unique domain organization and expansion of the class III cyclase family in M

22 Pages·2004·1.11 MB·English

ComparativeandFunctionalGenomics
CompFunctGenom2004;5:17–38.
PublishedonlineinWileyInterScience(www.interscience.wiley.com).DOI:10.1002/cfg.349
Primary Research Paper
A survey of nucleotide cyclases in
Actinobacteria: unique domain organization
and expansion of the class III cyclase family in
Mycobacterium tuberculosis
Avinash R.Shenoy1**,K. Sivakumar2, A. Krupa2, N. Srinivasan2 and Sandhya S.Visweswariah1*
1DepartmentofMolecularReproduction,DevelopmentandGenetics,IndianInstituteofScience,Bangalore560012,India
2MolecularBiophysicsUnit,IndianInstituteofScience,Bangalore560012,India
*Correspondenceto: Abstract
SandhyaS.Visweswariah,
Cyclic nucleotides are well-known second messengers involved in the regulation of
DepartmentofMolecular
important metabolic pathways or virulence factors. There are six different classes
Reproduction,Developmentand
Genetics,IndianInstituteof of nucleotide cyclases that can accomplish the task of generating cAMP, and four
Science,Bangalore560012, of these are restricted to the prokaryotes. The role of cAMP has been implicated in
India. the virulence and regulationofsecondary metabolites inthe phylum Actinobacteria,
E-mail: which contains important pathogens, such as Mycobacterium tuberculosis, M. leprae,
[email protected] M.bovis andCorynebacterium,andindustrialorganismsfromthegenusStreptomyces.
We have analysed the actinobacterial genome sequences found in current databases
**Correspondenceto:
AvinashR.Shenoy,Department for the presence of different classes of nucleotide cyclases, and find that only class
ofMolecularReproduction, IIIcyclasesarepresentintheseorganisms.Importantly,prominentmemberssuchas
DevelopmentandGenetics, M. tuberculosis and M. leprae have 17 and 4 class III cyclases, respectively, encoded
IndianInstituteofScience, in their genomes, some of which display interesting domain fusions seen for the
Bangalore560012,India. first time. In addition, a pseudogene corresponding to a cyclase from M. avium has
E-mail:[email protected] been identified as the only cyclase pseudogene in M. tuberculosis and M. bovis. The
Corynebacterium and Streptomyces genomes encode only a single adenylyl cyclase
each, both of which have corresponding orthologues in M. tuberculosis. A clustering
of the cyclase domains in Actinobacteria reveals the presence of typical eukaryote-
like,fungi-likeandotherbacteria-likeclassIIIcyclasesequenceswithinthisphylum,
suggesting that these proteins may have significant roles to play in this important
Received:7August2003 group of organisms.Copyright  2004 John Wiley & Sons, Ltd.
Revised:13October2003
Keywords: class III cyclase; adenylyl cyclase; genome analysis; comparative
Accepted:21October2003
genomics; Actinobacteria; Mycobacterium tuberculosis; Mycobacterium leprae
Introduction these six classes have evolved independently in
diverse organisms during evolution.
Cyclic AMP and cGMP are involved in diverse
The class I enzymes are present in E. coli and
signalling networks in all life forms. In bacteria,
related enteric bacteria (Danchin 1993), and are
cAMP is an important second messenger that
represented by single copy genes that code for
regulatesseveraloperonsandregulons(Caseset al.
an adenylyl cyclase involved in the phenomenon
1998). There are six different families of proteins
of catabolite repression (Cases et al. 1998). Class
that convert NTPs to either cAMP or cGMP
(Danchin 1993; Shenoy et al. 2002), and these II cyclases are represented by toxins secreted
are distinguished by differences in amino acid by Bacillus anthracis (Leppla 1982), Bordetella
sequence and catalytic mechanism, suggesting that pertussis (Weiss et al., 1984) and Pseudomonas
Copyright2004JohnWiley&Sons,Ltd.
