Phylogenetic relationships in class I of the superfamily of bacterial,
fungal, and plant peroxidases
Marcel Za
´
mocky
´
Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia
Molecular phylogeny among catalase–peroxidases, cyto-
chrome c peroxidas es, and ascorbate peroxidases was ana-
lysed. Sixty representative sequences covering all known
subgroups of class I of the superfamily of bacterial, fungal,
and plant heme peroxidases were selected. Each sequence
analysed contained the typical p eroxidase motifs evolved to
bind effectively the prosthetic heme group, enabling per-
oxidatic activity. The N-terminal and C-terminal domains of
catalase–peroxidases matching the ancestral tandem g ene
duplication event were treated separately in the phylogenetic
analysis to reveal their specific evolutionary history. The
inferred unrooted phylogenetic tree obtained by three dif-
ferent methods revealed the existence of four clearly
separated c lades ( C-terminal and N-terminal d omains of
catalase–peroxidases, ascorbate peroxidases, and cyto-
chrome c peroxidases) which were segregated early in the
evolution of this superfamily. F rom the results, it is obvious
that the duplication e vent in the g ene f or catalase–peroxid ase
occurred in the later phase of evolution, in which the indi-
vidual specificities of the peroxidase families distinguished
were already formed. Evidence is presented that class I of the
heme peroxidase superfamily is spread among prokaryotes
and eukaryotes, obeying the birth-and-death p rocess of
multigene family evolution.

dase superfamily [5]. They were further classified into three
subclasses according to their cellular localization and
function. All representatives possess the same heme pros-
thetic group containing high-spin f erric iron, so the reaction
specificity is a pparently determined by the protein surround-
ings of the heme. Catalase–peroxidases, which belong to
class I , are the only group of this superfamily that possess
notable catalase activity (i.e. they can oxidize and reduce
hydrogen peroxide; see [6] f or details). All other members of
the superfamily can only reduce hydrogen peroxide with
subsequent oxidation of a secondary substrate. These
ÔnoncatalaseÕ members of class I exhibit strong specificity
for electron donors: the preferred substrate is ascorbate in
the case of a scorbate peroxidases and cytochrome c for
cytochrome c peroxidases. Several crystal structures of heme
peroxidases have been solved, now covering all subgroups
of this superfamily. In clas s I, the crystal structure of
cytochrome c peroxidase (CCP) from Saccharomyce s
cerevisiae [7] a nd ascorbate peroxidase (APX) from Pisum
sativum [8] is known; the former has served as a benchmark
for peroxidase structures for two decades. APX was already
crystallized in a complex with its substrate [9]. After many
unsuccessful attempts, the crystals of several catalase–
peroxidases we re also obtain ed ( e.g. [10,11]). Recently, the
structure of catalase–peroxidase from the halophilic arch-
aeon Haloarcula marismortui was solved t o high resolution
[12], and this was followed b y the highly resolved structure o f
KatG from Burkholderia pseudomallei [13].
The phylogenetic relations of heme peroxidases have only
been analysed to a certain extent: the evolutionary analysis

genes are created by repeated gene duplication; some are
maintained in the genome for a long time, whereas others
are d eleted or become nonfunctional [18–20]. It is well
documented that almost all KatGs contain two fused copies
of the primordial peroxidase gene [5,15]. The copy trans-
lated into the N-terminal domain participates in catalysis
and possesses t he prosthetic heme group, but the c atalytic
function of the C-terminal domain is not apparent [12], and
thus the role of the corresponding part of the gene is also
unknown. These g ene f usions togeth er with single-copy
genes of APXs and CCPs are ideal for investigating the
evolution of a widespread multigene family. H ence, the
most probable evolutionary route leading to clades of extant
heme peroxidases will help to explain the occurrence and
function of multigene families present in both prokaryotic
and eukaryotic genomes.
Experimental Procedures
Sequence data
All protein sequences used in this study were obtained from
the U niProt database and are listed in Table 1 together with
their accession numbers and the organisms from which they
originate. The protein sequences were used to infer the
phylogenetic relationships. Both codon usage bias of
analysed sequences ranging from a rchaea to high er plants
and the presence of introns in only some of the members
analyzed (plant APXs) can cause serious problems with
analyzing the DNA sequences directly. Owing to the
currently unequal availability of the sequences of the
proposed groups of heme peroxidases (known from previ-
ous analysis in [15]), only representative sequences from

