American Journal of Molecular Biology, 2013, 3, 198-203 AJMB
http://dx.doi.org/10.4236/ajmb.2013.34026 Published Online October 2013 (http://www.scirp.org/journal/ajmb/)
Identification and preliminary characterization of novel
B3-type metallo-β-lactamases
Manfredi Miraula1,2, Conor S. Brunton1, Gerhard Schenk2, Nataša Mitić1
1Department of Chemistry, National University of Ireland—Maynooth, Maynooth, Co., Kildare, Ireland
2School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane, Australia
Email: schenk@uq.edu.au, natasa.mitic@nuim.ie
Received 30 May 2013; revised 23 June 2013; accepted 9 July 2013
Copyright © 2013 Manfredi Miraula et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
ABSTRACT
Antibiotic resistance has emerged as a major global
threat to human health. Among the strategies em-
ployed by pathogens to acquire resistance the use of
metallo-β-lactamases (MBLs), a family of dinuclear
meta lloenzyme s, is among the most potent. MBLs are
subdivided into three groups (i.e. B1, B2 and B3) with
most of the virulence factors belonging to the B1
group. The recent discovery of AIM-1, a B3-type
MBL, however, has illustrated the potential health
threat of this group of MBLs. Here, we employed a
bioinformatics approach to identify and characterize
novel B3-type MBLs from Novosphingobium pentaro-
mativorans and Simiduia agarivorans. These enzymes
may not yet pose a direct risk to human health, but
their structures and function may provide important
insight into the design and synthesis of a still elusive
universal MBL inhibitor.
Keywords: Antibiotic Resistance; β-Lactam Antibiotics;
Metallo-β-Lactamases; Sequence Homology;
Novosphingobium Pentaromativorans; Simiduia
Agarivorans
1. INTRODUCTION
The introduction of β-lactam antibiotics (Figure 1) in the
1940s has been considered as a breakthrough, if not the
most significant breakthrough in the history of medicine.
However, only a few years after introducing penicillin
resistance was observed in Staphylococcus aureus and
meanwhile a large and increasing number of pathogens
have acquired resistance to the most commonly used
antibiotics [1,2], triggering some experts to liken antibi-
otic resistance to terrorism in terms of its global impact.
http://www.bbc.co.uk/news/health-21737844
One of the most frightening forms of antibiotic resis-
(a) (b)
(c) (d)
Figure 1. Representatives for the most common β-
lactam antibiotic families. (a) Penicillin; (b) carba-
penem; (c) cephalosporin; and (d) monobactam.
tance occurs through the action of metallo-β-lactamases
(MBLs), and enzymes are capable of breaking down
most widely used β-lactam antibiotics [1-5]. These
Zn2+-dependent enzymes are not susceptible to any
known drugs [5]. MBLs have been divided into three
subgroups (i.e. B1, B2 and B3) based on their sequences
and metal requirements [1,4,5]. Despite only low levels
of sequence similarity these subgroups show homology
at the level of structure; they all exhibit α/β/β/α folded
with two metal binding sites located between the two
central β sheets (Figure 2). The key active site residues
responsible for binding the metal ions in the three sub-
groups show variations that result in differences in metal
requirements and catalytic mechanisms [5]. Most of the
known MBL virulence factors belong to subgroup B1
and include BCII from Bacillus cereus [6], CcrA from
Bacteroides fragilis [7], as well as IMP-1 and SPM-1,
both initially identified in Pseudomonas aeruginosa [8,9].
The recently identified NDM-1 (“New Delhi Imipene-
mase-1”) has acquired particular notoriety as it induces
resistance to virtually all known β-lactam antibiotics [10].
