 1° oscillation

were collected at MX2 of the Australian

Synchrotron from sodium acetate grown crystal B

and indexed in space group P4₁2₁2, with a unit cell

dimensions of 66.94, 66.94, 377.35 Å. Data were

integrated to 2.5 Å in XDS and scaled using Scala;

5% of reflections were reserved for the calculation

of the R_freestatistics (Table 2). The structure

obtained using crystal A was used for molecular

replacement yielding four monomers in the

asymmetric units. A higher resolution data set was

collected by the same method from a second

sodium acetate grown crystal (crystal C), and

indexed with parameters indistinguishable from

those for the preliminary data set from crystal B.

The programs of the CCP4 suite and Phenix were

used for structure solution, with the refined 2.5 Å

structure from crystal B used for molecular

replacement. Four monomers were placed in the

asymmetric unit. The active site iron and solvent

molecules were placed using Phaser-EP (MR-

SAD). Phenix Refine and Readyset were used for

refinement. The following residues were not

included in the final model (chain A: 1-4, 199-211;

chain B: 1-4, 198-211; chain C: 1-3, 200-211;

chain D:1-3, 202-211).

Iron Binding by PA2602 Protein. Iron binding

was investigated by adding various amounts of

ferrous iron sulfate to deoxygenated protein,

removing the excess iron by Chelex 100, and then

measuring the protein-bound iron by ferrozine-

based assay.(57) The data were fitted to(64,65): y

= 0.5(K_app+E+x) - (0.25(K_app+E+x)²- Ex)^1/2,

where x is the concentration of added iron, y is the

concentration of detected protein-bound iron, E is

the concentration of protein competent for iron

binding and K_appis the apparent dissociation

constant for the protein-iron complex.

Assessment of PA2602 Substrate Specificity.

p3MDO (170 µM active site iron) cysteine-

specific activity in the presence of 10 mM cysteine

was tested for inhibition by 10 mM cysteine

analogs (3-MPA, DL-homocysteine, N-Acetyl-L-

Cysteine or cysteamine) using the HPLC-ELSD

assay described above.

Quantitative

assessment

of

3-MPA

Dioxygenase Activity of PA2602 after Fe^II

Enrichment. A derivatization method (66) based

on the reaction between the free-thiol group of 3-

MPA and 5,5'-dithiobis-(2-nitrobenzoic acid)

(DTNB, or Ellmans reagent) was adapted to

quantify 3-MPA. PA2602 (9 µM) was incubated at

37 °C with 0.2-5 mM 3-MPA (100 mM phosphate

buffer, pH 7.5). Reaction was initiated by mixing

50 µL PA2602 with 450 µL temperate buffered

substrate. Reaction aliquots were withdrawn at

various time points and processed and analyzed as

Cysteine-dependent Oxygen Depletion Activity

of PA2602 Protein. Activities upon addition of

cysteine to protein samples were tested by

following oxygen depletion with a Clarke-type

5

Thiol Dioxygenase from Pseudomonas aeruginosa

previously

described.

Michaelis-Menten

version of the equation describing ES complex

formation under conditions of substrate depletion

was derived: y = 0.5(K+1+x) - (0.25(K+1+x)²–

x)^1/2, where y = [ES]/[E], x = [S]/[E] and K =

K_d/[E].

Synthesis of the Disodium Salt of 3-

sulfinopropionic Acid. 3-sulfinopropionic acid (3-

SPA) was synthesized based on the method by

Jollès-Bergeret(68) and a modified version by

Schurmann et al.(44) All chemicals for the

synthesis were used without further purification

from Sigma Aldrich. Briefly an intermediate bis-

(2-carboxyethyl)sulfone was formed by the

condensation of acrylic acid and sodium

formaldehyde sulfoxylate in aqueous solution. The

parameters were obtained and analyzed in the

same way as described for cysteine dioxygenation

above.

Mössbauer Spectroscopy. ⁵⁷Ferrous iron stock

solutions for Mössbauer spectroscopy experiments

were prepared as previously described.(67)

Anaerobic preparations of protein (0.8-1.5 mM) in

100 mM Tris-HCl (pH 8.0) and 50 mM NaCl were

iron-saturated anaerobically by exogenously

adding ferrous iron in the presence of 1 eq

dithionite followed by treatment with Analytical

Grade Chelex 100 sodium form, 200-400 mesh

(Bio-Rad). Protein samples were frozen in liquid

nitrogen in a glovebox (Belle Technology). ⁵⁷Fe

Mössbauer spectra of frozen liquid samples in a

custom teflon sample holder (approximately 400

µL volume) were recorded on a Mössbauer

spectrometer from SEE Co. (Science Engineering

& Education Co., MN) equipped with a closed-

cycle refrigerator system from Janis Research Co.

and SHI (Sumitomo Heavy Industries Ltd.) and a

intermediate

was

precipitated

through

acidification, dried and then cleaved under basic

conditions. The product 3-sulfinopropionate was

precipitated as a disodium salt with a large excess

of an ethanol-acetone (5:1) mixture with a final

yield of 61% after a second cleavage step.

Elemental analysis was carried out at the Campbell

Micro-analytical Laboratory, University of Otago.

Elemental analysis calcd. for C₃H₄O₄SNa₂: C

= 19.8; H = 2.2; S = 17.6; Na = 25.3. Found: C =

18.5; H = 2.3; S = 12.4; Na = 23.0; m.p. 308 °C;

HRMS (-ve ESI) m/z calcd. For [M-H]-: 136.9914,

temperature

controller

from

Lakeshore

Cryotronics, Inc. Data were collected in constant

acceleration mode in transmission geometry with

an applied field of 47 mT parallel to the γ-rays.

The zero velocity of the Mössbauer spectra refers

to the centroid of the room temperature spectrum

of a 25 µm metallic iron foil. Analyses of the

spectra were conducted using the WMOSS

program (SEE Co., formerly WEB Research Co.

Edina, MN). The Mössbauer spectra of

p3MDO:Fe^IIresting state (n=10), cysteine-

saturated (n=4) and 3-MPA-saturated (n=2)

samples were recorded and average p3MDO

Mössbauer parameters determined. All spectra

shown in this work were fitted using a parameter

file containing constrained average p3MDO

Mössbauer parameters such that only relative areas

of p3MDO:Fe^IIand p3MDO:Fe^II:substrate species

were allowed to vary.

