The crystal structure of a homodimeric Pseudomonas glyoxalase I enzyme reveals asymmetric metallation commensurate with half-of-sites activity

Article abstract of DOI:10.1002/chem.201405402

The Zn inactive class of glyoxalase I (Glo1) metalloenzymes are typically homodimeric with two metal-dependent active sites. While the two active sites share identical amino acid composition, this class of enzyme is optimally active with only one metal per homodimer. We have determined the X-ray crystal structure of GloA2, a Zn inactive Glo1 enzyme from Pseudomonas aeruginosa. The presented structures exhibit an unprecedented metal-binding arrangement consistent with half-of-sites activity: one active site contains a single activating Ni²⁺ ion, whereas the other contains two inactivating Zn²⁺ ions. Enzymological experiments prompted by the binuclear Zn²⁺ site identified a novel catalytic property of GloA2. The enzyme can function as a Zn²⁺/Co²⁺ -dependent hydrolase, in addition to its previously determined glyoxalase I activity. The presented findings demonstrate that GloA2 can accommodate two distinct metal-binding arrangements simultaneously, each of which catalyzes a different reaction.

Full text of DOI:10.1002/chem.201405402

DOI: 10.1002/chem.201405402
Communication
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Enzymes
The Crystal Structure of a Homodimeric Pseudomonas Glyoxalase I
Enzyme Reveals Asymmetric Metallation Commensurate with
Half-of-Sites Activity
Rohan Bythell-Douglas,[a] Uthaiwan Suttisansanee,[b] Gavin R. Flematti,[a] Michael Challenor,[a]
Mihwa Lee,[a] Santosh Panjikar,[c, d] John F. Honek,[b] and Charles S. Bond*[a]
tural studies have shown that Glo1 enzymes are typically ho-
modimeric, with two identical metal-binding active sites relat-
ed to one another by two-fold rotational symmetry.[3] Glo1 en-
zymes can be categorized into Zn active and Zn inactive class-
es[1b,4] (Figure S2, Supporting Information). Both classes require
octahedral coordination of the active site metal for their can-
onical lyase activity, converting methylglyoxal-glutathione
hemithioacetal to S-d-lactoylglutathione.[5] Zn inactive Glo1 en-
zymes are inactive with Zn2+ bound, and are optimally active
with Ni2+ bound.[1b,4] These enzymes demonstrate half-of-sites
activity, with optimal activity at a ratio of one metal ion per ho-
modimer.[1b,4] Furthermore, isothermal titration calorimetry and
15N-1H HSQC NMR spectroscopic studies on the Zn inactive Es-
cherichia coli Glo1 demonstrated asymmetry in metal-binding
affinity.[6] Despite these results, the crystal structure of the Zn
inactive Glo1 enzyme from E. coli possesses full occupancy
Ni2+ ions at each of the two active sites.[3a] All published Zn in-
active Glo1 structures are essentially symmetrical, providing no
structural insight into half-of-sites activity.[3a,7]
Abstract: The Zn inactive class of glyoxalase I (Glo1) met-
alloenzymes are typically homodimeric with two metal-de-
pendent active sites. While the two active sites share iden-
tical amino acid composition, this class of enzyme is opti-
mally active with only one metal per homodimer. We have
determined the X-ray crystal structure of GloA2, a Zn inac-
tive Glo1 enzyme from Pseudomonas aeruginosa. The pre-
sented structures exhibit an unprecedented metal-binding
arrangement consistent with half-of-sites activity: one
active site contains a single activating Ni2+ ion, whereas
the other contains two inactivating Zn2+ ions. Enzymolog-
ical experiments prompted by the binuclear Zn2+ site
identified a novel catalytic property of GloA2. The enzyme
can function as a Zn2+/Co2+-dependent hydrolase, in ad-
dition to its previously determined glyoxalase I activity.
The presented findings demonstrate that GloA2 can ac-
commodate two distinct metal-binding arrangements si-
multaneously, each of which catalyzes a different reaction.