Subgroup B2 enzymes share only ~11% sequence ho-
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M. Miraula et al. / American Journal of Molecular Biology 3 (2013) 198-203 199
Figure 2. Representative MBL structures. Left: B1-type
CcrA from B. fragilis; center: B2-type CphA from A.
hydrophila; right: B3-type L1 from S. maltophilia (only one
subunit shown). The protein main chains are color-ramped
from the N-(blue) to the C-terminus (red). Zinc ions are
rendere d a s gray spher e s.
mology with B1-type MBLs, hydrolyze exclusively car-
bapenems (e.g. meropenem and imipenem) and require
only one metal ion for catalysis [5,11]. Representative
B2-type MBLs are CphA from Aeromonas hydrophila
[12], ImiS from A. veronii [13] and Sfh-1 from Serratia
fonticola [14]. Subgroup B3 is closer related to B1-type
MBLs rather than B2-type MBLs, requiring two bound
metal ions for catalysis (Figure 2). The most studied
representative is the tetrameric L1 from Stenotropho-
monas maltophilia [15]. Other members include FEZ-1
from Legionella gormanii [16], GOB-1 from Eliza-
bethkingia meningoseptica (of which to date 18 variants
have been reported) [17] and SMB-1 from Serratia
marcesens [18], the most recently identified MBL, which
has a higher hydrolytic activity against a wide range of
β-lactams than other B3-type MBLs [18]. Of clinical re-
levance is in particular the enzyme AIM-1 from Pseu-
domonas aeruginosa, which has been identified re-
cently in multi-drug resistant isolating in a hospital in
Adelaide, Australia (hence, the nomenclature of “Ade-
laide Imipenemase-1”—AIM-1) [19].
Despite rather modest homology across their full
length amino acid sequences MBLs share considerable
similarities in their active site structures [5]. All MBLs
provide binding sites for two closely spaced metal ions
that are invariably Zn2+ in vivo (i.e. Zn1 and Zn2). Some
variations are observed among the ligands that bind to
the zinc ions in the active site. B1- and B3-type MBLs
have three histidines (His) on the Zn1 site. In the B2-type
MBLs one of these histidines (His116) is replaced by an
asparagine (Asn). For B1- and B2-type MBLs the second
metal binding site is conserved with an aspartate (Asp), a
histidine and a cysteine (Cys) coord inating the metal ion.
In the B3 subclass the cysteine is replaced by another
histidine (Figure 3). A standard numbering scheme for
residues in MBLs has been developed based on sequence
alignments for B1, B2 and B3 MBLs, and is used
throughout this work to simplify comparisons between
different MBLs [4,20,21].
In light of the rapid spread of antibiotic resistance and
Figure 3. Active site st ruct ure s of representative MBLs. Left:
B1-type BcII from B. cereus; center: B2-type CphA from A.
hydrophila; right: B3-type L1 from S. maltophilia. Zinc ions
are rendered as grey spheres, and water molecules are shown
as red spheres. Coordination bonds are shown as solid lines.
the increasing emergence of novel virulence factors (ex-
emplified by NDM-1 and AIM-1). It is essential to iden-
tify novel putative MBLs, ideally before they become a
threat to health care. Furthermore, novel MBLs may also
provide essential insight into the structure and/or func-
tional aspects relevant to the design and synthesis of
universal inhibitors that may be clinically useful to com-
bat antibiotic resistance. Here, our focus was on the B3
subgroup because they appear to be less known than their
counterparts in the other two subgroups, but they are a
danger to be reckoned as the recent emergence of AIM-1
and SMB-1 illustrated.
2. METHODS
2.1. Selection of the Query Sequence and Protein
Database Search Using BLAST
The B3-type AIM-1 from P. aeruginosa was used as
query sequence for the protein database search. The pro-
tein sequence of AIM-1 was obtained from the Protein
Data Bank (PDB; accession code: 4AWY), and the Basic
Local Alignment Search T ool (BLAST;
http://blast.ncbi.n lm.nih .gov/Blast.cgi) was used to iden-
tify homologues. The two most promising candidates,
from Novosphingobium pentaromativorans and Simiduia
agarivorans were selected for multiple sequence com-
parisons including known B3-type MBLs.