18 A.R.Shenoyetal.
aeruginosa (Yahr et al., 1998) that elevate cAMP (Tesmer et al., 1998). Two metal atoms are bound
levels within the host cells on infection. The by two conserved aspartate residues, and the reac-
crystal structure of the anthrax oedema factor, the tion involves theabstraction of the proton from the
(cid:1)
calmodulin-activated adenylyl cyclase, shows that 3 OH group of ATP and the nucleophilic attack
the catalytic mechanism of the class II cyclases by the negatively charged oxygen on the phospho-
appears to require a single divalent metal ion diester bond (Tesmer et al., 1997). The abstraction
(Drum et al., 2002). of the proton is facilitated by one of the two metal
The class III enzymes are the most widely atomsintheclassIIIcyclases(Tesmeret al.,1997),
distributed and are found in bacteria, archaea and and this function is interestingly carried out by
eukaryotes (Danchin, 1993; Shenoy et al., 2002). a suitably placed histidine residue in the class II
The mammalian G-protein coupled, receptor- cyclases (Drum et al., 2002). A pentavalent phos-
activated adenylyl cyclases are members of the phate group is believed to be the transition state
class III adenylyl cyclase family (Defer et al., species that is stabilized by critical asparagine and
2000), as are the receptor and soluble guanylyl arginine residues, which have been shown to be
cyclases (Wedel et al., 2001). Crystal structures essential for activity by mutational analysis (Tes-
of the class III adenylyl cyclases (Tesmer mer et al., 1997; Yan et al., 1997). The binding of
et al., 1997; Bieger et al., 2001) and subsequent ATP requires a lysine and an aspartate, which are
homology modelling and mutational analysis of replaced by glutamate and cysteine in the guanylyl
the guanylyl cyclases (Liu et al., 1997) have cyclases to allow utilization of GTP as substrate
led to the identification of amino acid residues (Liu et al., 1997).
responsible for substrate selectivity amongst these The Actinobacteria include several bacteria of
enzymes (Sunahara et al., 1998; Tucker et al., medical or industrial interest. Streptomyces is a
1998; Hannenhalli et al., 2000). Classes IV, V ubiquitous soil organism known for its versatile
and VI of the cyclase family have only a single metabolic prowess, especially in forming useful
representative each,foundinAeromonas (Sismeiro secondary metabolites, the production of some
et al., 1998), Prevotella (Cotta et al., 1998) and being regulated by cAMP (Susstrunk et al., 1998;
Rhizobium (Tellez-Sosa et al., 2002), respectively. Horinouchi et al., 2001). Mycobacterium tubercu-
Class III cyclases have been the most exten- losis is the aetiological agent of tuberculosis and
sively studied, both structurally and biochemi- belongs to an order that includes other pathogens
cally. The mammalian 12-transmembrane adenylyl such as M. leprae and several members in other
cyclases act as functional dimers of the C1 and genera, such as Corynebacterium, Nocardia and
C2 regions contained within a single polypeptide Actinomyces (NCBI http://www.ncbi.nlm.nih.
chain (Tang et al., 1998). The C1 and C2 regions gov/entrez/query.fcgi?db=Taxonomy). Interes-
are class III cyclase domains, hence mammalian tingly, M. tuberculosis infection of macrophages
membrane-bound adenylyl cyclases have two class has been shown to lead to increased cAMP in
III domains within their polypeptide chains. Crys- macrophages (Lowrie et al., 1979) and M. microti
tal structures have revealed that the C1–C2 dimer has been thought to escape macrophage killing by
interface gives rise to the active site, and hence the release of cAMP (Lowrie et al., 1975). Since
dimerization and correct juxtaposition of residues studies with Bordetella pertussis (Confer et al.,
from both protomers is essential for activity of 1982),Bacillusanthracis (Hoover et al.,1994) and
these enzymes (Tang et al., 1998). The guanylyl Trypanosoma (Wirth et al., 1982) have demon-
cyclases, on the other hand, have only a single stratedtheimportanceofcAMPinreducingphago-
class III cyclase domain per polypeptide and act cyte activity, it would be interesting to investigate
by homodimerization, as is seen in the receptor whether cAMP plays any role in compromising
guanylyl cyclases, or as heterodimers of α and the functioning of the macrophage in the case of
β subunits in the case of the soluble guanylyl Mycobacterium infection. An analysis of the M.