1

found: 136.9919; H-NMR (400 MHz, D₂O): δ

(ppm) 2.45 (2H, s, H2), 2.55 (2H, s, H3); ¹³C

NMR (125 Mhz, D₂O): δ (ppm) 30.05 (C3), 57.23

(C2), 181.04 (C3); IR ν_max(ATR, cm^-1): 2960 (C-

H stretch weak), 1678 (C=O asy. stretch medium),

1420 (C=O sy. stretch strong), 1020, 1014, 975

(S=O stretch strong).

1

Product Determination by H-NMR and ¹³C-

NMR Analysis. PA2602 (200 µM) was incubated

for 30 min in 10 mM phosphate buffer (pH 7.5) at

37°C in the presence of 10 mM 3-MPA. The

solution was diluted 6.5 fold and measured by

nuclear magnetic resonance (NMR) using a TSP-

1

d4-capillary for H-NMR 500 MHz and ¹³C-NMR

p3MDO:Fe^II:substrate

125 MHz analysis at 25 °C on a Varian 500 MHz

AR NMR spectrometer using VNMRJ 2.3A

Determination

of

Complex Dissociation Constants. Formation of ES

complex as a function of substrate concentration

was followed using Mössbauer spectroscopy and

substrate bound fraction was determined assuming

that the Lamb-Mössbauer factors of bound and

non-bound protein were the same. The fraction

bound was plotted against cysteine concentration

normalized to active site ferrous iron concentration

present in the sample. Consequently, a modified

1

software. H-NMR and ¹³C-NMR spectra 3-SPA

dissolved in D₂O served as reference spectra for

enzymatically produced 3-SPA. Considering the

relatively high concentrations of reactants used in

this experiment the possibility of 3-MPA

autoxidation to 3-MPA disulfide was investigated.

To this end, ¹H-NMR spectra of room temperature

buffered solutions of 3-MPA alone, and 3-MPA

6

Thiol Dioxygenase from Pseudomonas aeruginosa

disulfide produced with H₂O₂were measured.

Using these spectra allowed estimation of 3-MPA

disulfide levels in enzymatic mixtures, which

represented less than one percent.

Untargeted shotgun tandem mass spectrometry

failed to identify peptides of p3MDO suggesting

that the protein is either absent or only present at

very low abundance (data not shown). To further

assess this we performed PRM assays on 7

predicted proteotypic p3MDO peptides as a more

sensitive targeted approach. PRM in conjunction

with a Mascot sequence database search identified

5 p3MDO peptide sequences out of the 7 recorded

peptide spectra with ion scores between 36 and 85

(Fig 4). The identification is highly significant and

specific for p3MDO within the 48 million

sequence entries in the non-redundant NCBI

sequence database. Fig 4 shows the raw fragment-

ion spectrum of the doubly charged precursor at

m/z 648.8433 and the Mascot fragment-ion

assignment for the sequence LEPGEVEALSPR as

a representative example of a significant peptide

identification. One of the 7 fragment-ion spectra

was assigned to isoprenoid biosynthesis protein

from P. aeruginosa (gi|489228560) indicating that

a false positive signal and retention time had been

considered for the scheduled PRM assay and

another peptide did not match any sequence in the

database. The PRM assay clearly confirmed the

presence of p3MDO. However, the failure to

identify the protein by shotgun LC-MS/MS and

the requirement of a highly sensitive targeted

approach suggests that the p3MDO protein is

present at very low abundance and this would

explain the lack of activity observed.

Crystallographic model. Initial crystallization

of p3MDO was achieved from ammonium sulfate

at pH = 7.5 (crystal A) and a 2.7 Å resolution

structure obtained. Crystallization using sodium

acetate conditions similar to those used to

crystallize rat CDO (56) produced crystals with a

different space group and which diffracted to

higher resolutions, 2.5 Å (crystal B) and 2.1 Å

(crystal C). Data were complete and moderately

redundant, with acceptable merging statistics

(Table 2). Refinement yielded good geometry and

good agreement with data. The four independent

copies in the asymmetric unit are similar (pairwise

RMSD of 0.24 to 0.40 Å) and similar to the two

copies in our preliminary 3USS structure (pairwise

RMSD of 0.27 to 0.44 Å, crystal A versus either

crystal B or C). All monomers in both crystals

participate in similar dimers, mediated by a β-

strand interaction between L189 across the

interface and hydrogen bonds between the side

Results

Protein Sequence Identification. BlastP

search(48) of putative bacterial CDO sequence

(YP_299237.1) from R. eutropha (strain JMP134)

identified twenty P. aeruginosa strains which each

contained a single possible CDO homologue. The

strains originated from a variety of habitats

including human hosts (eg. PAO1), plant (E2),

industrial water (ATCC700888) and soil

(ATCC14886). The P. aeruginosa CDO

homologues exhibited high sequence similarity

with variations observed at only three residues: 32,

35 and 42. BlastX search (48) of P. aeruginosa

PAO1 genome (NC_002516.2) using PA2602

nucleotide sequence (gi|110645304:2945264-

2945869) revealed only one putative gene coding

for CDO homologue. No other related gene was

found. Alignment of this homologue with other

CDOs including putative bacterial CDO sequence

(YP_299237.1) from R. eutropha, highlighted

conserved residues and location of the highly

conserved cupin motif.(23) Like that of R.

eutropha and other bacteria, the P. aeruginosa

sequence lacks a cysteine which participates in the

cysteine-tyrosine crosslink observed in all

mammalian CDOs. Comparison with the

previously determined CDO fingerprint motif (21)

allowed unambiguous identification of all other

key residues within the PA2602 sequence

suggesting a possible thiol dioxygenase function

(Fig 2). Purified, recombinant PA2602 protein was

assessed for ligand binding and catalytic ability.

As we show later it is a 3-mercaptopropionate

dioxygenase and therefore name it p3MDO.

p3MDO is expressed in low levels in P.

aeruginosa. To investigate whether p3MDO is

expressed by P. aeruginosa we initially assayed

thiol dioxygenase activity in cell lysate. Selected

growth conditions were tried, including media

containing disulfides as the sole carbon source.