2.2. Multiple Sequence Alignments
Multiple sequence alignments including the novel B3-
type MBLs and well known members of this group of
enzymes (i.e. AIM-1 [19], L1 [15] and SMB-1 [18])
were carried out using ClustalW2, a multiple sequence
alignment program, available via The European Bioin-
formatics Institute website.
http://www.eb i.ac.uk /Tools/msa/clustalw2/
3. RESULTS AND DISCUSSION
3.1. Protein Database Search, Nomenclature and
Classification of Novel MBLs
Using the BLAST search engine with AIM-1 as the
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M. Miraula et al. / American Journal of Molecular Biology 3 (2013) 198-203
200
query two promising candidate sequences were retrieved,
i.e. MBL-like sequences from N. pentaromativorans (ac-
cession code: ZP_09194167.1) and S. agarivorans (ac-
cession code: YP_006917856.1). These microorganisms
are both Gram-negative. N. pentaromativorans is a poly-
cyclic aromatic hydrocarbon-degrading bacterium [22],
while S. agarivorans is a heterotrophic marine bacterium
[23]. None of these organisms poses a direct current
threat to human health, however, the observation that
they harbor a potential MBL may not only foreshadow a
future problem, they also could represent a genetic pool
from which horizontal genetic transfer can occur and
they could lead to the design and development of univer-
sally applicable inhib itors against MBLs. It is thus essen-
tial to investigate the prop erties of these novel MBL-like
proteins and compare them with those of well-known
MBLs (e.g. AIM-1, L1 or SMB-1).
As a step towards their characterization, the sequences
of the N. pentaromativorans and S. agarivorans MBL-
like proteins were compared with those of well-charac-
terized MBLs from the B3 subgroup, i.e. AIM-1 [19], L1
[15] and SMB-1 [18]. The results from pairwise se-
quence comparisons are summarized in Table 1. Not sur-
prisingly, the two sequences are most closely related to
AIM-1. The MBL-like protein from N. pentaromativo-
rans shares 53% sequence identity and 65% homology
(including conserved amino acid substitutions) with
AIM-1. The sequence identity/homology to the other two
well characterized B3-type MBLs, L1 and SMB-1, is
smaller (38%/54% and 41%/58%, respectively) but still
strongly indicative that th e N. pentaromativorans protein
is indeed a B3-type MBL. In comparison, pairwise se-
quence comparisons with the B1-type NDM-1 and B2-
type CphA indicate only 26%/39% and 23%/42%, re-
spectively. A similar conclusion can be drawn for the
MBL-like sequence from S. agarivorans. Its similar-
ity/homology with AIM-1 (47%/64 %) is less than that of
the N. pentaromativorans MBL, but it appears to be
closer related to SMB-1 instead (Table 1). The two
MBL-like proteins share 47%/63% identity/homology in
a direct pairwise sequence comparison. In summary,
these pairwise comparisons strongly support the classifi-
cation of these novel proteins sequences from N. pen-
taromativorans and S. agarivorans as MBLs from the B3
subgroup. In accordance with frequently applied no-
menclature procedures (e.g. “Adelaide Imipenemase-1”
or AIM-1) the MBL-like sequences from N. pentaroma-
tivorans and S. agarivorans are labeled here “Maynooth
Imipenemase-1” (MIM-1) and “Maynooth Imipenemase-
2” (MIM-2), respectively.
3.2. Important Amino Acid Residues
The above discussion demonstrated that the MBL-like
sequences in the genomes of N. pentaromativorans and
Table 1. Pairwise sequence comparisons between MBL-like
sequences from N. pentaromativorans (MIM-1; see text for
details) and S. agarivorans (MIM-2) and selected MBLs from
the B1 (NDM-1), B2 (CphA) and B3 (AIM-1, L1, SMB-1)
subgroups.
MBL Identity (%) Homology (%)
MIM-1AIM-1 53 65
L1 38 54
SMB-1 41 58
NDM1 26 39
CphA 23 42
MIM-2MIM-1 47 63
AIM-1 47 64
L1 33 51
SMB-1 43 63
NDM-1 37 59
CphA 24 44
S. agarivorans are likely members of the B3 subgroup in
the MBL family. However, in order to substantiate this
interpretation it is essential to ascertain that amino acid
residues that are essential for MBL function are con-
served in the amino acid sequences of MIM-1 and
MIM-2. The most relevant amino acid residues with re-
spect to MBL function are those that form the metal
binding site. Other residues in the proximity of the metal
ion binding sites may be important for substrate or in-
hibitor binding or bo th. In order to evaluate the function -
ality of MIM-1 and MIM-2 a multiple sequence align-
ment between these sequences and the structurally
well-characterized AIM-1 [19], L1 [15] and SMB-1 [18]
was carried out (Figure 4).
Importantly, the six amino acids that form the metal
ion binding site are also invariant in both MIMs (i.e.