cyclases (Wedel et al., 2001). Mutagenesis studies tuberculosis genome showed the presence of sev-
(Zimmermann et al., 1998) and structural similar- eral cyclases and cNMP binding proteins (Cole
ity of the adenylyl cyclase core to the palm fold of et al., 1998; McCue et al., 2000) and recently at
DNA polymerases suggested that the cyclase reac- least two gene products that code for adenylyl
tion mechanism involves two-metal ion catalysis cyclases from M.tuberculosis have been the focus
Copyright2004JohnWiley&Sons,Ltd. CompFunctGenom2004;5:17–38.
ClassIIInucleotidecyclasesinActinobacteria 19
of biochemical studies (Guo et al., 2001; Reddy (NCBIhttp://www.ncbi.nlm.nih.gov/BLAST/)for
et al., 2001; Linder et al., 2002; Shenoy et al., additional actinobacterial sequences from organ-
2003). However, no comprehensive analysis of the isms other than those mentioned above. These
genomes from these bacteria has been performed sequences included completed and unfinished
to date in terms of the domain fusions and critical whole genomes and plasmids from these organ-
catalytic residues present in these proteins. isms. At the time of analysis (May 2003), 67 dif-
In this study, we have analysed the completed ferent genomes were in various stages of sequenc-
genome sequences of members of the Actinobac- ing amongst the Actinobacteria. The preliminary
teria for the presence of nucleotide cyclases repre- sequence information of the M. avium genome
sented by all the six classes of nucleotide cyclases. was made available from the Institute for Genomic
Interestingly,onlysequencessimilartotheclassIII Research (TIGR http://www.tigr.org/tigr-scripts/
cyclases are present in these bacteria, and promi- ufmg/ReleaseDate.pl).
nentpathogenssuchasM.tuberculosis andM.lep-
rae have more than one class III cyclase encoded
Searches
in their genomes. Many of the putative cyclase
geneshaveinterestingdomainfusionsthathavenot PSI–BLAST(Altschulet al.,1997)wasperformed
been identified in other class III cyclases to date, with an inclusion (h) value cut-off of 10−4 till
suggesting that biochemical analysis of these pro- convergence, using catalytic domain sequences of
teins and their regulation could be a fruitful area representative adenylyl and guanylyl cyclases as
of research in the future. queries. The catalytic domain of the M. tuber-
culosis Rv1625c gene, shown to have adenylyl
cyclase activity (Guo et al., 2001; Reddy et al.,
Materials and methods 2001), was also used as the seed sequence in
PSI–BLAST using amino acids 212–443 (Shenoy
et al., 2003). This performed better than other
Dataset
cyclase domain sequences. A position-specific
The completed genome sequences of Mycobac- scoring matrix (PSSM) generated using the align-
terium tuberculosis H37Rv (Cole et al., 1998), ment of the cyclase domains of the 16 M.tubercu-
M. tuberculosis CDC1551 (Fleischmann et al., losis cyclases was also used in the searches. The
2002), M. leprae (Cole et al., 2001), M. bovis large number of pseudogenes in M. leprae (Cole
AF2122/97 (Garnier et al., 2003), Streptomyces et al., 2001) prompted us to search for the possible
coelicolor A3(2) (Bentley et al., 2002), S. avermi- existence ofcyclase pseudogenes. Thiswas carried
tilis MA-4680 (Omura et al., 2001), Tropheryma out using the same PSSM in a PSI–TBLASTN
whipplei str. twist (Raoult D, Audic S, Robert C search on the nucleotide sequence of the M. lep-
et al. 2002. Tropheryma whipplei illustrates the rae genome. The amino acid sequences were
diversity of gene loss patterns in small genome then mapped on to the annotated genome and all
bacterial pathogens; unpublished MS), T. whip- eightofthepseudogenesidentifiedcorrespondedto
plei TW08/27 (Bentley et al., 2003), Bifidobacter genes labelled as pseudogenes earlier (Cole et al.,
longum NCC2705(Schellet al.,2002),Corynebac- 2001).
