MINIREVIEW THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 288, NO. 19, pp.

13178 –13185, May 10, 2013

© 2013 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

Biosynthesis of the Urease teria, which can lead to unproductive volatilization of ammonia
and harmful soil alkylation (4). Also of interest, urease from jack
Metallocenter* bean (Canavalia ensiformis) seeds was the first enzyme to be
Published, JBC Papers in Press, March 28, 2013, DOI 10.1074/jbc.R112.446526 crystallized (5) and the first protein shown to contain nickel (6).
Mark A. Farrugia‡, Lee Macomber§, and Robert P. Hausinger‡§1
Finally, as described in subsequent sections, urease is a signifi-
From the Departments of ‡Biochemistry and Molecular Biology and
§
Microbiology and Molecular Genetics, Michigan State University, cant model enzyme that has advanced our understanding of the
East Lansing, Michigan 48824 mechanisms of metallocenter assembly.
This enzyme catalyzes the hydrolysis of urea to ammonia and
Metalloenzymes often require elaborate metallocenter carbamic acid, which subsequently decomposes to another
assembly systems to create functional active sites. The medically molecule of ammonia and bicarbonate (7–9): H2N-C(O)-NH2
important dinuclear nickel enzyme urease provides an excellent ⫹ H2O 3 NH3 ⫹ H2N-COOH and H2N-COOH ⫹ H2O 3
model for studying metallocenter assembly. Nickel is inserted NH3 ⫹ H2CO3.
into the urease active site in a GTP-dependent process with the Regardless of the source of the enzyme, the overall protein
assistance of UreD/UreH, UreE, UreF, and UreG. These acces- structures exhibit extensive similarities. Most bacterial ureases
sory proteins orchestrate apoprotein activation by delivering
have three subunits in a (UreABC)3 configuration (Fig. 1A), as
the appropriate metal, facilitating protein conformational
exemplified by proteins from Klebsiella aerogenes (10, 11) and
changes, and possibly providing a requisite post-translational
Sporosarcina (formerly Bacillus) pasteurii (12). In Helicobacter
modification. The activation mechanism and roles of each
species, a fusion of two genes (corresponding to K. aerogenes
accessory protein in urease maturation are the subject of ongo-
ing studies, with the latest findings presented in this minireview.
ureA and ureB) results in only two subunits, yielding a
((UreAB)3)4 structure (Fig. 1B) (13). In fungi and plants, all ure-
ase domains are encoded by a single gene such as JBURE-I for
Metallocenters serve essential biological functions such as jack bean, with the trimeric protein forming back-to-back
transferring electrons, stabilizing biomolecules, binding sub- dimers, ((␣)3)2 (Fig. 1C) (14). The metallocenter structures of
strates, and catalyzing desirable reactions. Synthesis of these these proteins are identical (Fig. 1D), with two Ni2⫹ ions
sites must be tightly controlled because simple competition bridged by a carbamylated Lys residue and water; one metal
between metals may lead to misincorporation with loss of func- additionally coordinates two His residues and a terminal water
tion and because excess cytoplasmic concentrations of free molecule, and the second Ni2⫹ ion also coordinates two His
metal ions can have toxic cellular effects. In many cases, cells residues, one Asp residue, and water. Aspects of the enzyme
have evolved elaborate metallocenter assembly systems that mechanism remain controversial (7, 9, 15–17), but most pro-
sequester metal cofactors from the cellular milieu, thus offering posals suggest that the urea carbonyl oxygen displaces the ter-
protection from adventitious reactions while ensuring the fidel- minal water from the Ni2⫹ ion shown on the left (Fig. 1D), with
ity of metal insertion. In addition to maintaining metal home- another Ni2⫹-bound water molecule acting as a nucleophile to
ostasis, these assembly systems facilitate protein conforma- achieve catalysis. Although much is known about the enzyme
tional changes and active site modifications that are required active site, the mechanism of nickel insertion into the protein is
for full enzymatic activity. Several metalloproteins have been still poorly understood.
investigated as models to understand the mechanisms and
dynamics of active site assembly and the complex orchestra- Prototypical Urease Activation Pathway
tions of their metallocenter assembly systems. In this minire-
view, we discuss recent findings related to maturation of the The biosynthesis of the urease dinuclear nickel metallo-
nickel-containing enzyme urease. center generally requires the participation of several accessory
proteins (7, 9). For example, the canonical urease system of
Introduction to Ureases K. aerogenes (involving ureDABCEFG expression in Esche-
Urease is of great medical, agricultural, and historical signif- richia coli) utilizes UreD, UreE, UreF, and UreG to facilitate
icance. The gastric pathogen Helicobacter pylori uses urease for activation of the UreABC apoprotein (Table 1) (18, 19). Many
localized neutralization of pH, allowing it to flourish in the other ureolytic bacteria contain these four auxiliary genes
stomach (1), whereas the uropathogen Proteus mirabilis uses it flanking the enzyme subunit genes (7–9, 20), although the gene
to colonize and form stones in the urinary tract (2). In agricul- order often changes, and ureD is renamed ureH in Helicobacter
ture, urea is both a plant metabolite and a fertilizer degraded by spp. (21). Homologs of ureD/ureH, ureF, and ureG exist in ure-
plant ureases (3); however, urea is also metabolized by soil bac- ase-containing eukaryotes (3, 22, 23); however, the eukaryotic
accessory genes are not adjacent to the urease structural genes,
* This work was supported, in whole or in part, by National Institutes of Health and sequences related to ureE have not been identified. Below,
Grant DK045686 (to R. P. H.). This is the third article in the Thematic Mini- we describe the four prototypical urease accessory proteins,
review Series on Metals in Biology 2013.
1
To whom correspondence should be addressed. E-mail: hausinge@ their complexes with each other and with urease, and their
[Link]. proposed roles in urease activation.