Under all growth conditions tried, no activity was

detected. It was therefore decided to use mass

spectrometry to determine whether the protein

itself is expressed. P. aeruginosa was grown in

rich media, lysed and protein separated by SDS-

PAGE before analysis by mass spectrometry.

7

Thiol Dioxygenase from Pseudomonas aeruginosa

chains of D68 and S69 on opposite molecules.

These interactions may influence the orientation of

these loops in these crystals. We know of no

evidence that the dimer exists under physiological

conditions or has biological significance.

induced by the iron coordination. Interestingly,

dioxygen is proposed to bind iron at the site trans

to this histidine.(74)

Surprisingly, superposition of the bacterial and

rat CDO structures based on the full polypeptide

chain places the catalytic iron in a different

position in the two structures, although the relative

position of iron, the hydroxyl O of Y159 and the

δ-N of H157 remain constant (numbering from

PA2602). This suggests that a more informative

structural comparison is possible if these three

centers are aligned. Superposition of these three

conserved features (Fig 5D) displaces the β-strand

formed by residues 153-159 demonstrating that the

key elements of the bacterial active site are

displaced by approximately 3 Å relative to the β-

barrel, if compared to the mammalian enzyme.

This shift correlates with the presence or absence

of a cis-peptide bond following the residue C-

terminal to Y159. All structures of mammalian

CDO show both the cysteine-tryrosine crosslink

and a serine-proline cis-dipeptide immediately

following the tyrosine. Both our structure and the

R. eutropha structure (4QMA) lack this sequence

and the corresponding bond is in the trans

conformation. The resulting shift of the mainchain

would open a void, which is filled in the bacterial

enzymes by the indole sidechain of W94,

immediately preceding the glycine that replaces

the cysteine observed in the mammalian crosslink.

This alternate mainchain conformation allows the

triangle formed by the catalytic iron, the hydroxyl

O of Y159 and the δ-N of H157 to superimpose

closely, with the hydroxyl of S153 moved by 2.3

Å. This rotation of the active site relative to the β-

barrel results in Q62 in p3MDO occupying a very

different position relative to corresponding residue

R60 in the rat structure. R60 has been shown in

CDO to interact with the carboxyl group of

cysteine substrate when cysteine is bound as a

chelate to the active site iron via its thiol and

amine groups.(71)

Initially, the structure can be compared with

that from R. eutropha, with which it shares high

identity (62%), and which has been suggested to

be a 3MDO.(69) The structures are indeed very

similar with an overall RMSD of 0.66 Å. This

similarity is also true of the active site (Fig 5A)

where identical distances between the iron and the

second sphere residues such as the hydroxyl of

Y159 are observed (4.1 Å). The only notable

difference is the absence of a dioxygen molecule

trans to H142 that was modeled in the equivalent

position in R. eutropha (4QMA). Although

increased electron density was observed at that

position, this was modeled as a sulfate in our

original structure of p3MDO (crystal A) and in the

original analysis of R. eutropha (2GM6). In our

subsequent higher resolution structures presented

here (crystals B and C), where sulfate was not

present, we attempted modeling dioxygen and

other molecules in solution but were unable to

produce convincing fits in all four chains in the

asymmetric unit. Without further supporting

evidence, this is modeled conservatively as a water

molecule.

Given the close similarity and lack of kinetic

data for R. eutropha 3MDO comparisons of the

bacterial structures offered only limited insights

into the structural basis of function. A useful

comparison was made with the structure of rat

CDO (4KWJ) for which there is considerable

crystallographic

and

kinetic

data.(70-73)

Alignment of the structures based on the backbone

confirms the structure similarity suggested by

sequence homology (RMSD of 0.86 Å, Fig 5B).

This similarity is also exhibited by the

organization of the active site with conserved

residues occupying similar positions in both

structures, even though p3MDO does not contain a

cysteine-tyrosine crosslink observed in all

mammalian CDO structures (Fig 5C). The iron,

coordinated by three histidines and three waters

has slightly distorted octahedral geometry (176 ±

3°, 159 ± 4° and 175 ± 3°, average angles as seen

in all four chains). The sidechain of H142 occupies

an unfavorable rotamer, as it does in all other

CDO X-ray crystal structures, suggesting strain

Iron binding and active site titration. The

relationship between exogenously added iron and

iron bound to p3MDO is presented in Fig 6A. The

initial slope of the fit (~1) reveals stoichiometric

binding between iron and p3MDO. The data points

were fitted to a quadratic equation(64) to estimate

the upper limit(75) of the p3MDO:Fe^IIdissociation

constant as 3 ± 1 µM. Currently, the only known

enzymes with His₃iron coordination with a

8

Thiol Dioxygenase from Pseudomonas aeruginosa

reported K_dare CDO, and A. johnsonii Diketone-

cleaving dioxygenase (Dke1). In both cases the K_d

is ~5 µM,(57,76) which is comparable with the

estimate of the p3MDO affinity for iron presented

here.

We then measured the cysteine-dependent

oxygen depletion activity by following time

dependent O₂uptake in the presence of p3MDO

with increasing concentrations of bound iron. The

relationship between protein-bound iron and initial

velocities of oxygen consumption exhibited a

linear correlation with the slope of the best-fit line

corresponding to 0.007 ± 0.001 s^-1(Fig 6B)

reflecting the catalytic turnover number per active

site bound iron. Activity plateaued when bound

iron equaled total enzyme concentration

experimental variables, the data obtained are

similar to those for rat CDO with cysteine(72) and

strongly support cysteine being a viable substrate

for p3MDO, even if not its preferred substrate.

