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Critical Review
Common Themes and Variations in the Rhodanese Superfamily
Rita Cipollone1, Paolo Ascenzi1,2 and Paolo Visca1,2
1
2
Summary
The rhodanese homology domain is a ubiquitous fold found in
several phylogenetically related proteins encoded by eubacterial,
archeal, and eukaryotic genomes. Although rhodanese-like proteins
share evolutionary relationships, analysis of their sequences highlights that they are so heterogeneous to form the rhodanese
superfamily. The variability occurs at dierent levels including
sequence, active site loop length, presence of a critical catalytic Cys
residue, and domain arrangement. Even within the same genome,
multiple genes encode rhodanese-like proteins presenting with
variably arranged rhodanese domain(s): as single or tandem
domain(s), or combined with other protein domain(s). Given the
highly variable organization of the rhodanese domain(s) and the
context where it is found, here we review the structural organization
and function of the rhodanese-like proteins. The overview of the
most recent ndings about rhodanese allow us to depict a
superfamily of versatile proteins relying on persulde chemistry to
accomplish cellular functions spanning from resistance to environmental threats, such as cyanide, and key cellular reactions related to
sulfur metabolism and progression of cell cycle.
IUBMB Life, 59: 5159, 2007
Keywords
INTRODUCTION
In 1933, Lang reported that certain tissues of mammals
contained an enzyme able to catalyze the reaction between
thiosulfate and cyanide leading to the formation of thiocyanate and sulte (1). The responsible enzyme was designed
Received 19 December 2006; accepted 8 January 2007
Address correspondence to: Rita Cipollone, Department of
Biology, University Roma Tre, Viale Guglielmo Marconi 446, I-00146
Roma, Italy. Tel: 39 (0)6 5517 6346. Fax: 39 (0)6 5517 6321.
E-mail: mic.stud@uniroma3.it
ISSN 1521-6543 print/ISSN 1521-6551 online 2007 IUBMB
DOI: 10.1080/15216540701206859
rhodanese, from the German name for thiocyanate (rhodanid), with the ending ese indicating that this compound was
formed through the enzymatic reaction. According to the
ocial nomenclature rules, the proper name of the enzyme is
thiosulfate:cyanide sulfurtransferase (TST, EC 2.8.1.1), based
on the reaction catalyzed in vitro; however, the trivial name of
rhodanese is still commonly used.
The rst and best characterized rhodanese is the mitochondrial bovine (Bos taurus) TST, conventionally referred to as
Rhobov (2 4). Analysis of the Rhobov structure (293 amino
acids long), highlights that the protein consists of two equallysized globular domains, the inactive N-terminal and the
catalytic C-terminal rhodanese domain, showing identical a/b
topology. Each domain is about 120 amino acids long with a
low sequence similarity. Only the C-terminal domain carries
the six-amino acid active site loop hosting at the rst position
the Cys residue involved in the catalytic process. The counterpart of the Cys residue in the N-terminal domain is an Asp
residue which has no involvement in catalysis (5) (Fig. 1).
The peculiarity of rhodanese resides in two patterns of
amino acid sequences that can be recognized at the N-terminal
region ([F/Y]-X3-H-[L/I/V]-P-G-A-X2-[L/I/V/F]) and at the
C-terminal end of the protein ([A/V]-X2-[F/Y]-[D/E/A/P]-G[G/S/A]-[W/F]-X-E-[F/Y/W]) (Fig. 1). These sequences, called
rhodanese signatures, have a remarkable degree of conservation among rhodaneses and therefore are used to recognize
these proteins encoded by dierent genomes. Rhodaneses
are present in organisms belonging to dierent phyla, and
although these proteins can dier signicantly at the sequence
level (Fig. 1), the tandem domain three-dimensional structure
resembling that of Rhobov is highly constrained. For instance,
Pseudomonas aeruginosa rhodanese shows 79% and 22%
sequence identity with Azotobacter vinelandii rhodanese and
Rhobov respectively, but no striking dierences emerge by
comparing their three-dimensional structures (Fig. 2). Thus,
the highly conserved double domain architecture of Rhobov
has been assumed as the reference structure of the entire
family of evolutionary conserved tandem-domain TSTs, which
can be addressed as rhodaneses sensu stricto.
52
CIPOLLONE ET AL.
Figure 1. Partial sequence alignment of the rhodanese domains from representative members of the rhodanese superfamily:
Rhobov (P00586), E. coli SseA (P31142), A. vinelandii RhdA (P52197), P. aeruginosa RhdA (Q9HUK9), human Cdc25A
(P30304), E. coli GlpE (P0A6V5), W. succinogenes Sud (Q56748), and E. coli ThiI (P77718). Rhodanese signatures are
highlighted in grey. Residues forming the six-amino acid active-site loop of the catalytic domain are underlined. Amino acid
sequences were retrieved from Swiss-Prot/TrEMBL database (www.expasy.org/sprot/) and aligned with the program
CLUSTAL_W with default parameters (55). Identical residues, conserved and semi-conserved substitutions are indicated by
asterisks, colons, and periods, respectively. Note that translation of Rhobov reading frame diers from chemically determined amino acid sequence by an N-terminal Met and the three residue C-terminal extension Gly-Lys-Ala (not shown in the
gure).