We present the first conspicuously asymmetric Zn inactive
Glo1 crystal structure, GloA2 from P. aeruginosa. The enzyme
possesses a single octahedrally coordinated Ni2+ at one active
site and two Zn2+ ions at the other site, congruent with half-of-
sites activity. We also present a novel hydrolytic function of
GloA2, related to this metal-binding arrangement. These data
suggest that GloA2 is capable of simultaneously possessing two
completely different and catalytically active metal-binding ar-
rangements, addressing the biological economy of this enzyme.
In all four presented structures, the two monomers that
form the GloA2 homodimer are related to one another by
noncrystallographic symmetry, as in other Glo1 enzyme struc-
tures[3a,b,d,7] (Figure 1). The overall fold of GloA2 is almost iden-
tical to the E. coli Glo1 enzyme[3a] (RMSD 0.39 ꢀ for all 117 Ca
atoms for chain A and 0.93 for chain B (114/117)) (Table S2,
Supporting Information) sharing the babbb architecture.
The structures confirmed active metal-binding residues His5,
Glu56, His74*, and Glu122*,[8] in which asterisks denote the
other chain of the homodimer. Metal ions were unambiguously
identified by using anomalous difference density maps gener-
ated from diffraction data sets collected below and above the
Ni and Zn X-ray absorption edges (Table S3, Supporting Infor-
mation).
Half-of-sites activity is a phenomenon where only one of two
essentially identical active sites of an enzyme is functional at
any one time.[1] This type of mechanism seems wasteful, as
though cellular resources are spent producing an effectively
nonfunctioning active site. A case-in-point is the Zn inactive
glyoxalase I (Glo1) class of enzyme.
Glo1 is a metalloenzyme involved in the methylglyoxal de-
toxification system[2] (Figure S1, Supporting Information). Struc-
[a] Dr. R. Bythell-Douglas, Dr. G. R. Flematti, M. Challenor, Dr. M. Lee,
Prof. C. S. Bond
School of Chemistry and Biochemistry, The University of Western Australia
35 Stirling Highway, Crawley 6009, Western Australia (Australia)
E-mail: charles.bond@uwa.edu.au
[b] Dr. U. Suttisansanee, Prof. J. F. Honek
Department of Chemistry, University of Waterloo
200 University Avenue West, Waterloo, Ontario, N2L 3G1 (Canada)
[c] Dr. S. Panjikar
Department of Biochemistry and Molecular Biology, Monash University
Clayton Campus, Melbourne 3800, Victoria (Australia)
[d] Dr. S. Panjikar
Australian Synchrotron, 800 Blackburn Road, Clayton
Victoria 3168 (Australia)
The electron density in all models was consistent with at
least one full-occupancy metal ion at both of the metal-bind-
ing active sites (Figure 2). Structures were determined with
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201405402.
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ion in NiZn-GloA2.a and NiZn-GloA2.b is bound in octahedral
coordination geometry, appropriate for Glo1 activity. GloA2 has
no Glo1 activity with Zn2+ bound[8] hence the Zn2+-bound site
is inactive.
The binuclear Zn site is a novel metal-binding arrangement
for a Glo1 enzyme, and as such the role of this site was initially
unclear. The two Zn2+ ions are 3.2 and 3.3 ꢀ apart in structures
NiZn-GloA2.a and NiZn-GloA2.b, respectively. These ions coor-
dinate a bridging water molecule, an arrangement typical of
a metallohydrolase.[10] The second reaction of the methylglyox-
al detoxification pathway is the hydrolysis of S-d-lactoylgluta-
thione by the unrelated binuclear metallohydrolase Glo2.[11]
Based on the binuclear metal arrangement in the X-ray crystal
structure, we generated the hypothesis that GloA2 can act as
a Glo2.
Figure 1. GloA2 (NiZn-GloA2.a; left) and Ni2+ bound E. coli Glo1[3a] (right).