The steady state parameters for cysteine were

determined and Fig 7 illustrates the dependence

and saturation behavior of the rate of CSA

formation as a function of cysteine substrate

concentration. The summary of Michaelis-Menten

kinetic parameters for p3MDO is shown in Table

3. p3MDO exhibited a K_min the millimolar range

(of cysteine) which is in accordance with

previously published values for other bacterial

CDOs (21). However, p3MDO had a significantly

slower steady state rate of catalysis than other

bacterial CDOs.(21)

suggesting

a

stoichiometric,

catalytically

The relatively slow rate of cysteine

dioxygenation led us to investigate other thiols as

potential inhibitors or substrates of the thiol

dioxygenase activity of p3MDO. Cysteamine,

homocysteine, N-acetyl-L-cysteine and 3-MPA

were chosen. In our HPLC assays we detected

only minor reduction of cysteine dioxygenase

activity in the presence of stoichiometric

concentrations of N-acetyl-L-cysteine, D,L-

homocysteine and cysteamine, 86 ± 15% (n = 6),

79 ± 14% (n = 8) and 75 ± 19% (n = 10),

respectively. We previously showed that D,L-

homocysteine binds to the active site of rat

CDO.(57) However, the reported extent of

inhibition of mammalian CDOs by homocysteine

remains ambiguous(17,25,79) and very inefficient

use of homocysteine as a substrate has recently

been reported.(80) Comparable inhibition by

cysteamine has been reported for other bacterial

CDOs.(21) N-acetyl-L-cysteine, homocysteine and

cysteamine dioxygenation product was not

observed either in the competition assays or when

incubated with P. aeruginosa CDO alone. In

contrast, incubation of P.aeruginosa p3MDO with

cysteine in the presence of stoichiometric 3-MPA

resulted in no detectable levels of CSA within the

measured time window. However, HPLC

chromatograms revealed a new peak (retention

time ~6 min) which was later assigned to the

presence of 3-SPA (see below), suggesting the

possibility that 3-MPA not only competed

effectively with cysteine for binding in the active

site, but could be utilized as a substrate for

dioxygenase reaction. This is in contrast to

bacterial CDOs which showed only a modest

competent relationship between p3MDO and iron.

Measurement of P. aeruginosa thiol

dioxygenase activities. We tested p3MDO for

cysteine dioxygenase activity using an HPLC

assay, previously described.(77,78) Incubation

reduced the cysteine-associated peak and produced

a peak in the HPLC chromatogram with a

retention time (~16 min) identical to that of CSA

standards. In addition, samples analyzed by

HPLC-ELSD were also analyzed by mass

spectrometry (-ve ESI) and showed m/z peak of

152.0029 which compared well to the calculated

profile (152.0023) and the CSA standard

(152.0029).

Fidelity of cysteine utilization as a substrate

for p3MDO was tested in a number of ways.

Firstly, the rate of cysteine depletion and CSA

formation could be followed by our HPLC based

assay in the same experiment. The relative rates

using 10 mM cysteine at 25 °C (0.005 s^-1CSA

formation versus 0.007 s^-1cysteine depletion, n=2)

suggested ~70% conversion of substrate cysteine

into CSA. Importantly, this rate of cysteine

depletion mirrors O₂uptake measured using an

oxygen electrode described above (0.007 s^-1).

Finally, we took a Mössbauer sample (774 µM

p3MDO) with 10 equivalents cysteine added to

ensure all protein has substrate bound and added

one equivalent of O₂dissolved in buffer in a sealed

system. The amount of CSA produced was

determined by HPLC (560 ± 20 µM, n=9) and this

also corresponds to ~70% use of dioxygen to

produce CSA. Althought these ratios are

dependent on enzyme concentration, pH and other

9

Thiol Dioxygenase from Pseudomonas aeruginosa

(<6%) inhibition of CSA production by 3-MPA

and no enzymatic turnover.(21)

other non-heme mononuclear-iron enzymes, the

Mössbauer spectrum of p3MDO:Fe^IIexhibited

minor heterogeneity which was substantially

removed by adding 1 eq of dithionite, a common

treatment of protein samples for Mössbauer

spectroscopy. The resulting Mössbauer spectrum

of the p3MDO:Fe^IIresting state consists of a

single broad quadrupole doublet (Fig 8A, top

spectrum) with parameters (δ = 1.24 mm s^-1; ΔE_Q

= 2.91 mm s^-1) typical for high-spin Fe^IIin

octahedral coordination.

To confirm the formation of 3-SPA, we

synthesized the disodium salt of 3-SPA by an

1

established method (44,68) and compared H- and

¹³C-NMR spectra of it and enzymatically produced

3-SPA. This, with reference to published

spectra(44,81,82) unambiguously showed that

PA2602 readily oxidizes 3-MPA. Further evidence

was provided by mass spectrometry (-ve ESI)

which showed a m/z peak of 136.9918 that

compared well to the calculated profile (136.9914)

and the 3-SPA standard (136.9919).

Our kinetic data identified both cysteine and

3-MPA as substrates for p3MDO undergoing the

dioxygenation reaction. Therefore, we used

An Ellmans based method was optimized to

monitor 3-MPA to 3-SPA conversion. Fig 7

illustrates the dependence and saturation behavior

of the initial rate of 3-MPA substrate utilization as

a function of its concentration. The summary of

Michaelis-Menten kinetic parameters for PA2602

is shown in Table 3. Our kinetic data show that

p3MDO exhibits K_mvalues in the millimolar range

for both cysteine and 3-MPA. A 5-fold faster

turnover rate of 3-MPA catalysis combined with a

lower K_mresults in a 40-fold higher overall

efficiency of dioxygenation of 3-MPA as

compared with cysteine. Therefore, in combination

with the competition data presented above, we

concluded that 3-MPA is the kinetically preferred

substrate for p3MDO.

The efficiency of this reaction was also

investigated. Time dependent ¹H NMR spectra

were taken as described previously for CDO and

cysteine.(77) The rate of 3-SPA appearance was

seen to be ~95% of 3-MPA disappearance. This

relationship was further confirmed by measuring

substrate disappearance through Ellman’s assay

(0.038 ± 0.007 s^-1, n=4) and O₂electrode (0.041 ±

0.008 s^-1, n=4) at 25 °C, with 5 mM 3-MPA (i.e.

well above K_m). This confirms that both substrates

are converted to product during this reaction with

3-MPA.