His116, His118 and His196 in the Zn1 site and Asp120,
His121 and His263 in the Zn2 site; see also Figure 3).
Other amino acid side chains that were identified as im-
portant in MBL function are well conserved, including
those in positions 221 (Ser) and 223 (Ser/Thr) that line
the pocket where the β-lactam substrate may bind [5].
Tyr228, a residue that aids the polarization of the β-lac-
tam carbonyl oxygen as a means to increase the suscep-
tibility of the carbonyl bond for a nucleophilic attack by
the “bridging” hydroxide is conserved in all MBLs com-
pared here except AIM-1 (Figure 4). Conserved is also
Trp39, another residue that has been shown to play an
important role in substrate binding [24]. Furthermore, in
AIM-1 and to a large extent SMB-1 (but not L1) the
structure of the enzyme is stabilized by the presence of
three disulfide bridges (i.e. the pairs Cys32-Cys66,
Cys208-Cys213 and Cys256-Cys290 [18,19]). These six
ysteine residues are conserved in both MIM-1 and c
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M. Miraula et al. / American Journal of Molecular Biology 3 (2013) 198-203
Copyright © 2013 SciRes.
201
39
6
6
AIM-1 DDAGWNDPAMPLKVYGNTWYVGTCGISALLVTSDAGHILVDAATPQAGPQILANIRALGFR
L1 VDASWLQPMAPLQIADHTWQIGTEDLTALLVQTPDGAVLLDGGMPQMASHLLDNMKARGVT
SMB-1 QDRDWSSPQQPFTIYGNTHYVGTGGISAVLLSSPQGHILVDGTTEKGAQVVAANIRAMGFK
MIM-1 GREGWSHPAPPAHIYGNTWYVGTCGIASILVTSDDGHVLIDSGPADAAPLVLANIRKLGFD
MIM-2 DWDAWDKPGPPFRVLGNTYYVGTCGIAAILITGDAGHVLIDSGTDRGAVIVRDNIARLGFS
* * * : .* :** ::::*: * :*:*. . : *: *.
116 – 121 152 164
AIM-1 PEDVRAIVFSHEHFDHAGSLAELQKATGAPVYARAPAIDTLKRGLPDRTDPQFEVAEPVA
L1 PRDLRLILLSHAHADHAGPVAELKRRTGAKVAANAESAVLLARG--GSDDLHFGDGITYP
SMB-1 LSDVKYILSTHSHEDHAGGISAMQKLTGATVLAGAANVDTLRTGVSPKSDPQFGSLSNFP
MIM-1 PADVRWILTSHEHHDHAGSIAELQKATGAQIAAVASARQVLESGKPSADDPQSGLIEGFP
MIM-2 LSDVKILLHSHEHIDHVGGMASLQSLSGATLYASPAAAAVMRNGTAGEDDPQAGALASFP
*:: :: :* * **.* :: :: :** : * : * * :
196 208 213 221/223
AIM-1 PVANIVTLADDGVVSVGPLALTAVASPGHTPGGTSWTWRSCEG-DDCRQMVYADSLTAIS
L1 PANADRIVMDGEVITVGGIVFTAHFMAGHTPGSTAWTWTDTRN-GKPVRIAYADSLS---
SMB-1 GSAKVRAVADGELVKLGPLAVKAHATPGHTEGGITWTWQSCEQ-GKCKDVVFADSLTAVS
MIM-1 PVHVARVLVDGDSVTLGRLALTVRETPAHSPGSASWTWQACDEAFTCRMIAYADSATTIS
MIM-2 VARVGGLVNDGDQIALGNLRLTAYATPGHTPGALSWQWRACEE-DRCTTLVYADSLSPVS
: * : :* : ... .*: *. :* * :.:*** :
228 263
AIM-1 DDVFRYSDDAAHPGYLAAFRNTLARVAALDCDILVTPHP
L1 APGYQLQGNPRYPHLIEDYRRSFATVRALPCDVLLTPHP
SMB-1 ADSYRFSD---HPEVVASLRGSFEAVEKLSCDIAIAAHP
MIM-1 ADDYRFSD---HPDRIARIRTGLSRIAQLPCDILVTPHP
MIM-2 AEGYRFNA---HPEYLQAYRLGLATLADLECDLLLTPHP
:: . :* : * : : * **: :: **
Figure 4. Multiple sequence alignment between known (AIM-1, L1 and SMB-1) and
putative (MIM-1 and MIM-2) B3-type MBLs. Amino acid side chains involved in
Zn2+ binding are shown in yellow. Other relevant residues are also indicated in color
and described in the text.
