DOI: 10.1002/chem.201405402
Communication
&
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)
[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.
Chem. Eur. J. 2014, 20, 1 – 5
1
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
&
&