13178 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288 • NUMBER 19 • MAY 10, 2013
This is an Open Access article under the CC BY license.
 MINIREVIEW: Biosynthesis of the Urease Metallocenter

FIGURE 1. Urease structures. A, three-subunit bacterial ureases (UreA, red; UreB, blue; UreC, green; with two more copies, yellow) assemble into a trimer of
trimers (Protein Data Bank code 1FWJ). B, two-subunit Helicobacter ureases (a fusion of the two small domains, blue; large subunit, green; with two more copies,
yellow) form a trimer of dimers, which interacts with three more trimers (gray surface view) to form a dodecamer of dimers (code 1E9Z). C, single-subunit urease
of fungi and plants (a fusion of all three domains, green; with two more copies, yellow) forms a trimer that stacks back-to-back with a second trimer (gray surface
view) (code 3LA4). D, dinuclear Ni2⫹ metallocenter of urease (Ni2⫹, magenta; solvent, red).

TABLE 1
Selected proteins needed for urease activation
K. aerogenesa H. pylori Plants Function
UreA Enzyme subunit
UreB UreAb Enzyme subunit
UreC UreBc Ureased Enzyme subunit
UreD UreH UreD Scaffold protein
UreE UreE —e Metallochaperone
UreF UreF UreF Potential fidelity enhancer
UreG UreG UreG GTPase
a
A similar set of proteins is present in many other bacteria, including S. pasteurii.
b
Equivalent to a fusion of UreA and UreB of K. aerogenes.
c
Equivalent to UreC of K. aerogenes.
d
Equivalent to a fusion of UreA, UreB, and UreC of K. aerogenes.
e
—, no UreE ortholog has been detected in plants.

UreD/UreH: Scaffold for Recruitment of Other Accessory Pro-

teins and Facilitator of Activation—Characterization of UreD/
UreH is most advanced for the K. aerogenes and H. pylori pro-
teins, which exhibit only 25% identity. Heterologous expression
of ureD or ureH in E. coli yields insoluble products (24, 25);
however, these problems were circumvented by different
approaches. For the K. aerogenes protein, a maltose-binding
protein (MBP)2 fusion variant of UreD is soluble and function-
ally replaces the native protein (26). In the case of H. pylori
FIGURE 2. Structure of UreH:UreF:UreG. Shown are two views of the (UreH:
ureH, solubilization is achieved by coexpression with ureF, UreF:UreG)2 complex from H. pylori (UreH, UreF, and UreG in shades of yellow,
which provides a UreH:UreF complex (25). The structure of gray, and magenta, respectively). A GDP molecule (cyan) is located in each UreG.
this complex (Protein Data Bank code 3SF5) and that of a UreH:
UreF:UreG complex3 reveal a novel ␤-helical fold for UreH,
with 17 ␤-strands and two ␣-helices (Fig. 2), that resembles
2
The abbreviation used is: MBP, maltose-binding protein. SufD (code 1VH4), a member of a scaffold protein complex that
3
K.-B. Wong, personal communication. functions in iron-sulfur cluster biosynthesis (27). Whereas no