In the case of catalase–peroxidases, two partial alignments
were performed, for the N-terminal and C-terminal domain.
Suitable parameters for all three partial alignments were:
gap opening penalty, 10.0; gap extension penalty, 0.2; and
gap separation distance, 8. The Blosum 62 series protein–
weight matrix was used in all three cases. These parameters
were the same as those used for the first alignment of class I
of the peroxidase superfamily [15]. Varying the gap opening
penalty setting in the range 5.0–20.0 did not change the
alignment output significantly. T he seq uence alignments
were displayed with
GENEDOC
[22] and refined manually
with respect to known structural homology.
Profile alignments
The profile alignment mode of
CLUSTALX
was used stepwise
on the partial alignments. Firstly, the N-terminal domains
of catalase–peroxidases were aligned with the prealigned
group of APXs and CCPs where the known secondary-
structure e lements were taken into account. This new profile
was final ly used to align the group of C-terminal domains of
catalase–peroxidases which share the lowest sequence
similarity with other superfamily members in catalytically
essential regions. Suitable parameters used for all profile
alignments were: gap opening penalty, 10.00; gap extension
penalty, 0.1; Blosum 30 protein–weight matrix; helix and
strand gap p enalty, 4; a nd loop gap penalty, 1. Finally, a reas
of extensive gaps (i.e. longer than 10 amino acid positions

the Stanford Genome Technology Center website at h ttp://www-sequence.stanford.edu/group/candida.
Abbreviation Accession number Enzyme Organism (strain)
ArathaAPXc Q05431 Ascorbate peroxidase 1 Arabidopsis thaliana
ArathaAPXt Q42593 Ascorbate peroxidase (thylakoid) Arabidopsis thaliana
ArchfulCP O28050 Catalase–peroxidase Archaeoglobus fulgidus
AspefumCP Q7Z7W6 Catalase–peroxidase Aspergillus fumigatus
AspenidCP Q96VT4 Catalase–peroxidase Emericella nidulans
BacihalCP Q9KEE6 Catalase–peroxidase Bacillus halodurans
BacisteCP P14412 Catalase I Geobacillus stearothermophilus
BlumgraCP Q8 · 1 N3 Catalase–peroxidase Blumeria graminis
BurkcepCP Q9AP06 Catalase–peroxidase Burkholderia cepacia
BurkpseCP Q939D2, pdb: 1MWV Catalase–peroxidase Burkholderia pseudomallei
CandalbCCP Contig19–10046* Cytochrome c peroxidase Candida albicans
CapsannAPX Q84UH3 Ascorbate peroxidase Capsicum annuum
CaulcreCP O31066 Peroxidase/catalase Caulobacter crescentus
ChlamspAPX Q9SXL5 Ascorbate peroxidase Chlamydomonas sp. W80
ChlareiAPX O49822 Ascorbate peroxidase Chlamydomonas reinhardtii
CucusatAPX Q96399 Ascorbate peroxidase (cytosolic) Cucumis sativus
CucurcAPXt O04873 Ascorbate peroxidase (thylakoid-bound)
Kurokawa Amakuri
Cucurbita cv.
DesulfiCP ZP_00096951 Catalase–peroxidase Desulfitobacterium hafniense
E_coliHPI P13029 Catalase HPI Escherichia coli
E_coliPCP P77038 EHEC-strain catalase peroxidase
(strain 0157:H7)
Escherichia coli
EuglgraAPX Q8LP26 Ascorbate peroxidase Euglena gracilis
FraganaAPX O48919 Ascorbate peroxidase Fragaria x ananassa
GaldparAPX Q8GT26 Hybrid-type ascorbate peroxidase
(Rhodophyta)