Mössbauer

spectroscopy

to

study

the

thermodynamics of formation of the enzyme

substrate complex at 20 °C (Fig 8). Addition of

cysteine led to the gradual conversion of the single

broad quadrupole doublet of the p3MDO:Fe^II

resting state (Fig 8A, top spectrum) to a narrow

symmetrical quadrupole doublet (Fig 5A, bottom

spectrum) with Mössbauer parameters (δ = 1.11

mm s^-1; ΔE_Q= 3.55 mm s^-1) also consistent with

homogeneous

high-spin

Fe^II.

Subsequent

experiments with resting state (n=10) and

saturating cysteine concentrations (n=4) allowed

unambiguous

identification

of

the

p3MDO:Fe^II:cysteine complex from which

average Mössbauer parameters of both species

were calculated (Table 4). The reduction in isomer

shift and increase in quadrupole splitting observed

upon anaerobic addition of cysteine to p3MDO

(∆δ = -0.12 and ∆∆E_Q= +0.63 mm s^-1) are

consistent with coordination of the thiol to iron.

Similar experiments were carried out with 3-

MPA. Interestingly, initial spectra measured in the

presence of dithionite used to remove

heterogeneity as described for cysteine above, led

to loss of iron from the protein (data not shown).

Unable to fully explain this observation yet, we

speculate that 3-MPA persulfide is formed in the

presence of 3-MPA and dithionite as we described

recently,(73) and this species is able to remove

iron. The spectra were therefore repeated without

pretreatment with dithionite (Fig. 8B) and are thus

significantly broader than those shown for cysteine

(Fig 8A). However, the 3-MPA bound species has

the same isomer shift as the cysteine bound form

suggesting that in both cases the thiol is

coordinated. The change in quadrupole splitting

upon binding of 3-MPA is quite different to

Substrate Binding to the Active Site.

Mössbauer spectroscopy has been successfully

applied by us to study active site iron interactions

in Rattus norvegicus CDO.(57) Iron binding

studies suggested a tight binding p3MDO:Fe^II

complex (see above), which allowed anaerobic

reconstitution of the active site with exogenously

added anaerobic ferrous ⁵⁷Fe during preparation of

protein samples for Mössbauer spectroscopy

experiments. Consistent with previous studies on

10

Thiol Dioxygenase from Pseudomonas aeruginosa

cysteine (ΔΔE_Q= -0.39 mm s^-1) and this is

attributable to a difference in overall charge of the

ES complexes or different binding geometries or a

combination of both effects.

There is a high level of sequence identity

(98.5%) within the twenty p3MDO homologues

identified across twenty strains of P. aeruginosa,

with variations occurring only outside the

Fitting spectra to a linear combination of

spectra with average Mössbauer parameters for

resting and ES complexes allowed quantitative

monitoring of p3MDO:Fe^II:substrate complex

formation (Fig 8). The proportion of substrate-

bound species was determined directly from the

spectra. Data from several titration experiments

exploring different E/S ratios were plotted versus

the concentration of cysteine added, normalized to

active site iron present and fitted to a modified

version of the quadratic equation (Fig 8C) (see

Experimental Procedures section). The use of a

quadratic equation is warranted due to comparable

concentrations of receptor and ligand. We report a

dissociation constant for the p3MDO:Fe^II:cysteine

complex in the lower millimolar range (K_d= 1.3 ±

0.1 mM), while the p3MDO:Fe^II:3-MPA shows

stoichiometric binding with a upper limit for K_dof

5 µM. In the case of cysteine the concentration of

p3MDO used in the experiments is approximately

the same as K_d. This, and the fact the p3MDO:Fe^II

concentration is known to 3 significant figures

means that in this case K_dis quite well defined. In

contrast, the stoichiometric binding of 3-MPA

means that only an upper limit is defined.

previously

motif.(21,23)

identified

Genomic

CDO

analysis

fingerprint

(BlastX)

identified only one copy of the gene coding for the

thiol dioxygenase homologue per genome. Mass

spectrometry has shown that the protein is indeed

expressed by the bacterium, albeit at very low

levels. Although we have not identified growth

conditions where this enzyme is up-regulated we

have shown that the thiol dioxygenase breakdown

pathway is present in P. aeruginosa. The

physiological role of p3MDO is unclear. P.

aeruginosa was unable to grow with 3MPA as the

sole carbon source; the bacteria were able to grow

using cysteine as sole carbon source but a

mutation in the p3MDO gene did not prevent

growth, showing that p3MDO is not essential for

thiol catabolism (data not shown). Whatever the

physiological role of this enzyme, it is present in

strains inhabiting a diverse spectrum of habitats

ranging from human hosts to plants, industrial

waters and soil indicating its biological

importance.

Our structural data provides insight into active

site organization in thiol dioxygenases and the role

of the cysteine-tyrosine crosslink found in

mammalian CDO. Interestingly, the active site

geometry is preserved independent of its

orientation to the polypeptide backbone: the

distances between the iron-to-hydroxyl of Y159,

iron-to-δ-nitrogen of H157 and hydroxyl of Y159-

to-δ-nitrogen of H157 are the same in the two

Discussion

In contrast to other non-heme mononuclear-

iron enzymes, mammalian and bacterial CDOs

display a remarkably high specificity for their

substrate cysteine. Mammalian ADO, bacterial

mercaptosuccinate dioxygenase and bacterial

3MDO are only able to dioxygenate cysteamine,

structures

(numbering

from

p3MDO).

Remarkably, the side chain rings of Y159 and

H157 exhibit almost perfect superposition in spite

of the fact that p3MDO is unable to be

crosslinked. The position of this tyrosine phenol

likely depends not only on the presence or absence

of crosslink, but also on the presence of a serine-

proline cis peptide bond in the mammalian enzyme

or W94 in the bacterial. This suggests that

interpretation of the role of the crosslink and of the

functional consequences of its removal must

account for this structural feature as well.

mercaptosuccinate

and

3-MPA

respectively.(6,83,84) Although recent work(80)

has shown mouse CDO is able to oxidize other

substrates, such as L-penicillamine, the coupling

between dioxygen consumption and product

formation is ≤ 5% suggesting that these substrates

interact with the enzyme active site and undergo

catalysis very differently from the authentic

substrate. In this work we identified and

characterized a P. aeruginosa CDO homologue

capable of oxidizing two related thiol-containing

substrates, cysteine and 3-MPA, to their respective

sulfinic acids with a high level of coupling to

dioxygen consumption.