terium glutamicum ATCC 13032 (Nakagawa S. Hidden Markov model (HMM) based searches
2002. Complete genomic sequence of Corynebac- were performed using HMMer (Eddy 2001 http://
terium glutamicum ATCC 13032; unpublished hmmer.wustl.edu/) in the global (ls) and local
MS) and C. efficiens YS-314 (Kawarabayasi Y, (fs) mode using the class III cyclase HMM
Yamazaki J, Hino Y et al. 2002. The entire (Accession No. PF00211) from the Protein Fam-
genomic sequence of Corynebacterium efficiens ilies (Pfam) database of alignments and HMMs
YS-314; unpublished MS) were downloaded from (Pfam http://www.sanger.ac.uk/Software/Pfam/;
the NCBI website (NCBI ftp://ftp.ncbi.nih.gov/ Batemanet al.,2002)atanExpect(E)valuecut-off
genomes/Bacteria/). The combined dataset, con- of 0.1. Additional cyclase sequences from bacte-
sisting of proteins predicted in the genomes, ria and eukaryotes were introduced or removed to
was analysed using the search methods described generate additional models for the searches. The
below.ThenrdbwassearchedontheNCBIwebsite cyclase domains ofthe16M.tuberculosis cyclases
Copyright2004JohnWiley&Sons,Ltd. CompFunctGenom2004;5:17–38.
20 A.R.Shenoyetal.
were also used to generate a model for HMM to the Molecular Evolutionary Genetics Analysis
search. software (Kumar et al., 2001) for generating and
Domain organizations of the cyclases were pre- bootstrapping the neighbour-joining tree.
dicted using the HMMs from the Pfam (Bateman
et al., 2002) and the Simple Modular Architec-
ture Research Tool (SMART) websites (SMART Results
http://smart.embl-heidelberg.de/; Letunic et al.,
2002) and the graphical outputs with modifica- Cyclases in Actinobacteria
tions have been used in the figures. RPS–BLAST
The presence of multiple classes of cyclases, with-
(Schaffer et al., 1999) using the Cluster of Orthol-
out sequence similarity to each other, indicates the
ogous Groups (Tatusov et al., 2001), Pfam and
independent evolution of proteins for catalysing
SMART matrices (NCBI; ftp://ftp.ncbi.nih.gov/
the conversion of ATP to cAMP. PSI–BLAST
pub/mmdb/cdd/) was also used along with Con-
searches with query sequences of the all classes
served Domain Architecture Retrieval Tool (CD
of adenylyl cyclases in Actinobacteria yielded
ART; http://www.ncbi.nlm.nih.gov/Structure/
hits above the E value cut-off defined in Materi-
lexington/lexington.cgi?cmd=rps) for assessing als and methods, for only the class III cyclases.
domain organization of the sequences. Trans- PSI–BLAST and HMM searches (see Materials
membrane spanning helices were predicted using and methods) for sequences similar to the cur-
TMHMM (http://www.cbs.dtu.dk/services/ rently known class III cyclases revealed the pres-
TMHMM/). Multiple sequence alignments were ence of several class III cyclase members within
computed using HMMalign (Eddy, 2001; http:// the genomes of manyActinobacteria (Tables 1–3).