SpinolAPXt O46921 Ascorbate peroxidase (thylakoid) Spinacia oleracea
Ó FEBS 2004 Molecular evolution of heme peroxidases (Eur. J. Biochem. 271) 3299
pairwise protein distance calculation method in which the
JTT protein matrix [24] was formed. This output was put in
the F itch–Margoliash Ôleast sq uaresÕ phylogenetic tree
estimation method, in which the search for the best trees
was allowed. T n addition, global rearrangement of the
sequence order after each cycle in the
FITCH
program was
activated. The series of tree s prod uced was analysed by the
Consense method to reveal the majority r ule c onsensus tree.
This tree was visualized with the program
TREEVIEW
[25].
The maximum likelihood unrooted phylogenetic tree was
also calculated using the program
PUZZLE
, version 5.0 [26].
The WAG model of amino acid substitution was applied
[27]. Slow and accurate parameter estimation and 50 000
puzzling s teps were used on the set of data subjected to the
above methods. The c-distribution of rate h eterogeneity
with parameter estimation from the actual data set was used
(value obtained for parameter Gamma ¼ 0.62). In total,
230 300 quartets were analysed, and an unrooted quartet
puzzling tree was produced. This tree was also visualized
with the program
TREEVIEW
[25]. The highest likelihood

be traced in the known 3D crystal structures of class I
peroxidases presented in Fig. 4 for members of each group
analysed.
The greatest s equence conservation is achieved in the area
on the distal side of the heme prosthetic group (known as
peroxidase consensus p attern PS00436 in the P rosite
database) surrounding the active s ite, where it reaches
76% (Fig. 1). The catalytic triad Arg92, Trp95, and His96
in HalomarCPn (abbreviations of all sequences analysed are
listed i n T able 1) located i n t he distal heme cavity is
invariantly conserved among all N-domains of catalase–
peroxidases, all CCPs and all A PXs. Whereas the essential
arginine (Arg92) and histidine (His96) are responsible for
compound I formation [4], the latter allowing the heterolytic
cleavage of the peroxide bond via acid-base catalysis [29],
the coessential tryptophan (Trp95) facilitates the two-
electron reduction of compound I by hydrogen peroxide
[30]. Site-directed mutagenesis in E_coliHPIn [31],
MycotubCPn [32], and SyncyspCPn [29], a s well a s in
PisusatAPX [33] and SacchceCCP [34], supported the role
of the catalytic triad by affecting the typical reactivity. The
level of decrease in the peroxidase activity (in contrast with
catalase activity) correlated with the ability o f the re spective
mutants t o bind heme. Residues corresponding to the
catalytic t riad are not conserved i n the C-domains of
catalase–peroxidases which do not bind heme. The position
corresponding to Arg92 (the numbering c orresponds to
HalomarCP, in which the residues c an also be found;
Fig. 4A,B) is variable in C-domains, but there a similar
basic residue occurs (e.g. Lys465; Fig. 4B). The position

His175 in SacchceCCP; Fig. 2) is less conserved. The o verall
sequence similarity around this iron ligand is only 48%.
Nevertheless, the corresponding peroxidase consensus pat-
tern PS00435 (Prosite database) is d iscernible in a ll the
peroxidases analysed. The lower sequence similarity com-
pared with the distal side can be e xplained by the fact that
this rather variable region contributes significantly to the
reaction specificity of the respective groups and therefore
each family has its own typical feature in this region. The
iron of the prosthetic heme group is invariantly
co-ordinated by the a bove essential proximal h istidine
(Fig. 4A,C,D). However, in all C-domains of catalase–
peroxidases, there is a conserved arginine (Arg622 in
HalomarCPc) in t he corresponding position, indicating that
these domains lost their ability to co-ordinate the heme.
Further, a conserved tryptophan (Trp311 in HalomarCPn,
Trp179 in PisusatAPX, and Trp191 in SacchceCCP) is
thought to participate in an important hydrogen-bond
network on the proximal side o f the heme. This residue is
not conserved in all C-domains of catalase–peroxidases and
some APXs (e.g. position P he171 in MesecryAPX), sup-
porting the theory that it is not essential for the reaction
mechanism o f A PXs [33]. In contrast, for CCPs, T rp191 has
been suggested t o be t he site of the free-radical formation of
the c orresponding CCP compound I [ 37]. Site-directed
mutagenesis was performed in the proximal heme cavity of
SyncyspCPn [38] and SacchceCCP [34] and focused on the
function of the two residues. In mutated catalase–peroxid-
ases, substitutions i n both residues had a pronounced effect:
a decrease in activity and loss of the prosthetic heme group.