Kinetic characterization of p3MDO using both

substrates, cysteine and 3-MPA, have shown that

3-MPA is the kinetically preferred substrate with a

five-fold greater k_catand eight-fold smaller K_m.

11

Thiol Dioxygenase from Pseudomonas aeruginosa

The enzyme is able to utilize both substrates,

although inhibition studies show that when both

substrates are present only 3-MPA is oxidized.

Preference for 3-MPA can be rationalized to a

certain extent through our structural studies of

p3MDO. Previous structural investigations of rat

CDO by us(73) and others(71) have shown that

R60 is integral to cysteine substrate binding. This

residue, which reorganizes upon substrate cysteine

forming a sulfur/nitrogen chelate with the ferrous

iron in the active site, forms an ionic bond with the

carboxyl group of cysteine and holds the substrate

in place. Driggers et al.(69) proposed that in the R.

eutropha homologue, replacement of this arginine

with Q67, along with presence of R173 (R.

eutropha numbering for both), supports 3-MPA

substrate specificity. Indeed, structural comparison

with p3MDO suggests that Q62 of the P.

aeruginosa p3MDO assumes the role of R60 of rat

CDO, or Q67 of the R. eutropha enzyme.

Significantly, glutamine is shorter than arginine

and Q62 is situated further away from the iron

atom and thus further from the site of substrate

binding. The active site can therefore

accommodate a more elongated structure of 3-

MPA bound by its thiol group alone, suggested by

our recent structure of 3-MPA-persulfide bound to

rat CDO.(73) Conversely, because of the weaker

interaction of its carboxylate with Q62, cysteine

binding would be expected to be destabilized

relative to rat CDO, with its cationic arginine in

this position. We do not observe for p3MDO

residue R168 the potential clash with amino group

of substrate cysteine suggested for R173 of the R.

eutropha enzyme.(69) It is possible that R168 of

p3MDO interacts favorably with the carboxylate

of a cysteine substrate, depending on conformation

of bound substrate. B. subtilis CDO resembles

could be due heterogeneity in number of water

molecules bound to the iron. This idea is supported

by crystallographic studies presented here and

previously(71,73) that show either one, two or

three well-defined water molecules. Further

support comes from the sharp symmetrical

quadrupole

doublet

exhibited

by

p3MDO:Fe^II:cysteine. Cysteine binding must

replace at least one of the three bound waters and

the lowering of the isomer shift supports thiolate

binding to the iron. The geometry of this species is

very well-defined as exhibited by significant

narrowing of the spectrum upon cysteine binding.

In contrast, although 3-MPA also binds via its

sulfur as shown by the lowering of the isomer

shift, the broadness of the spectrum is intriguing in

light of different dissociation constants described

below.

The large differences in the spectra observed

upon substrate binding allowed the proportion of

cysteine-bound p3MDO to be determined as a

function of substrate added in the absence of

dioxygen. These conditions preclude catalysis and

allow quantification of the enzyme-substrate

complex poised for oxygen binding. p3MDO

showed stoichiometric binding with 3-MPA

suggesting a low dissociation constant. In contrast,

we determined a K_dof 1.3 mM for the

p3MDO:Fe^II:cysteine

complex.

This

is

substantially weaker than the low micromolar

dissociation constants reported for ES complexes

within other non-heme mononuclear-iron enzymes

such as HPCD and TauD.(85,86) It is also very

much weaker than rat CDO (unpublished results).

This weaker binding constant can perhaps be

related to Q62 occupying the position of R60 in rat

CDO as discussed above.

In conclusion, dioxygenation occurs at an

octahedral Fe^IIion in p3MDO but unlike other

thiol dioxygenases, p3MDO has significant

substrate specificity towards more than one

substrate; 3-MPA and cysteine, the latter with

forty-fold lower catalytic efficiency but significant

efficiency of coupling to dioxygen consumption.

The preference for 3-MPA can be explained by a

proposed weaker binding of cysteine caused by a

weak interaction between Q62 and the carboxylate

of cysteine due to Q62 being uncharged, shorter

and placed further from the iron than the

equivalent arginine residue in rat CDO. Presence

of R168 or lack of a cis-proline following Y157

mammalian

CDOs

in

lacking

this

glutamine/arginine pair, and retains the

"mammalian" serine-cisproline noted above as

lying between Y157 and R168, despite lacking the

adjacent cysteine-tyrosine crosslink. This raises

the possibility that the cis dipeptide is correlated

with substrate specificity rather than crosslink

occurrence.

We investigated the iron site of catalysis and

substrate binding using Mössbauer spectroscopy.

The resting state, p3MDO:Fe^II, exhibits a broad

asymmetric quadrupole doublet with parameters

similar to rat CDO. The broadness in the signal

12

Thiol Dioxygenase from Pseudomonas aeruginosa

may also favor 3-MPA binding over cysteine.

Further studies to more clearly define how these

substrates bind are underway.

Acknowledgments

GNLJ (PI) and SMW (AI) thank the Marsden Fund of the Royal Society of New Zealand for funding.

EPT thanks the Canadian Institutes of Health Research Postdoctoral Fellowship. We thank Mervyn

Thomas, Ian Stewart and Jenny Lyburn (Department of Chemistry, University of Otago) for help with

NMR and MS experiments.

Conflict of interest

The authors declare that they have no conflicts of interest with the contents of this article.

Author contributions

GNLJ directed the study. GNLJ and SMW conceived experiments and wrote the paper. EPT and GNLJ

designed and analyzed the experiments in Figure 6 and 8. MF, ES and GNLJ designed and analyzed the

experiments in Figure 7. EPT, MF and SMW designed and analyzed the experiments in Figure 5. TK

designed and analyzed the experiments in Figure 4. LWM and ILL designed, performed and analyzed

growth studies in P. aeruginosa. SA carried out kinetic studies to investigate efficiency of reaction. All

authors reviewed the results and approved the final version of the manuscript.

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Thiol Dioxygenase from Pseudomonas aeruginosa

Footnotes:

^‡These authors contributed equally.