MAY 10, 2013 • VOLUME 288 • NUMBER 19 JOURNAL OF BIOLOGICAL CHEMISTRY 13179
MINIREVIEW: Biosynthesis of the Urease Metallocenter

FIGURE 3. Model of K. aerogenes urease activation. The trimer-of-trimers urease apoprotein (UreA, red; UreB, blue; UreC, green) either sequentially binds
UreD (yellow), UreF (gray), and UreG (magenta) or binds the UreDFG complex (only one protomer of each protein is shown, but the isolated complex contains
two protomers of each). Formation of the active enzyme requires CO2 to carbamylate Lys-217 at the native active site, GTP binding to and hydrolysis by UreG,
and nickel delivery by dimeric UreE (cyan). It remains unclear whether the accessory proteins are released as a UreDFG unit or as individual proteins.

obvious metal-binding sites are apparent in UreH, 2.7 Ni2⫹ ions (code 3SF5) and the UreH:UreF:UreG complex,3 in which a
bind per UreD protomer (26). UreF dimer (with bound UreG when present) bridges two UreH
Several studies have demonstrated that UreD/UreH binds to protomers (25). These structures confirm the proposed inter-
urease. Enhanced expression of K. aerogenes ureD in the pres- actions between UreF and UreH that were based on yeast two-
ence of the cognate structural proteins results in a UreABC: hybrid and tandem affinity purification studies (31, 32, 39).
UreD complex that contains zero to three molecules of UreD/ UreH stabilizes the N-terminal helix of UreF and exposes a
(UreABC)3 according to two-dimensional (native and de- conserved Tyr residue at position 48. This residue and the
naturing) gel electrophoresis (24). Although a crystal structure highly conserved C terminus make up one face of the three-
is not available, a model of this species has been created (Fig. 3) dimensional UreF dimer (shown to be the UreG-binding site;
based on several lines of evidence (28). For example, small angle see below). The interface between UreF and UreH is poorly
x-ray scattering studies of this complex yielded data that are conserved, likely due to the low similarities within the UreF and
best modeled with UreD binding to the vertices of the triangu- UreD/UreH sequences.
lar urease (29). Chemical cross-linking of this species con- The assembly model shows UreF binding to UreABC:UreD
firmed that UreD binds to the UreB and UreC subunits (30). to form UreABC:UreD:UreF (Fig. 3), a complex that can be
MBP-UreD associates in vivo with UreABC (26) but not with directly isolated from cells expressing the corresponding genes
UreAC (i.e. urease missing UreB) (40). Similarly, yeast two-hy- (35). Alternatively, in vitro incubation of UreE-UreF with Ure-
brid studies of H. pylori proteins identified interactions ABC:UreD provides UreABC:UreD:UreE-UreF (37). Native gel
between UreH and UreA (31, 32). Of great functional signifi- electrophoresis of UreABC:UreD:UreF revealed multiple spe-
cance, in vitro studies with purified K. aerogenes components cies, and zero to three pairs of UreD:UreF are suggested to bind
showed that UreD enhances the extent of activation of urease per (UreABC)3. Small angle x-ray scattering experiments sug-
apoprotein. For example, whereas ⬃15% of the urease apopro- gested a close proximity between UreD and UreF, with both
tein generates functional sites when incubated with 100 ␮M accessory proteins binding in the vicinity of UreB (29). Chem-
NiCl2 and 100 mM bicarbonate (needed to carbamylate the Lys ical cross-linking results support this configuration and also
metal ligand) (33), ⬃30% is made functional when using the provide evidence for a conformational change in urease within
UreABC:UreD species (34). Along with results from additional the UreABC:UreD:UreF complex (30); specifically, UreB is pro-
studies (see below), these findings led to the current hypothet- posed to undergo a hinge-like motion that enhances access to
ical role of UreD/UreH as both a scaffold for recruiting other the nascent active site (40). Following in vitro activation of Ure-
accessory proteins and a direct facilitator of nickel insertion ABC:UreD:UreF, the urease-specific activity is similar to that
into the active site. obtained with UreABC:UreD; however, much lower concentra-
UreF: Checkpoint for Metallocenter Fidelity—UreF proteins tions of bicarbonate are required, and the process is more
also are best characterized for K. aerogenes and H. pylori. Het- resistant to inhibition by Ni2⫹ (35). UreF serves as the binding
erologous expression of K. aerogenes ureF yields insoluble site for the UreG GTPase within the UreABC:UreD:UreF com-
product (35); however, MBP-UreF (36) and UreE-UreF (37) plex (see below). A role as a GTPase-activating protein was
fusion proteins are soluble, with the latter protein shown to suggested for UreF (41), but the UreH:UreF:UreG structure
function in cellular activation of urease. The native form of (which shows UreF binding UreG opposite the GTP site) (Fig.
H. pylori UreF is soluble and exhibits an equilibrium between 2) and experimental evidence derived from mutagenesis and
the monomeric and dimeric species (25). The protein crystal- GTPase activity studies (42) argue against this proposal. For
lizes as an all-␣-helical dimer (Protein Data Bank code 3CXN), example, a urease activation complex containing a UreF variant
but it lacks the C-terminal 21 residues due to proteolysis, and its exhibited enhanced GTPase activity compared with the com-
N-terminal 24 residues are disordered (38). The intact struc- plex with wild-type accessory protein. UreF thus appears to
ture of UreF (Fig. 2) is available from the UreH:UreF complex gate the GTPase activity of UreG so as to promote efficient