(low similarity) to black (highest similarity).
Functionally im port ant residue s invol ved
in the catalytic mechanism are marked with
an asterisk. This figure was constructed using
GENEDOC
[22].
3302 M. Za
´
mocky´ (Eur. J. Biochem. 271) Ó FEBS 2004
36 amino acid-long insertion (between residues Asp268 and
Thr304 in HalomarCPn) which has been suggested to have
a function in the strength of Fe–N co-ordination on the
proximal side [6]. This unique sequence motif in known
KatG structure(s) is in principle a large loop [12] leading
from the edge on the proximal side of heme to the molecular
surface on the distal side. Part of this loop on the surface,
around Glu271 of HalomarCP (Fig. 4A, shown in green),
forms an entrance to the substrate access channel, and the
remainder interacts with the C-domain of the neighboring
subunit [12]. This loop contributes to the typical organiza-
tion of the substrate channel to the active site (compare
Fig. 4A with Fig. 4C and Fig. 4D), not surprisingly as it is a
very flexible region which could not be located i n the
electron density map. The role of this unique insertion has
been examined in MyctubCPn by mutating Ser315 to a
threonine [39]. The mutated protein did not activate
isoniazid because o f th e introduction of a steric hindrance
in the access c hannel. Hence, it is very likely that this
extension from the proximal side to the substrate channel
guarantees efficient catalytic reaction of catalase–peroxid-

by the
PUZZLE
method revealed four main clades of heme
peroxidases belonging t o class I of the superfamily of
bacterial, fungal, and plant heme peroxidases. In Fig. 5 the
simplified inferred
FITCH
tree, is presented, and in Fig. 6
the same tree in a simplified form with an outgroup is
shown. The NJ-reconstructed tree exhibited identical topo-
logy with slightly different branch lengths, and a very similar
maximum likelihood tree was revealed b y
PUZZLE
.Thelatter
method w i th the Ô+GÕ option f or rates of heterogeneity
Fig. 4. Structural comparison of four representatives of class I of the superfamily of bacterial, fungal, and plant peroxidases. (A) N-terminal domain
and (B) C-terminal domain of catalase–peroxidase from Haloarcula marismortui (PDB code 1ITK); (C) S. cerevisiae CCP (PDB code 2CYP);
(D) cytosolic APX from Pisum sativum (PDB code 1APX). All figures are in solid ribbon presentation. In (A), (C) and (D) the prosthetic heme
group is presented in ball and stick presentation. Functionally important conserved r esidues discussed in the te xt and marked also in Fig. 1 are
shown with their corresponding number in the amino acid sequence. Those on the distal s ide of the prosthetic heme grou p are coloured yellow and
those o n the proxim al side blue. The large loop in catalase–peroxidases with an essential residue in the entrance of a substrate chann el is coloured
greenin(A).
3304 M. Za
´
mocky´ (Eur. J. Biochem. 271) Ó FEBS 2004
produced the parameter a ¼ 1.96 estimated from t he actual
data set of 94 a nalysed peroxidase sequences. The bootstrap
support in all main nodes is strong; only in some minor
nodes is refining of the particular species within groups
moderate. In the case of

Whereas in some complete genomes, no KatG is present
(as discussed below), some bacteria even contain t wo
different ones (e.g. Mycobacterium fortuitum). This unequal
distribution of KatGs can be attributed to a lateral gene
transfer [41] between otherwise phylogenetically unrelated
micro-organisms. In the case of KatGs, it was first
proposed to occur between archaea and eubacteria based
on the analysis of three archaeal and 16 bacterial KatGs
[42]. Later it was postulated that this phenomenon often
occurs in all lineages of hydroperoxidases capable of
catalytic reaction [43]. From the phylogenetic tree presen-
ted here (with 34 KatGs divided in the separate domains), it
is obvious that archaeal and eubacterial KatGsare
phylogenetically more closely related than the rest of the
genomes. Moreover, several lateral gene transfer events are
discernible in t he phylogenetic tree in both branches of
catalase–peroxidases (Fig. 5). Interestingly, the sequence of
ArchfulCP segregated very early on from the remaining
known KatGs. In th is paper, I focus on the analysis of
lateral gene transfer between KatGs of Firmicutes, Cyano-
bacteria, and Proteobacteria, which is also supported by
high bootstrap values for both domains. Their positions on
the branches indicate that KatGs from pathogenic proteo-
bacteria are d escendants o f gen es from Firmicute s and
Cyanobacteria. T here is also an obvious discrepancy
between the rather high GC c ontent o f p roteobacterial
KatGs a nd the GC content of the whole o rganism
(Table 2), s upporting the hypothesis on the direction of
the lateral gene transfer. Pathogenic and soil proteobacteria
could profit from such a mode of lateral gene transfer by