hmmer.wustl.edu/), ClutalX (Jeanmougin et al., Weidentified62proteinsinthisstudy,eachhaving
1998) and T-Coffee (Notredame et al., 2000) and a single class III cyclase domain. The genomes of
were subsequently manually edited. Clustal X Bifidobacter longum and Tropheryma whipplei did
was used to generate Figures 2 and 5. Align- not reveal the presence of members of any of the
mentsofthecyclasedomainswerethentransferred currently described classes of nucleotide cyclases,
Table1.CyclasesfoundinM.tuberculosisstrainsH37RvandCDC1551andM.bovis
M.tb Protein
Gene CDC1551 M.bovis length
Genenames identifiers gene gene (aa)
Solublecyclases Rv0891c gi1314030 MT0915 Mb0915c 285
Rv1120c gi2117214 MT1152 Mb1151c 164
Rv1264 gi480322 MT1302 Mb1295 397
Rv1647 gi1838999 MT1685 Mb1674 328
Rv1359 gi1419062 MT1403 Mb1394 253
Rv1900c gi2225960 MT1951 Mb1935c 462
Rv2212 gi1237065 MT2268 Mb2235 388
Membrane-boundcyclases Rv1625c gi2113909 MT1551 Mb1651c 443
Rv1318c gi1340084 MT1359 Mb1352c 541
Rv1319c gi1340085 MT1361 Mb1353c 535
Rv1320c gi1340086 MT1362 Mb1354c 567
Rv3645 gi2105049 MT3748 Mb3669 549
Rv2435c gi1666149 MT2509 Mb2461c 730
MultidomainDNA-bindingcyclases Rv0386 gi2909507 MT0399 Mb0393 1092
Rv1358 gi1419061 MT1402 Mb1393 1159
Rv2488c gi2791528 MT2563 Mb2515c 1199
ThegenomeofM.bovisishighlysimilartothatofM.tuberculosisandalsohasidenticalcyclasegenes.Theprimaryannotationsfor
theM.tuberculosisCDC1551andM.bovisgenomesforcorrespondinggenesfromM.tuberculosisH37Rvarelisted.Thegenesare
classifiedbasedonthedomainanalysisthathasbeenperformedanddescribedinthisstudy.Theproteinlengthsinaminoacids
(aa)fortheM.tuberculosisH37Rvproteinsaregiven,aspertheprimaryannotation.M.tuberculosisCDC1551hasonecyclase
morethantheothertwomycobacteria(MT1360).
Copyright2004JohnWiley&Sons,Ltd. CompFunctGenom2004;5:17–38.
ClassIIInucleotidecyclasesinActinobacteria 21
Table2.CyclasesidentifiedinM.leprae
Protein M.tb
Status Geneidentifiers Genenames length(aa) orthologue
Full-lengthgenes gi13093284 ML1399 324 Rv1647
gi13093492 ML1753 1106 Rv1358
gi13093954 ML2341 732 –
gi13092553 ML0201 530 Rv3645
M.lepraegene M.tborthologue
ML0465 Rv1320c
ML1111 Rv1264
ML1154 Rv1318c
Pseudogenes ML1242 Rv0386
ML1285 Rv1625c
ML1820 Rv1319c
ML1948 Rv3645
ML2016 Rv1900c
The full-length genes (gene identifiers and primary annotations are listed) represent those that code for
proteins that match genes in M. tuberculosis (M. tb). The ML2341 cyclase is unique to M. leprae. The
pseudogenescorrespondingtocyclaseswereidentifiedbyPSI–TBLASTN andhavebeenidentifiedinthe
originalgenomeannotation(Coleetal.,2001).Theproteinlengthisgiveninaminoacids(aa).
Table3.CyclasesidentifiedinthisstudyinvariousmembersoftheActinobacteria
Protein
Organisms Geneidentifiers Genenames length(aa)
CorynebacteriumefficiensYS-314 gi25026866 CE0310 538
CorynebacteriumglutamicumATCC13032 gi19551561 Cgl0311 486
StreptomycescoelicolorA3(2) gi21223302 SCK13.20 381
StreptomycesavermitilisMA-4680 gi29829871 SAV3328 405
Streptomycesgriseus gi11131886 399
Brevibacteriumliquifaciens gi117790 403
Thermobifidafusca gi23018870 358
Arthrobacternicotinovorans gi25169176 323
Actinosynnemapretiosumsubsp.auranticum gi21449364 913
Geneidentifiersandprimaryannotationsforcompletedgenomesequencesareshown.Theproteinlengthsare
giveninaminoacids(aa).
and therefore these organisms may have no need of the nucleotide cyclase family, using methods
of cAMP as a regulatory molecule, or have novel described above. Searches with queries from
proteins that need to be identified through classical families other than the class III cyclase family
screening procedures. did not yield hits with scores above cut-off, as
described in Materials and methods. The largest
number (17) of cyclases is seen in the genome
Cyclases in M. tuberculosisand M. bovis
of M. tuberculosis CDC1551, followed by the M.