It is now essential to characterize the properties of
these novel B3-type MBLs to determine which of these
variations are functionally relevant and in particular
which of those may affect the mode and magnitude of
inhibition by known inhibitors (such as MCR). Ulti-
mately, it is anticipated that the functional and structural
comparison of a multitude of related MBLs will identify
residues that are suitable targets to develop universally
applicable inhibitors that may be resistant to frequent
mutational changes characteristic of this family of en-
zymes.
MIM-2, suggesting that these enzymes possess a similar
overall fold as AIM-1 and SMB-1.
Some observed sequence variations may, however, de-
serve mentioning here as they may be significant for dif-
ferences in substrate preference, inhibitor binding and/or
catalysis. The region between residues 152 and 164
forms a flexible loop that may clamp down on the bound
substrate and thus assist catalysis [5]. In this loop, the
degree of sequence conservation is low (Figure 4) which
may indicate variations in substrate selection, catalytic
efficiency and possibly also interactions with potential
enzyme inhibitors.
While there is currently no clinically useful MBL in-
hibitor available, mercaptoacetates (MCRs) are known to
be potent in vitro inhibitors of some MBLs [25]. A recent
crystallograph ic study with SMB-1 has shown th at MCR
interacts with active site residues Ser221 and Thr223 and
has a Ki = 9.4 ± 0.4 µM (Figure 4) [18]. MIM-1 and
AIM-1 have a sequence identical to that of SMB-1 in this
so-called “MCR binding” region, whereas L1 and MIM-
2 have a Ser instead of Thr. Although this represents a
conserved substitution, it may nonetheless be of signifi-
cance as the different sizes of these side chains may af-
fect the modes of substrate/inhibitor binding. Of particu-
lar interest may also be the residue in position 162, oc-
cupied by the bulky and nonpolar Phe residue in AIM-1,
L1 and SMB-1, but by the small and polar Ser in MIM-1
and the small and nonpolar Ala in MIM-2. This residue
lies within the flexible loop mentioned in the previous
paragraph and may thus play an essential role in sub-
strate and inhibitor binding.
3.3. Conclusion
The main finding of this study is the identification of two
novel MBLs from N. pentaromativorans (MIM-1) and S.
agarivorans (MIM-2) that belong to the B3 sub-group of
this family of enzymes. Both proteins containing the amino
acid ligands necessary to bind two zinc ions in their ac-
tive sites and various residues in the vicinity of the cata-
lytic center are invariant or highly conserved, indicating
that MIM-1 and MIM-2 should be efficient catalysts for
the hydrolysis of β-lactam antibiotics. While MIM-1 and
MIM-2 are not expected to represent an immediate threat
to human health, they may harbor information that is
crucial for 1) our understanding of the reaction mecha-
nism(s) MBLs may employ; and 2) the development of
universal MBL inhibitors that are resistant to frequent
mutational variations observed among members of this
family of enzymes. This study is an initial step towards
the characterization of these novel MBLs. Steps toward
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M. Miraula et al. / American Journal of Molecular Biology 3 (2013) 198-203
202
their recombinant expression and purification, as well as
their catalytic and structural characterization are cur-
rently in progress. Importantly, we and others have de-
veloped an arsenal of in vitro inhibitors, mainly against
B1-type MBLs [26], and these compounds will be tested
for their effects against MIM-1 and MIM-2. It is hoped
that in due course a universal inhibitor may emerge from
these and related studies to combat successfully the
threat of antibiotic resistance which poses an immediate
threat to global health.
4. ACKNOWLEDGEMENTS
N. M. thanks the Science Foundation Ireland (SFI) for financial support
in form of a President of Ireland Young Researcher Award (PIYRA)
and G. S. acknowledges the award of a Future Fellowship from the
Australian Research Council (FT120100694) and is grateful to the
National Health and Medical Research Council of Australia for fund-
ing.
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