Download J. Mol. Evol., 54

J Mol Evol (2002) 54:411–415
DOI: 10.1007/s00239-001-0035-8
© Springer-Verlag New York Inc. 2002
Letters to the Editor
Archaeal Signal Peptidases from the Genus Thermoplasma: Structural and
Mechanistic Hybrids of the Bacterial and Eukaryal Enzymes
Jerry Eichler
Department of Life Sciences, Ben Gurion University, P.O. Box 653, Beersheva 84105, Israel
Received: 1 June 2001 / Accepted: 24 September 2001
Type I signal peptidases (EC 3.4.21.89) are integral
membrane proteins responsible for the removal of signal
sequences from preproteins following protein translocation across a variety of membranes (Dalbey et al. 1997).
Insight into the behavior of signal peptidase has come
from the recent description of the crystal structure of the
ectodomain of the Escherichia coli enzyme (Paetzel et al.
1998). In E. coli, signal peptidase is composed of two
structural components, referred to as domain I and domain II. Corresponding to the “catalytic core” of the
enzyme, domain I encompasses boxes A–E, five regions
of significant sequence homology preserved throughout
evolution (Fig. 1). Box B contains the nucleophilic Ser90
residue (E. coli numbering) and the conserved Met91
residue, while box D contains the proposed general base
Lys145 and the conserved Arg146. Ser90 and Lys145 are
believed to form the catalytic dyad responsible for enzyme activity (Paetzl et al. 2000). Domain II, corresponding to the stretch of amino acid residues between
box D and box E (residues 154–271), folds as a large
␤-sheet. Sequence alignment studies of signal peptidases
from various Gram-negative bacteria (Salmonella typhimurium, Pseudomonas fluorescens, Haemophilus influenzae, Bradyrhizobium japonicum, Borrelia burgdorferi,
Phormidum laminosum, Azotobacter vinelandii, Rickett-
Correspondence to: Jerry Eichler; email: [email protected]
sia prowazekii, Thermotoga maritima, and Rhodobacter
capsulatus) reveal the presence of domain II sequences
containing between 39 and 152 residues. Examination of
the corresponding regions of Gram-positive bacteria (Bacillus subtilis, Staphylococcus aureus, Streptococcus
pneumoniae, Aquifex aeolicus, Myobacterium leprae,
and Myobacterium tuberculosis) reveals the presence of
smaller domain II regions. Despite their omnipresence,
the role of domain II in bacterial signal peptidase activity
is currently not understood.
In contrast to their bacterial homologues, eukaryal
signal peptidases do not contain domain II regions. Eukaryal signal peptidases also differ from their bacterial
counterparts in that the eukaryal enzyme functions as
part of a multisubunit signal peptidase complex (SPC). In
Saccharomyces cerevisiae, this complex is comprised of
the Sec11, Spc1, Spc2, and Spc3 proteins (YaDeau et al.
1991). Both Sec11 and Spc3 are required for signal sequence cleavage and indeed can be coimmunoprecipitated (Meyer and Hartmann 1997; Fang et al. 1997).
While Sec11 contains the evolutionarily conserved boxes
A–E sequences (Fig. 1), it has been proposed that Spc3
provides the lysine residue of the catalytic serine/lysine
dyad conserved in bacteria (K145) yet absent in eukarya,
where it is replaced by a conserved histidine residue.
Site-directed mutagenesis of conserved and nonconserved lysine residues as well as of other selected Spc3
amino acids, however, failed to affect enzyme activity
(Van Valkenburgh et al. 1999). It appears, therefore, that
412
Fig. 1. The evolutionarily conserved signal peptidase boxes B–E.