Chain A is colored blue and chain B is colored red. Ni2+ and Zn2+ ions are
shown as green and grey spheres.
a single Ni2+ at both sites (NiꢀGloA2, 2.17 ꢀ) and a single Zn2+
at both sites (ZnꢀGloA2, 1.83 ꢀ). Two further structures were
determined with a novel asymmetric metal-binding arrange-
ment, with a Ni2+ ion at one site and two Zn2+ ions at the
Classical Glo1 and Glo2 enzymes are structurally unrela-
ted[11c,12] (Figure S6, Supporting Information). However, super-
imposing GloA2 and Glo2 structures by the coordinates of the
second site (Figure 3); one structure at high resolution (NiZnꢀ binuclear metal centers demonstrates that the solvent accessi-
GloA2.a, 1.80 ꢀ) and the other with accompanying anomalous
difference maps for metal identification (NiZnꢀGloA2.b, 2.17 ꢀ).
The additional Zn2+ is bound adjacent to the predicted posi-
tion. As far as we are aware, this is the first time that a homodi-
meric metal-binding enzyme has been observed to bind a dif-
ferent metal at each active site. In these structures, residues
Ala100 to Val109 (Loop-8) also demonstrate asymmetry. One
monomer adopts a “folded in” conformation, orienting His105
appropriately to form the novel metal-binding site along with
Glu122 (Figure S3, Supporting Information). Loop-8 on the
other chain adopts a similar conformation to the E. coli Glo1
structure.
bility and substrate-binding site of GloA2 is similar to Glo2 en-
zymes (Figure S7, Supporting Information).
Using HPLC-MS and established Glo2 kinetic protocols,[12a]
we determined that GloA2 consumes S-d-lactoylglutathione to
produce lactate and glutathione, the products of the Glo2 re-
action (Figure 5, Figure S8, Supporting Information). GloA2 has
a Km for the Glo2 reaction of 260ꢁ27 mm (Figure S9, Support-
ing Information), similar to other characterized Glo2 enzy-
mes.[11b,12a,13] However, GloA2s Vmax of 55ꢁ3 nmolminꢀ1 mgꢀ1
,
kcat of 0.027 sꢀ1 and kcat/Km of 100mꢀ1 sꢀ1 are all three orders of
magnitude lower than established Glo2 enzymes.[11b,12a,13]
As the novel metal-binding site is comprised of His105 and
Glu122, we generated the variants H105A and E122Q by site-
directed mutagenesis to determine the influence of these resi-
The coordination geometry of the active-site metals
(Figure 4) provides insight into half-of-sites activity. The Ni2+
Figure 2. The coordination geometry of each active site metal is shown as a gray polyhedron. Lines between carbon atoms from chains A and B are colored
blue and red, respectively. Zn2+ ions, Ni2+ ions and water-molecule oxygen atoms are colored as grey, green, and red spheres, respectively.
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Zn2+, a metal ion that GloA2
cannot use to catalyze the Glo1
reaction. These data demon-
strate that despite identical
amino acid composition for each
substrate-binding site, GloA2 is
capable of an asymmetric metal-
binding arrangement.
While Loop-8 provides His105
to coordinate the novel metal
binding site (Figure S3, Support-
ing Information), there is no sig-
nificant difference between wild-
type and H105A variant GloA2
enzymes in terms of Glo2
enzyme kinetics. This result dem-
onstrates that His105 is not es-
sential for Glo2 activity. Our re-
sults do not delineate whether
GloA2 requires a binuclear or
mononuclear Zn2+ site for hy-
drolytic activity.
The slow rate at which GloA2
catalyzes the Glo2 reaction sug-
gests that this capability is not
likely physiologically relevant, as
P. aeruginosa possesses a canoni-
Figure 3. Anomalous difference electron-density maps of NiZn-GloA2.b collected at X-ray energies either side of
the Ni and Zn X-ray absorption edges. NiZn-GloA2.b is shown as a ribbon with the metals shown as spheres. Dark
blue mesh represents anomalous difference density (contoured at 3.5 s) for data collected at 8233, 8433, 9565,
and 9765 eV. The bottom panel relates X-ray energy to the theoretical anom-
alous signal strength f“ of Ni and Zn[9]
.
dues on GloA2 hydrolytic activity. Similar kinetic data were ob-
served for H105A as the wild type, whereas the E122Q variant
showed a significant decrease in activity relative to the wild
type (Km 780ꢁ880 mm, Vmax 6.9ꢁ5.6 nmolminꢀ1 mgꢀ1
;
Figure 6).