13180 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288 • NUMBER 19 • MAY 10, 2013
 MINIREVIEW: Biosynthesis of the Urease Metallocenter

coupling of GTP hydrolysis and metallocenter biosynthesis, mass spectrometric studies.4 The structure of the analogous
thereby enhancing the fidelity of urease activation. UreH:UreF:UreG complex from H. pylori (Fig. 2)3 reveals two
UreG: GTPase for Urease Activation—In contrast to UreD/ UreG protomers binding to one face of the UreF dimer, with
UreH and K. aerogenes UreF, which are insoluble, UreG is sol- each UreG interacting with both UreF protomers and with each
uble and has been characterized from several sources (43– 47). UreH interacting with a single UreF. A GDP is bound opposite
The K. aerogenes protein is a monomer that binds 1 eq of Ni2⫹ of UreF within each UreG, confirming that the former protein is
or Zn2⫹ (Kd ⬃ 5 ␮M for either metal) (48). Mycobacterium not a GTPase-activating protein. A potential metal-binding site
tuberculosis (45) and S. pasteurii (44) possess dimeric UreG is deeply buried and bridges the two UreG molecules, with each
proteins, with the latter binding two Zn2⫹ ions (Kd ⬃ 40 ␮M) or, protomer providing His and Cys residues.
more weakly, larger numbers of Ni2⫹. The H. pylori protein UreE: A Nickel Metallochaperone—A hint that UreE might
dimerizes in the presence of Zn2⫹ (Kd ⬃ 0.3 ␮M, 1/dimer) but be involved in nickel delivery to urease is given in the sequence
not with Ni2⫹, which binds more weakly (Kd ⬃ 10 ␮M, 1.8/ of the K. aerogenes protein, which reveals 10 His residues in the
monomer) (46). X-ray absorption spectroscopy of the zinc- C-terminal 15 residues, thus resembling a His-tagged protein
bound protein revealed a trigonal bipyramidal site including (18). Indeed, the purified protein binds ⬃6 eq of Ni2⫹/dimer
two His and two Cys residues, likely positioned at the subunit (57). Not all UreE proteins contain this His-rich extension (58),
interface (49). The soybean (Glycine max) protein also exhibits and a truncation variant of the K. aerogenes protein lacking this
a monomer/dimer equilibrium, with the dimer stabilized by region (H144* UreE) retains its ability to facilitate urease acti-
Zn2⫹, but the binding thermodynamics are quite complex (47). vation (59). The crystal structure of copper-bound K. aerogenes
No crystal structure is available for free UreG, perhaps related H144* UreE (Protein Data Bank code 1GMW) reveals three
to its intrinsic disorder (50); however, UreG homology models metal-binding sites, including an interfacial site with His-96
(44 – 47) have been created by using HypB (required for nickel from each subunit and peripheral sites in each protomer involv-
insertion into [Ni-Fe] hydrogenase) (51, 52) from Methanocal- ing His-110 and His-112 (60). Equilibrium dialysis measure-
dococcus jannaschii (a dimeric GTPase with a dinuclear zinc ments confirm the binding of Ni2⫹ to multiple distinct sites in
the truncated protein (61). Mutagenesis studies demonstrated
site at the subunit interface (53)) as the template, and the struc-
that only the interfacial site is required for UreE function (62).
ture of the H. pylori UreH:UreF:UreG complex is known (Fig.
The full-length zinc-bound S. pasteurii UreE dimeric structure
2).3 The latter complex has two protomers of each peptide, with
(code 1EAR) exhibits close similarity to the K. aerogenes pro-
the two UreG molecules in contact as expected for a protein
tein, lacks the C-terminal His-rich region and the two periph-