Rhodanese catalytic process occurs via a double displacement (ping-pong) mechanism involving the stable formation of
a persulde-containing intermediate (ES) (6 8):
53
54
CIPOLLONE ET AL.
Table 1
Values of catalytic parameters for TST actiona
Substrate, Km (mM)
Enzyme
Cyanide
Thiosulfate
0.063
8.7
16
7.0
1.0
7.4
17
4.6
78
27
7.4
1.1
Vmax
600 mmol min71 mg71
1250 mmol min71 mg71
815 mmol min71 mg71
230 s71
0.0067 s71
4 s71
10.7 s71
Values of catalytic parameters were obtained between pH 8.5 and 9.0, and between 20.08C and 25.08C.
From 56.
c
From 57.
d
From 8.
e
From 15.
f
From 16.
g
Values of catalytic parameters were obtained at [cyanide] 60 mM. From 58.
b
55
56
CIPOLLONE ET AL.
Cyanide Detoxification
It has been understood for nearly a century that most of the
sub-lethal quantities of cyanide absorbed by either ingestion
or inhalation is detoxied by cyanide reaction with reactive
sulfur. In eukaryotes, rhodaneses and MSTs are supposed to
be involved in cyanide detoxication. As far as tandemdomain TSTs are concerned, the hypothesis is supported by
the high concentration of these enzymes in tissues and organs
exposed to cyanide (47). Although the distribution pattern of
tandem-domain TSTs in dierent tissues is species specic,
rhodanese levels are usually high in hepatocytes that are in
close proximity to the blood supply of the liver, in epithelial
cells surrounding the bronchioles (a major entry route for
gaseous cyanide) and in proximal tubule cells of the kidney
(serving to facilitate cyanide detoxication and elimination as
thiocyanate). In cattle, rhodanese is maximally expresses in the
stomach epithelium of rumen, omasum, and reticulum. In
these districts, where cyanide is released following ingestion of
plant cyanogenic glucosides, rhodanese activity is signicantly
higher than in liver, arguing for a role of rhodanese in cyanide
detoxication (48). Moreover, rhodanese has recently been
detected also in the mouse brain (49), and found to be
localized in areas which synthesize cyanide possibly acting as a
neuromodulator (50). Thus, a possible regulatory role of
57
CONCLUDING REMARKS
No unique reaction can be written at present to completely
dene the biological function(s) of the rhodanese superfamily
members. The three-dimensional structure of some of the
representative members has been obtained from both prokaryotic and eukaryotic species, highlighting the underlying
invariant features as well as the range of possible structures
for other superfamily members. On the other hand, there are
still numerous unanswered questions regarding the physiological functions of individual proteins and their mechanisms of
action. The increasing number of novel rhodanese-like
proteins with diverse and highly specialized functions supports
the notion that rhodaneses are involved in dierent cellular
processes, and coexist within the same organism since they
accomplish essential cell functions.
However, the large body of work carried out in vitro and
in vivo on dierent rhodanese superfamily members indicates
that the determinants responsible for functional selectivity of
the rhodanese domain reside in the features of the active-site
loop and in the domain arrangement (see Figs 1 and 2).
58
CIPOLLONE ET AL.
Both elements, in fact, concur to ensure the formation, interconversion, and transport of the highly reactive sulfane sulfur
species to the proper sulfur acceptor, whose involvement in
some cellular processes (e.g., biosynthesis of cofactors
and thionucleosides) and in environmental adaptation (e.g.,
cyanide detoxication) is preponderant.
Undoubtedly, much of research on rhodanese has been
focused on cyanide resistance, as rst suggested after the discovery of the Rhobov. However, increasing body of evidence
suggest that Nature has settled on protein persulde groups the
privileged sulfur source for several biochemical processes. The
identity of the primary sulfur source is still questionable,
but whatever it is, the unexpected occurrence of so many
rhodanese-like proteins suggests that cells use sulfane sulfur to
tune sulfur ux through pathways involving rhodanese
domains. The rhodanese domain seems to be the key enzymatic
withholder of the persulde group, which can be transferred
directly from the enzyme that incorporates it into a substrate,
or can be passed to another protein acceptor before nal
delivery.
Although Rhobov was discovered more than 70 years ago,
the list of open issues about the rhodanese-like protein superfamily is still long. The availability of large-scale genomic
databases holds out the prospect of discovering novel
rhodanese classes which can infer an even wider range of
functions than those presently known. Hopefully, the investigation of these new proteins will help to answer impelling
questions regarding the identication of possible sulfur
donor(s) and/or acceptor(s), and will oer fascinating possibilities for examining the not fully resolved regulatory role of
sulfur in cellular metabolism.
ACKNOWLEDGEMENTS
This work was supported by grants from Istituto Superiore per
la Prevenzione e la Sicurezza sul Lavoro (B1-39/DML/04),
Ministero dellUniversita` e della Ricerca (COFIN 2004), and
Ministero della Salute (Ricerca Corrente INMI Lazzaro
Spallanzani 2005) to P.A. and P.V.
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