The amino acid sequences of signal peptidase boxes B–E of a Gramnegative bacterium (E.coli), Gram-positive bacteria (B.sub, M.lep,
M.tub, S.aur), Archaea (A.ful, A.per, H.NRC-1, M.jan, M.th, P.aby,
P.hor, S.sol, T.aci, T.vol), and a representative eukaryote (Sec11) are
shown. The positions of Ser90 (E. coli numbering) and Lys/His145 are
shown in boldface. A.ful, Archaeoglobus fulgidus; A.per, Aeropyrum
pernix; B.sub, Bacillus subtilis; E.coli, Escherichia coli; H.NRC-1,
Halobacterium species NRC-1; M.jan, Methanococcus jannaschii;
M.th, Methanococcus thermoautotrophicum; M.lep, Myobacterium
leprae; M.tub, Myobacterium tuberculosis; P.aby, Pyrococcus abysii;
P.hor, Pyrococcus horikoshii; S.aur, Staphylococcus aureus; Sec11, S.
cereivisiae Sec11; S.sol, Sulfolobus sulfataricus; T.aci, Thermoplasma
acidophilum; T.vol, Thermoplasma volcanium. In those cases where
more than one archaeal signal peptidase exists, the name or accession
number of the gene is given.
eukaryal signal peptidases rely on a different catalytic
mechanism than do their bacterial counterparts. This
catalytic mechanism, as well as the contribution of Spc3
to that activity, remains unclear.
Recent studies on the signal peptidases of Bacillus
subtilis may shed light on the relationship between bacterial and eukaryal signal peptidases. B. subtilis contains
five chromosomal genes and two plasmid genes that encode for seven signal peptidases (Tjalsma et al. 1998). Of
these, six genes encode for bacterial-like signal peptidases, containing the catalytic serine/lysine dyad. In contrast, the chromosomally encoded signal peptidase SipW
contains a histidine residue in place of the bacterially
conserved lysine residue of the catalytic dyad and lacks
the domain II region found in bacterial signal peptidases,
including the other six B. subtilis signal peptidases. It
appears, therefore, that SipW assumes a eukaryal-like
structure and invokes a eukaryal-like catalytic mechanism. It is not known whether SipW enlists a bacterial
version of Spc3 for its function. To date, B. subtilis rep-
resents the sole example of a bacterium simultaneously
containing both bacterial and eukaryal signal peptidases,
although a partial sequence of a SipW-like protein has
been detected in Clostridium perfringens (Tjalsma et al.
1998). It is unclear whether the signal peptidases of B.
subtilis evolved from a common precursor, reflect convergent evolution of bacterial and eukaryal enzymes, or
result from a horizontal gene transfer event.
The relation between bacterial and eukaryal signal
peptidases may, however, be more complicated. Amino
acid alignments have revealed the existence of conservation between the E. coli domain II and the lumenal
domain of S. cerevisiae Spc3 (Fang et al. 1997; Van
Valkenburgh et al. 1999). Such studies raise the possibility that eukaryal Sec11 and Spc3 may functionally
correspond to the catalytic and noncatalytic domains I
and II of the bacterial signal peptidase, respectively.
Therefore, to investigate further the relation between the
bacterial domain II and the Spc3 lumenal domain, as well
as the origin of signal peptidases, a broader comparison
413
Fig. 2. Domain II regions of archaeal signal peptidases. The amino
acid sequences lying between the conserved signal peptidases boxes D
and E, referred to as domain II, were compared in a variety of archaeal
strains. A.ful, Archaeoglobus fulgidus; A.per, Aeropyrum pernix;
H.NRC-1, Halobacterium species NRC-1; M.jan, Methanococcus jan-
naschii; M.th, Methanococcus thermoautotrophicum; P.aby, Pyrococcus abysii; P.hor, Pyrococcus horikoshii; S.sol, Sulfolobus sulfataricus; T.aci, Thermoplasma acidophilum; T.vol, Thermoplasma volcanium. In those cases where more than one signal peptidase exists, the
accession number of the gene is given.
of signal peptidases along evolutionary lines was performed.