An analysis of the M.tuberculosis H37Rv genome tuberculosis H37Rvgenomewhich,inouranalysis,
for the presence of cNMP binding proteins and shows 16 putative cyclase genes (Table 1). The
cNMP metabolizing enzymes has been reported presence of one extra cyclase in the CDC1551
earlier, and predicted 15 cyclases in the genome strain compared to the H37Rv strain is explained
(McCue et al., 2000). Recently, the genome by expansion of the Rv1318c cyclase gene family
sequence of the CDC1551 strain has been made (Fleischmann et al., 2002). All M. tuberculosis
available (Fleischmann et al., 2002) and we have putative cyclase genes have only a single class III
analysed this genome as well for the members cyclase domain per polypeptide chain, in contrast
Copyright2004JohnWiley&Sons,Ltd. CompFunctGenom2004;5:17–38.
22 A.R.Shenoyetal.
to the eukaryotic adenylyl cyclases. Highly similar A close examination of Rv1120c (MT1152)
genes (>99% sequence identity) are present in reveals that the protein is only 164 amino acids
the CDC1551 strain (the gene names from the long, which is smaller than a typical class III
CDC1551 strain are mentioned in parentheses cyclase catalyticdomain(∼200–250 aminoacids).
in the text). A variety of domains are found A number of conserved class III cyclase-like
fused to the cyclase domain in these 16 genes residues are present in Rv1120c, but it lacks
(Figure 1) and some of these fusions are unique the more C-terminal residues responsible for sub-
to the actinobacterial cyclases (see below). The strate selectivity and transition-state stabilization
genome of M. bovis reveals the presence of (Figure 2). A BLASTN search on the M. avium
16 cyclases identical to those in M. tuberculosis genomerevealsthepresenceofanorthologue(80%
H37Rv (Table 1). identity to the first 149 amino acids of Rv1120c)
whose protein length is long enough to encode a
complete cyclase domain. Wetherefore aligned the
Genes that code for proteins with only an
nucleotidesequenceintheH37Rvgenome,beyond
identifiable cyclase domain
the predicted stop codon of the Rv1120c, against
There are six genes in the M.tuberculosis genome the corresponding region of the unfinished genome
that, upon prediction, seem tocode for only aclass ofM.avium andfoundthatasinglebasedeletionin
III cyclase domain, with no additional domains theM.tuberculosis genomehasledtoaframeshift
identifiable from current databases. These are and premature truncation of the Rv1120c polypep-
Rv0891c (MT0915), Rv1120c (MT1152), Rv1264 tide (Figure 4). The putative Rv1120c orthologue
(MT1302),Rv1359(MT1403),Rv1647(MT1685), in M. avium extends to the end of an ORF that
and Rv2212 (MT2268) (see Table 1, Figure 1). As is contained within the Rv1119c gene in M. tuber-
shown in Table 1, Rv0891c and Rv1359 contain culosis. Therefore, the putative gene in M. avium
the number of amino acids minimally required possesses functional class III cyclase C-terminal
for a functional cyclase domain. Rv1264, Rv1647 residues that are lacking in the M. tuberculosis
and Rv2212 are longer proteins and contain >100 protein. It is possible therefore, that a loss of the
amino acids adjacent to the cyclase domain. full-length cyclase, seen in the Rv1120c cyclase
However,thesesequencesdonotcontainadditional gene in M. tuberculosis H37Rv (and CDC1551),
domains that are listed in Pfam currently. has occurred and that Rv1120c could represent the
Rv0891cappearstohavealltheresiduesrequired only cyclase pseudogene in M. tuberculosis. The
forcatalyticactivityinclassIIIcyclases.However, status of the Rv1120c orthologue in M. bovis is
ithasnon-conservedresidues(arginineandleucine) identical to that in M. tuberculosis.
at positions responsible for substrate selectivity, in The Rv1264 (MT1302) adenylyl cyclase was
contrast to those seen in the mammalian enzymes recently characterized biochemically and its N-
(Figure 2). A clustering based on the sequence terminal region was found to downregulate the
alignment of representative actinobacterial cyclase activityofthecatalyticdomain(Linderet al.2002).