ICP-MS analysis of GloA2 demonstrated that Zn is the major
metal species bound to the enzyme as isolated and purified
from the E. coli expression host (Table S6, Supporting Informa-
tion). Treating GloA2 with the chelating agent dipicolinic acid
(DPA) resulted in a 66% decrease in Glo2 enzyme activity, con-
firming that GloA2 Glo2 activity is metal dependent (Figure 6).
ICP-MS analysis of GloA2 incubated with excess ZnCl2 demon-
strated that the enzyme can bind three metal ions per homo-
dimer in solution, validating the metal-binding stoichiometry
observed in the crystal structure.
To identify the optimal Glo2 activity metal for GloA2, Glo2
activity was measured for protein spiked with a range of metal
cations. Only Co2+ stimulated Glo2 activity (Figure 6). Attempts
to use Zn2+ to metallate Apo-GloA2, however, resulted in inac-
tive protein. The same effect has been observed previously in
other binuclear Zn2+ active Glo2 enzymes[11a,b] and has been
attributed to mis-metallation.
Figure 4. Stereoview of the two metal-bound active sites of NiZn-GloA2.a.
a) The binuclear Zn bound active site. b) The Ni bound active site. Chains A
and B are colored blue and red, respectively. Metal-binding residues and
glycerol are shown in stick representation. Zn2+ and Ni2+ ions are shown in
grey and green. Waters are shown as red spheres. 2mjFoj-DjFcj, electron-
density maps (dark-blue mesh) are contoured to 1.0 s.
We have determined the first crystal structure of a Zn inac-
tive Glo1 enzyme that provides insight into half-of-sites activi-
ty. One of the two active sites is bound to Ni2+ in a configura-
tion appropriate for Glo1 activity. The other site is bound to
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provide novel insight in to the functional flexibility of metal-
loenzymes.
Acknowledgements
We acknowledge the use of beamlines MX1 and MX2 of the
Australian Synchrotron, funded by the Western Australian
Foundation Investor consortium. We thank the undergraduate
students of UWA’s CHEM3307 for participating in X-ray data
collection. We thank Agata Sadowska, Denise Lupton and Paul
Kirkwood for technical assistance. J.H. gratefully acknowledges
the financial support of NSERC (Canada). U.S. gratefully ac-
knowledges the financial support of the University of Waterloo
(Ontario, Canada) and the Government of Thailand.
Figure 5. Extracted ion chromatograms of d-lactate ([MꢀH]ꢀ m/z: 89) and
glutathione ([MꢀH]ꢀ m/z: 306) from a sample of GloA2 incubated with S-d-
lactoylglutathione.
Keywords: crystal-structure determination
metalloenzymes · proteins · zinc
·
enzymes
·
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Figure 6. Glo2 activity of GloA2. WT: wild-type, H105A: H105A variant,
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It is of particular note that GloA2 can catalyze two complete-
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fold merely by switching the bound metal species. These re-
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Received: September 25, 2014
Published online on && &&, 0000
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COMMUNICATION
Half-of-sites activity is a puzzling phe-
nomenon where only one of two essen-
tially identical active sites of an enzyme
is catalytically active. Structural and ki-
netic studies on the metalloenzyme
GloA2 demonstrated that the two sites
are capable of adopting two completely
different metal-binding arrangements,
which catalyze two different reactions
(see figure).
&
Enzymes
R. Bythell-Douglas, U. Suttisansanee,
G. R. Flematti, M. Challenor, M. Lee,
S. Panjikar, J. F. Honek, C. S. Bond*
&& – &&
The Crystal Structure of
a Homodimeric Pseudomonas
Glyoxalase I Enzyme Reveals
Asymmetric Metallation
Commensurate with Half-of-Sites
Activity
Chem. Eur. J. 2014, 20, 1 – 5
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These are not the final page numbers! ÞÞ