In many instances possessing a mosaic of bacterial
and eukaryal traits, archaea offer unique insight into a
variety of biological processes and, in certain cases,
serve to connect seemingly disparate processes in the
other two domains of life. While containing the evolutionarily conserved signal peptidase boxes A–E, archaeal
signal peptidases have a box D lacking the conserved
lysine of the bacterial serine/lysine catalytic dyad and,
like eukarya, have replaced the bacterially conserved lysine (E. coli K145) with a histidine residue (Fig. 1).
Thus, in terms of catalysis, archaeal signal peptidases are
seemingly more reminiscent of their eukaryal than of
their bacterial counterparts. Further support for the similarity of archaeal and eukaryal enzymes comes with the
observation that the signal peptidases of Methanococcus
jannaschii, Methanobacterium thermoautotrophicum,
Archaeoglobus fulgidus, Sulfolobus solfataricus, Pyrococcus abyssi, and Pyrococcus horikoshii lack significant domain II regions (Fig. 2). Not all archaea, however,
lack a domain II region. The recently released genomic
sequences of Thermoplasma acidophilum (Ruepp et al.
2000) and Thermoplasma volcanium (Kawashima et al.
2000) reveal the presence of genes encoding for signal
peptidases containing domain II stretches of 51 residues
each.
The archaeal domain II regions are not very similar to
their bacterial counterparts. Amino acid alignments show
that the Thermoplasma domain II regions bear little homology to Gram-positive domain II regions of similar
lengths (6% identity, 18% similarity, and 22% conserved
changes) (Fig. 3A). Moreover, like the Gram-positive
domain II regions, the Thermoplasma domain II regions
do not show substantial sequence similarity to the much
longer E. coli domain II region (16% identity, 16% simi-
larity) (Fig. 3B). Furthermore, alignment of the Thermoplasma [and Gram-positive (not shown)] domain II sequences with the lumenal domain of S. cerevisiae Spc3
reveals only a low degree of similarity. Still, it remains
possible, as proposed in the case of the E. coli enzyme,
that the Thermoplasma domain II may fulfill the role
played by Spc3 in the eukaryal SPC. Indeed, BLAST
searches of the Thermoplasma genomes failed to reveal
the existence of homologues to Spc3 or other components of the eukaryal SPC, suggesting that, like the bacterial enzyme, archaeal signal peptidases do not function
as a multisubunit complex.
The finding that archaea such as Thermoplasma
acidophilum and Thermoplasma volcanium [and, to a
lesser extent, Halobacterium sp. NRC-1 (Ng et al. 2000)]
contain signal peptidases that possess a bacteria-like domain II region, that apparently rely on a eukaryal catalytic mechanism, and, as in bacteria, that function as a
single polypeptide suggests that archaeal signal peptidases may represent an evolutionary intermediate between present-day eukaryal and bacterial signal peptidases. Moreover, the observation that signal peptidases
of some archaea, such as Thermoplasma species, contain
a bacteria-like domain II region whereas others do not
could suggest that ancient archaeal signal peptidases
contained the domain II region and that, during later
diversification of archaea, this domain was lost. Further
examination of archaeal signal peptidases could serve to
clarify this point, to direct future studies aimed at discerning the roles of domain II and Spc3 in signal peptidase function, and to describe the catalytic mechanism of
eukaryal signal peptidases.
Acknowledgments. This work was supported by the Israel Ministry
of Absorption and the Israel Science Foundation founded by the Israel
414
Fig. 3. Alignment of Thermoplasma signal peptidase domain II regions with bacterial domain II regions and the lumenal domain of Spc3.
Domain II regions of Thermoplasma acidophilum (T.aci) and Thermoplasma volcanium (T.vol) signal peptidases were aligned with (A) domain II regions of Gram-positive bacterium signal peptidases (B.sub,
Bacillus subtilis; M.lep, Myobacterium leprae; M.tub, Myobacterium
tuberculosis; S.aur, Staphylococcus aureus), (B) the domain II region
of E. coli signal peptidase, and (C) S. cerevisiae Spc3 using ClustalW
1.8, with settings set at default. Asterisks correspond to identical residues, colons correspond to well-conserved residues, and dots correspond to less well-conserved residues. In A, similarities are based on at
least six of the eight bacterial sequences and two of the three archaeal
sequences at any given position, while pound signs correspond to positions where at least six bacterial sequences contain the identical residue and all three archaeal sequences share a different residue, referred
to as conserved changes in the text.