able to dimerize. Although UreG is a GTPase, the free protein
eral sites, and binds a single Zn2⫹ ion (presumably substituting
exhibits slow (44, 45, 47) or no (43, 46, 48) GTPase activity.
for Ni2⫹) at the interfacial site (63). Structures of several forms
When present in urease activation complexes, GTPase activity
of H. pylori UreE are known, including the nickel-bound spe-
is observed (54), and substitution of a key residue in the GTP-
cies (codes 3L9Z and 3TJ8) (64, 65), in which the Ni2⫹ is coor-
binding P-loop motif of K. aerogenes or H. pylori UreG abol-
dinated at the interfacial site with an additional His residue
ishes the cell’s ability to make active urease (43, 55).
provided from the C terminus. These highly soluble proteins
A UreABC:UreD:UreF:UreG complex (Fig. 3) forms in are proposed to bind metal ions in the cytoplasm and specifi-
K. aerogenes cultures grown without Ni2⫹ (56). The complex cally deliver nickel to urease within the complex of other acces-
can also be accessed by mixing UreG with UreABC:UreD:UreF sory proteins.
(54). Furthermore, UreABC:UreD:UreE-UreF:UreG is made in UreE forms UreG:UreE and UreABC:UreD:UreF:UreG:UreE
cells producing the UreE-UreF fusion protein (38). Mutagene- complexes, with the latter species likely to serve as the ultimate
sis studies identified Asp-80 as a key UreG residue involved in urease activation machinery (Fig. 3). For the H. pylori compo-
this interaction (48) and defined several residues along one face nents, two UreG protomers bind the UreE dimer, with the
of UreF as the UreG-docking site (42). Using standard activa- interaction stabilized by Zn2⫹ but not Ni2⫹ (66). In contrast,
tion conditions, ⬃60% of the nascent active sites in UreABC: one UreG monomer from K. aerogenes binds to its cognate
UreD:UreF:UreG become active (54). Significantly, when using UreE dimer, with the interaction stabilized by either Zn2⫹ or
more physiological levels of bicarbonate and Ni2⫹, the resulting Ni2⫹ (48). Ligand identities in the metal-stabilized UreE:UreG
activity is decreased, but activation is greatly facilitated by GTP. complexes have not been reported. The transient formation of
UreG is active as a GTPase when present in this complex. a UreABC:UreD:UreF:UreG:UreE complex is suggested by the
UreD/UreH:UreF:UreG: Molecular Chaperone Complex for generation of fully active urease when UreABC:UreD:UreF:
Urease Activation—As an alternative to sequentially adding UreG is incubated with UreE, bicarbonate, Ni2⫹, and GTP (67).
each accessory protein to the urease apoenzyme, the hetero- In addition, UreABC:UreD:UreF:UreG:UreE can be directly
trimer may bind as a unit to urease (Fig. 3). A UreD:UreF:UreG isolated from cells that synthesize a G11P UreB variant (29) or a
complex forms in vivo when the corresponding K. aerogenes Strep tag II variant of UreG when the culture contains Ni2⫹
genes are expressed independently of the structural compo- (48).
nents (43); however, this species is poorly soluble and not well The current working model for urease activation with the
characterized. This solubility problem is overcome in the MBP- prototypical accessory proteins (Fig. 3) involves the binding of
UreD:UreF:UreG complex, and this species binds to urease but
not to urease lacking UreB (40). MBP-UreD:UreF:UreG con- 4
M. A. Farrugia, L. Han, Y. Zhong, J. L. Boer, B. J. Ruotolo, and R. P. Hausinger,
tains two copies of each protomer according to gel filtration and unpublished data.