†

Abbreviations: CDO, cysteine dioxygenase; CSA, cysteine sulfinic acid; 3-MPA, 3-mercaptopropionic

acid; 3-SPA, 3-sulfinopropionic acid, 3MDO, 3-mercaptopropionate dioxygenase; p3MDO, P.

aeruginosa 3-mercaptopropionate dioxygenase; CoA, coenzyme A; BLAST, Basic Local Alignment

Search Tool, SIB, Simple is Beautiful; PRM, parallel reaction monitoring; HILIC, hydrophobic

interaction liquid chromatography.

18

Thiol Dioxygenase from Pseudomonas aeruginosa

Figure Legends:

Figure 1. Structural formula of thiol-containing compounds tested as substrates for P. aeruginosa thiol

dioxygenase (p3MDO).

Figure 2. Alignment of the P. aeruginosa CDO homologue with selected family members. Numbering of

P. aeruginosa residues is at top, extent of each sequene is indicated at left. Residues of the CDO

fingerprint proposed by Stipanuk et al. are shown in white on black. White on grey indicates the possible

divergence of the fingerprint in substrate specificity at position 62 in PA2602 (glutamine vs arginine), the

difference in uncrosslinked residue at position 95 (glycine or alanine vs cysteine), and at position 168

(argenine vs various). Residues adjacent and possibly influencing the crosslink black on light grey. The

conserved cupin motif residues are indicated on the bottom. PA2602 from Pseudomonas aeruginosa

PAO1, NP_251292.1 is aligned with characterized 3-mercaptopropionic dioxygenases (3MDOs) (6)

(Variovorax paradoxus TBEA6, ACB72254; Ralstonia eutropha H16, YP_841375); characterized

cysteine dioxygenases (CDOs) (20,21,25) (Homo sapiens, NCBI protein sequence accession number

BAA12873; Rattus norvegicus, BAA11925; Streptomyces coelicolor A3(2) [SCO3035], NP_627257;

Streptomyces coelicolor A3(2) [SCO5772], NP_629897; Bacillus subtilis, NP_390992; Bacillus cereus,

NP_832375) and putative CDOs (23) with a putative cysteine-tyrosine crosslink (Vibrio splendidus

12B01, EAP93565.1; Chromobacterium violaceum ATCC, NP_902852.1).

Figure 3. Purified samples of P. aeruginosa thiol dioxygenase (p3MDO) were loaded on a 15% (w/v)

polyacrylamide gel, resolved by SDS-PAGE and detected by staining with Coomassie Blue dye. A series

of p3MDO dilutions (1, 1:10, 1:20, 1:40). A 40-fold difference in protein sample loaded illustrates at least

97.5% purity of the protein preparation.

Figure 4. Significant identification of a proteotypic peptide sequence of p3MDO by PRM. A:

Representative raw spectrum of fragment ions after collision induced dissociation of the doubly charged

precursor at m/z 648.8433. Peaks annotated as y-ions (fragment ions carrying the C-terminus) were

assigned to fragment ions of the peptide sequence LEPGEVEALSPR by the Mascot software. B:

Detected sites of peptide fragmentation that confirm the sequence. Only y-ions were identified.

Figure 5. Structural comparison of p3MDO (4TLF, blue) with proposed 3MDO from R. Eutropha

(4QMA, green) and rat CDO (4KWJ, red). Selected conserved sidechains are shown with oxygen,

nitrogen, sulfur and iron atoms highlighted in red, blue, yellow and orange. Comparison with proposed

3MDO. (A) shows the close similarities between the active site of the two enzymes. Slightly larger

electron density trans to H142 was modeled as O₂in 3MDO but as water in p3MDO. Comparison with

rat CDO (B) shows the overall cupin fold aligned on all backbone atoms. (C) displays the active site when

the peptide backbones are aligned (iron-bound water molecules have been removed for clarity). (D)

shows an alignment based on Fe, the hydroxyl O of Y159 and the δ-N of H157 (the iron-bound waters of

only 4TLF are shown). This last alignment clearly shows the difference in position of Q62 compared to

the corresponding residue of rat CDO, R60*. This difference can explain the difference in reactivity of

p3MDO towards cysteine and 3-MPA. To aid comparison, * refers to the rat CDO sequence numbering.

Figure 6. Affinity and kinetic characterization of p3MDO:Fe^IIcomplex. Protein (70 µM) was mixed

anaerobically with varying concentrations of ferrous sulfate. Unbound-iron was removed by Chelex under

anaerobic conditions and the concentration of bound iron measured using the ferrozine assay. A) The

protein-bound iron is plotted vs. total iron added and fitted according to the Experimental Procedures. B)

Aliquots of 70 µM p3MDO with various amounts of iron bound were reacted with 10 mM cysteine at

25°C and O₂uptake measured by oxygen electrode in an air tight system. The initial velocities of O₂

depletion were calculated by linear regression and plotted vs. the concentration of protein-bound iron. The

best-fit line is plotted with a slope of 0.007 s^-1.

19

Thiol Dioxygenase from Pseudomonas aeruginosa

Figure 7. Michaelis-Menten kinetics of the dioxygenation reaction catalyzed by p3MDO using 3-MPA

(open circles) and cysteine (full circles) as substrates measured at 37°C. Michaelis-Menten constants

obtained through fitting (black lines) are reported in Table 2. Error bars represent standard deviation of at

least n=3 data points.

Figure 8. Determination of p3MDO:Fe^II:cysteine complex dissociation constant. A) Mössbauer spectra of

anaerobic p3MDO resting state (top spectrum) and upon anaerobically added increasing concentrations of

cysteine (spectra below). Spectra represent data from two separate titration experiments. Cysteine

concentrations were normalized to the concentration of the p3MDO active site ferrous iron present in the

sample and are illustrated by the ratios. The spectra can be fitted to two quadrupole doublets employing

average Mössbauer parameters (Table 3) for unbound p3MDO:Fe^II(red line) and substrate-bound

p3MDO:Fe^II:cysteine (blue lines) complexes as described in Experimental procedures. B) Similar titration

experiment using 3-MPA. The same color scheme is used to label bound and unbound species as for

cysteine. C) The relative area occupied by cysteine- (full circles) and 3-MPA-bound (open circles) species

is plotted vs. final thiol concentration (normalized to the active site concentration) and fitted according to

the Experimental procedures.