Academy of Sciences and Humanities. The author is the incumbent of
the Murray Shusterman Career Developmental Chair in Microbiology.
Fang H, Mullins C, Green N (1997) In addition to SEC11, a newly
identified gene, SPC3, is essential for signal peptidase activity in
the yeast endoplasmic reticulum. J Biol Chem 272:13152–
13158
Kawashima T, Amano N, Koike H, Makino S, Higuchi S, KawashimaOhya Y, Watanabe K, Yamazaki M, Kanehori K, Kawamoto T,
Nunoshiba T, Yamamoto Y, Aramaki H, Makino K, Suzuki M
(2000) Archaeal adaptation to higher temperatures revealed by ge-
References
Dalbey RE, Lively MO, Bron S, van Dijl JM (1997) The chemistry and
enzymology of the type I signal peptidases. Protein Sci 6:1129–
1138
415
nomic sequence of Thermoplasma volcanium. Proc Natl Acad Sci
USA 97:14257–14262
Meyer HA, Hartmann E (1997) The yeast SPC22/23 homolog Spc3 is
essential for signal peptidase activity. J Biol Chem 272:13159–
13164
Ng WV, Kennedy SP, Mahairas GG, Berquist B, Pan M, Shukla HD,
Lasky SR, Baliga NS, Thorsson V, Sbrogna J, Swartzell S, Weir D,
Hall J, Dahl TA, Welti R, Goo YA, Leithauser B, Keller K, Cruz R,
Danson MJ, Hough DW, Maddocks DG, Jablonski PE, Krebs MP,
Angevine CM, Dale H, Isenbargerf TA, Peck RF, Pohlschroder M,
Spudich JL, Jung KH, Alami M, Freitasi T, Hou S, Daniels CJ,
Dennis PP, Omer AD, Ebhardt H, Lowe TM, Liang P, Riley M,
Hood L, DasSarma S (2000) Genome sequence of Halobacterium
species NRC-1. Proc Natl Acad Sci USA 97:12176–12181
Paetzel M, Dalbey RE, Strynadka NCJ (1998) Crystal structure of a
bacterial signal peptidase in complex with a ␤-lactam inhibitor.
Nature 396:186–190
Paetzel M, Dalbey RE, Strynadka NCJ (2000) The structure and
mechanism of bacterial type I signal peptidases. A novel antibiotic
target. Pharmacol Ther 87:27–49
Ruepp A, Graml W, Santos-Martinez ML, Koretke KK, Volker C,
Mewes HW, Frishman D, Stocker S, Lupas AN, Baumeister W
(2000) The genome sequence of the thermoacidophilic scavenger
Thermoplasma acidophilum. Nature 407:508–513
Tjalsma H, Bolhuis A, van Roosmalen ML, Wiegert T, Schumann W,
Broekhuizen CP, Quax WJ, Venema G, Bron S, van Dijl JM (1998)
Functional analysis of the secretory precursor processing machinery of Bacillus subtilis: Identification of a eubacterial homolog of
archaeal and eukaryotic signal peptidases. Genes Dev 12:2318–
2331
Van Valkenburgh C, Chen X, Mullins C, Fang H, Green N (1999) The
catalytic mechanism of endoplasmic reticulum signal peptidase appears to be distinct from most eubacterial signal peptidases. J Biol
Chem 274:11519–11525
YaDeau JT, Klein C, Blobel G (1991) Yeast signal peptidase contains
a glycoprotein and the Sec11 gene product. Proc Natl Acad Sci
USA 88:517–521