MAY 10, 2013 • VOLUME 288 • NUMBER 19 JOURNAL OF BIOLOGICAL CHEMISTRY 13181
MINIREVIEW: Biosynthesis of the Urease Metallocenter

UreD, UreF, and UreG to the urease apoprotein, either sequen- biosynthesis of [Ni-Fe] hydrogenases, but they appear to serve a
tially or as a molecular chaperone unit, followed by interaction dual role here of still undefined function.
with the metallochaperone UreE. This activation complex car- Missing Accessory Proteins—Plants appear to lack homologs
ries out metallocenter assembly by steps that include Lys car- to ureE (3), and a large number of ureolytic microorganisms
bamylation, nickel incorporation, and GTP hydrolysis. No evi- lack one or more of the standard set of urease genes (9, 79). A
dence indicates that accessory proteins facilitate the interaction dramatic example of this situation exists in Bacillus subtilis,
of Lys with carbon dioxide at the nascent active site, but this where the genome reveals the presence of only the structural
possibility is not excluded, and it is reasonable to suspect that urease genes; nevertheless, the cells synthesize an active nickel
the nearby His residues assist in this reaction. The structure of urease, although with poor efficiency (80). In many other cases,
UreABC bound to UreD/UreH:UreF:UreG is not defined, but however, the sequenced microorganisms were not examined
two distinct models have been proposed. Fig. 3 shows a com- for urease activity.
putational model (28) derived from studies using the K. aero- Iron Urease—Helicobacter mustelae, a gastric pathogen of
genes components. In this case, each vertex of the urease trimer ferrets, contains two urease gene clusters: ureABIEFGH and
binds a single molecule of UreD, UreF, and UreG, requiring ureA2B2 (81). The former cluster, closely related to that found
dissociation of (UreD:UreF:UreG)2. By contrast, (UreH:UreF: in H. pylori, is induced by Ni2⫹ and encodes two structural
UreG)2 of H. pylori (Fig. 2) has been proposed to bind at 2-fold genes, a proton-gated urea channel (82), and the four standard
symmetry sites of its cognate urease dodecamer so that the maturation proteins. The latter cluster is inversely regulated by
accessory protein complex remains intact (25). Such an inter- Ni2⫹ and encodes only the two structural genes (83). Urease
action is precluded for the three-subunit bacterial ureases and activity is retained in ureB and ureB/ureG mutants, indicating
the single-subunit eukaryotic ureases (Fig. 1), suggesting possi- that ureA2B2 encodes an active urease, and its activation does
ble species-specific differences in the properties of this acces- not require the standard urease-specific GTPase. This finding
sory protein complex. It remains unclear how UreE binds to the was confirmed and extended by results showing that heterolo-
urease activation complex and how nickel is transferred from gous expression of ureA2B2 in E. coli generates active enzyme
UreE to the active site; one proposal suggests intermediate (84). Purified UreAB is a conventional nickel urease, whereas
binding sites on UreG and UreD (26). The function of GTP isolated UreA2B2 is an oxygen-labile iron-containing enzyme.
hydrolysis by UreG in this process remains poorly understood. The structure of oxidized UreA2B2, a dodecamer like that
The net outcome from the activation machinery is to transform shown in Fig. 1B, reveals a dinuclear active site that is remark-
urease apoprotein into the holoprotein, with dissociation of the ably similar to the metallocenter of conventional ureases (84).
accessory proteins for possible reuse. This finding is consistent with the high degree of similarity in
their sequences, e.g. UreA is 57% identical to UreA2, and UreB
Variations in Urease Activation Systems is 70% identical to UreB2. Two other strains of Helicobacter,
Additional Accessory Proteins—Additional genes have been Helicobacter felis and Helicobacter acinonychis, have similar