20

Thiol Dioxygenase from Pseudomonas aeruginosa

Tables:

Table 1. Peptides identified by PRM and software assisted spectrum interpretation using Mascot.

Predicted m/z (charge state)^a

gi|372467082 Chain A, Crystal Structure of Cysteine Dioxygenase from P. aeruginosa

Peptide sequence identified by Mascot

Score

Modification

474.5772 (3+)

648.8433 (2+)

515.5866 (3+)

829.4547 (2+)

725.6833 (3+)

YQQYLLHVDSR

LEPGEVEALSPR

IGDVHQVSNAFSDR

TSISIHVYGANIGAVR

AVFSAEGEEKPFISGYSNSR

36

85

40

48

70

gi|489228560 isoprenoid biosynthesis protein, partial [P. aeruginosa]

755.7236 (3+)

No identification

764.3578 (3+)

LVTTPAYMLAQSIAEAASGINK

85

-

Oxidation (M)

GAEYSQPYAFDAGGRPHPSGAR

^aPredicted m/z (charge state) is the m/z of the strongest charge state predicted by the Skyline software that

was used to build the scheduled PRM assay. Score: Mascot ion score at a significance threshold of

p<0.05. An ion score >56 indicates a significant peptide identification in a search against 48 million

sequence entries of the non redundant NCBI sequence database. All peptide sequences assigned to

cysteine dioxygenase were unique for the identified protein.

Table 2. Data collection, reduction and crystallographic refinement statistics.

Crystal A ((NH₄)₂SO₄)

Crystal B (NaOAc)

Crystal C (NaOAc)

Resolution range (highest

shell)

48.9 – 2.70 Å (2.85-2.70 Å) 47.3 – 2.50 Å (2.64-2.50 Å)

47.2 – 2.14 Å (2.25-2.14 Å)

Observed reflections

Unique reflections

Redundancy

112,263 (16,607)

16,270 (2,339)

6.9 (7.1)

238,593 (31,848)

31,044 (4,391)

7.7 (7.3)

665,020 (92,078)

49,221 (6,940)

13.5 (13.3)

Completeness

I/σ

R_merge

99.9% (99.7%)

13.9 (4.2)

99.9% (99.5%)

16.6 (2.8)

99.8% (98.7%)

16.5 (7.7)

a

0.100 (0.464)

16,261 (1,180)

0.208 (0.283)

0.283 (0.375)

0.017Å

0.099 (0.664)

28,051 (4132)

0.183 (0.312)

0.245 (0.391)

0.009Å

0.114 (0.371)

45,220 (5,937)

0.198 (0.271)

0.249 (0.332)

0.009Å

Reflections used in refinement

b

R_cryst

R_free

c

Deviation from ideal bond

lengths

Deviation from ideal bond

angles

Average B factor

Macromolecules

Iron atoms

1.753°

1.258°

1.168°

41.64 Å²

79.04 Å²

41.69 Å²

3USS

40.91 Å²

41.02 Å²

42.95 Å²

37.42 Å²

-

26.13 Å²

25.63 Å²

26.10 Å²

31.31 Å²

4TLF

Solvent

PDB

^aR_merge= Σ|I_obs-I_ave|/ΣI_ave

^bR_cryst= Σ|F_obs-F_calc|/ΣF_obscomputed over a working set composed of 95% of data.

^cR_free= Σ|F_obs-F_calc|/ΣF_obscomputed over a test set composed of 5% of data.

21

Thiol Dioxygenase from Pseudomonas aeruginosa

Table 3. Michaelis-Menten parameters of p3MDO measured at 37°C.

3-MPA

Cysteine

0.021 ± 0.003

8 ± 3

k_cat(s^-1)

0.11 ± 0.02^a

1.0 ± 0.4

110 ± 50

K_m(mM)

k_cat/K_m(M^-1s^-1)

3 ± 2

^aStandard error represents the error associated with the fit to Michaelis-Menten equation.

Table 4. Mössbauer parameters of P. aeruginosa thiol dioxygenase measured at 5.2 K.

δ (mm s^-1)

1.23 ± 0.02

1.11 ± 0.01

1.11 ± 0.02

ΔE_Q(mm s^-1)

2.92 ± 0.09

3.6 ± 0.2

Γ_L(mm s^-1)

0.57^a± 0.04

0.39^a± 0.01

0.90^b± 0.2

Γ_R(mm s^-1)

0.80 ± 0.06

Γ_L=Γ_R

Fe^II:p3MDO

Fe^II:p3MDO:cysteine

Fe^II:p3MDO:3-MPA

2.5 ± 0.3

Γ_L=Γ_R

^aSpectra fitted using a Lorentzian lineshape.

^bSpectra fitted using Voigt lineshape (dithionite was not added).

c

Standard deviation represent errors in parameters calculated using data from at least four independent

experiments. n=10 for Fe^II:CDO parameters.

22

Thiol Dioxygenase from Pseudomonas aeruginosa

Figures:

Figure 1

23

Thiol Dioxygenase from Pseudomonas aeruginosa

Figure 2

24

Thiol Dioxygenase from Pseudomonas aeruginosa

Figure 3

25

Thiol Dioxygenase from Pseudomonas aeruginosa

A

B

y9

y6

y10

y5

y2

y7

y1

y3

y4

y8

m/z

y7

y1

y6 y5 y4 y3 y2

y10 y9 y8

L

E

P

G

E

V

E

A

L

S

P

R

Figure 4

26

Thiol Dioxygenase from Pseudomonas aeruginosa

Figure 5

27

Thiol Dioxygenase from Pseudomonas aeruginosa

Figure 6

28

Thiol Dioxygenase from Pseudomonas aeruginosa

Figure 7

29

Thiol Dioxygenase from Pseudomonas aeruginosa

Figure 8

30

Article Doi

The cysteine dioxygenase homologue from Pseudomonas aeruginosa is a 3-mercaptopropionate dioxygenase

DOI: 10.1074/jbc.M114.635672

Source and publish data:

Authors:

Article abstract of DOI:10.1074/jbc.M114.635672

Full text of DOI:10.1074/jbc.M114.635672

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