domains identified in this study and other class III Rv1264c is more closely related to the Strepto-
cyclases(Figure 3)showsthattheRv0891ccyclase myces cyclases in having a short deletion just N-
domain is more related to the fungal and para- terminal to the substrate selective aspartate residue
sitecyclasesandagroupofActinobacteria-specific (Figure 2). Another related cyclase is the Rv2212
NB-ARC (nucleotide-binding–common to Apaf1, (MT2268) cyclase, which is as yet uncharacterized
plant resistance gene products and CED4) domain biochemically. The clustering shown in Figure 3
(van der Biezen et al., 1998) containing cyclases reveals that it positions in a group of cyclases
as described below. that includes proteins from Streptomyces, Bre-
The Rv0891c (MT0915) gene is adjacent to the vibacterium and Thermobifida. Rv2212 has all the
Rv0890c gene which is predicted to have a NB- residues required for catalytic activity and appears
ARC domain and a C-terminal helix-turn-helix to be specific for ATP as a substrate (Figure 2).
(HTH) DNA binding domain and the operonic Rv1264 has sequence similarity (29% identity in
nature of these two genes (TIGR http://www.tigr. the cyclase domain) to the cyclase from Brevibac-
org/tigr-scripts/operons/pairs.cgi?taxon id=89) terium, which has been shown to be activated by
hints at their possible functional interplay. pyruvate. However, Rv1264 was not stimulated by
Copyright2004JohnWiley&Sons,Ltd. CompFunctGenom2004;5:17–38.
ClassIIInucleotidecyclasesinActinobacteria 23
Figure 1. Domain organizations of the M. tuberculosis cyclases. Schematic domain organizations drawn approximately
to scale are shown for the 16 proteins identified in the M. tuberculosis H37Rv strain. The corresponding M. tuberculosis
CDC1551 gene names are given in parentheses. It is interesting to note that M. tuberculosis has four cyclases (MT1359,
MT1360,MT1361,MT1362)similartothreecyclasesfoundinM.tuberculosisH37Rv(Rv1318c,Rv1319c,Rv1320c)
pyruvate or other small molecules (Linder et al., domain. However, Rv1359 has higher sequence
2002). similarity to the Rv0386 (MT0399) cyclase than to
BothRv1359(MT1403)andtheadjacentRv1358 its neighbour (47% identity to Rv0386 compared
(MT1402) genes contain the class III cyclase to 38% to Rv1358). In fact, Rv0386 is an enzyme
Copyright2004JohnWiley&Sons,Ltd. CompFunctGenom2004;5:17–38.
24 A.R.Shenoyetal.
Copyright2004JohnWiley&Sons,Ltd. CompFunctGenom2004;5:17–38.
ClassIIInucleotidecyclasesinActinobacteria 25
ngm;ng(cid:1))
MprofileusiC.glutamicuotations,aloapproach(
seHMs;C.glu,meanntational
anou
yclcieenmp
sIIIcC.effithegaco
clasC.eff,per↓(),
e sy
tothciens;mesamicall
edefanahe
nsequenceswerealignA.pretiosum;B.liq,B.liquT.fus,Tfusca.Thegeneresiduesidentifiedbioc
engthproteiorans;A.pre,e,S.griseus;eselectivity
omains.Full-lc,A.nicotinovoelicolor;S.gr∗),substrat(
cyclaseddited.A.niS.coe,S.caspartatesated
alignmentofactinobacterialwithT-CoffeeandmanuallyeM.leprae;S.ave,S.avermitilis;enused.Themetalbindingž)areindiczationresidues(
quencealignedM.lep,havebeestabili
2.MultiplesegnandthenreM.tuberculosis;neidentifiers,transitionstat
FigureHMMaliM.tub,withgeandthe
Copyright2004JohnWiley&Sons,Ltd. CompFunctGenom2004;5:17–38.
26 A.R.Shenoyetal.
Copyright2004JohnWiley&Sons,Ltd. CompFunctGenom2004;5:17–38.