shown to facilitate urease activation in some microorganisms. arrangements of urease genes. The hosts of these pathogens,
For example, located just 5⬘ of the typical urease genes in Yers- both in the Felidae (cat) family, are carnivores like the ferret,
inia pseudotuberculosis is yntABCDE, which encodes an ATP- leading to speculation that these bacteria have evolved an iron
binding cassette-type metal transporter; deletion of these genes urease because of their association with meat diets that are rich
eliminates urease activity and reduces the Ni2⫹ uptake rate in iron and depleted in nickel (83). When the three UreA2B2
(68). Evidence for a similar Ni2⫹ transporter dedicated to ure- sequences are aligned and compared with the sequences of
ase in Actinobacillus pleuropneumoniae comes from recombi- nickel ureases, a prominent cluster of distinct residues are seen
nant expression of the urease gene cluster with or without its to encircle the channel into the active site (84). These results are
adjacent cbiKLMQ genes in E. coli (69). In the same manner, compatible with an interaction between UreA2B2 apoprotein
heterologous expression of the Bacillus sp. TB-90 urease gene and an iron delivery protein. One possibility is that activation of
cluster and its deletion mutants indicates a nickel-dependent iron urease makes use of a general iron delivery system that is
role for ureH (unrelated to ureH of H. pylori) in urease activa- used for maturation of the many iron proteins in the cell. The
tion; Bacillus ureH is suspected to encode a Ni2⫹ permease (70). oxidized state of UreA2B2, suggested to be a ␮-oxo-bridged
Many other microorganisms contain Ni2⫹ transporters and Fe(III)-O-Fe(III) species, probably forms naturally within the
Ni2⫹ permeases (with their levels often controlled by nickel-de- microaerophilic microorganism, and the cell likely has a mech-
pendent transcriptional regulators) that enhance urease activ- anism to regenerate the active diferrous species (85). The ability
ity by providing the essential metal ion (52, 71, 72), but gener- to form active iron urease in E. coli using only ureA2B2 implies
ally the corresponding genes are distant from the urease genes. that urease-specific accessory proteins are not required for Lys
For example, H. pylori uses NixA, AbcABCD, and the outer carbamylation.
membrane transporter HP1512 to take up Ni2⫹, and deletion of
these genes leads to reductions in urease activity (73–76). Of Future Directions
additional interest, hypA and hypB of this microorganism are As should be clear from the preceding discussion, many
required for urease activity, but the corresponding gene prod- questions remain to be answered about how the urease metal-
ucts are not involved in Ni2⫹ uptake (77). A direct competition locenter is synthesized. Significantly, these questions also often
is observed between HypA and UreG for binding UreE (78). apply to the biosynthesis of other types of metallocenters such
HypA and HypB are generally associated with metallocenter as that found in [Ni-Fe] hydrogenases, which utilize the HypB

13182 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 288 • NUMBER 19 • MAY 10, 2013
 MINIREVIEW: Biosynthesis of the Urease Metallocenter

GTPase along with SlyD and HypA metallochaperones for their 13. Ha, N.-C., Oh, S.-T., Sung, J. Y., Cha, K. A., Lee, M. H., and Oh, B.-H.
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Acknowledgments—We thank Prof. Kam-Bo Wong for coordinates of urease apoprotein and role of UreD as a chaperone required for nickel
the structure in Fig. 2 and Prof. Célia Carlini for coordinates of the metallocenter assembly. Proc. Natl. Acad. Sci. U.S.A. 91, 3233–3237
computational model in Fig. 3. 25. Fong, Y. H., Wong, H. C., Chuck, C. P., Chen, Y. W., Sun, H., and Wong,
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