occur in Ascomycetes. However, they are not present in all
members of this phylum (e.g. they are not present in the
genome of S. cerevisiae). From Fig. 5, it is obvious that
these eukaryotic KatGs do not represent a separate evolu-
tionary branch but arose from proteobacterial catalase–
peroxidase genes via a single lateral gene transfer. Again, t he
GC content of s ome ascomycetous KatG s is slightly higher
than that of the corresponding genome, but for s ome
representatives (e.g. BlumgraCP) the relatively low number
of coding seq uences currently in the database may affect this
value. Interestingly, none of the known fungal genomic
DNA sequences in which KatG is included (listed in
Table 1) contain an intron, so the KatGs in these genomes
were not subjected to rearrangements cause d by these
mobile genetic elements. More data on the distribution of
introns in ascomycetous genomes and more genomic
sequences of KatGs are needed to analyse this phenomenon.
APXs are known to exist in Rhodophyta, Viridiplantae and
Euglenozoa. No bacterial APX gene has yet been found
(although t here have been repo rts of APX activity in
Cyanobacteria; see [15] for a detailed discussion). In the
evolutionary branch of the APX family (Fig. 5, upper part),
we can see that one APX from Rhodophyta and one from
Euglenozoa segregated early i n the diversification of this
clade. The remaining APXs can b e divided into two distin ct
subbranches. The subbranch of cytosolic APXs currently
only has members from the cytoplasm of higher plants.
Only selected examples are presented in this tree, but in
several plant species multiple forms of genes coding for
cytosolic and chloroplast APXs exist as a rule, indicating a

[46] is not sufficient to determine the distribution and
evolution of such genes in higher animals. No complete
APX sequences from the kingdom Animalia are y et known.
Only three closely re lated ascomycetous CCPs are
currently known that belong to this peroxidase superfamily
(bacterial CCPs are clearly unrelated). Of t hese, only
S. cerevisiae CCP has been studied in detail, and its
structure [7] has served as a paradigm for structural biology
for two decades. Because of their high sequence similarity,
the two remaining C CPs are expected to have similar
structures and functions. The number o f similar CCPs will
grow with the ongoing sequencing projects, but, at t he
moment, w e c an only be s ure a bout the presence o f t his type
of CCP gene in Ascomycota. Most probably, the ancestral
peroxidase gene which was already adapted to APX and
CCP specificity was introduced o nce into the fungal
progenitor, and all extant ascomycetous CCPs are offspring
of this single gene transfer. It is also evident that, in a
complete fungal genome, two independent evolutionary
lines of class I of this superfamily occur, i.e. the CCP line
and the KatG fusion line, as is the c ase for Neuro spora crassa
[47].
Evolution by the birth-and-death process in class I of the
superfamily of bacterial, fungal, and plant peroxidases
Gene duplication is one of the most common evolutionary
processes b y which new genes arise. From the results
presented, it is obvious that gene duplication must have
occurred repeatedly in the superfamily of bacterial, fungal,
and plant heme peroxidases. In class I, this event apparently
occurred i n e ach i mportant period of evolution, firstly in the

etic diversity) was generated by the introduction of new
Ó FEBS 2004 Molecular evolution of heme peroxidases (Eur. J. Biochem. 271) 3307
variants from different lo ci t hrough interlocus recombi-
nation or gene conversion. M ultiple sequence alignment
of the whole of class I, as well as the high conservation
of the secondary-structure elements for ming the typical
peroxidase structural fold (Fig. 4) for all known 3D
structures, does not indicate a robust conversion of
analysed peroxidase genes or indications for recombina-
tion with different loci. In contrast, the basic criteria for
evolution by the birth-and-death process are fulfilled: (a)
the gene duplication event must have occurred s everal
times; (b) the representatives of the respective evolution-
ary branches in one organism are more distantly related
to each other than their counterpar ts from d ifferent
species (tracked in Fig. 5 for sequences of APX and
KatG,andinthecaseofN. crassa two divergent evolu-
tionary lines occur in its genome); (c) at least some of
the duplicated genes subsequently became nonfunctional
(evidence in all currently sequenced catalase–peroxidases:
the catalytically inactive C-domains with as yet unknown
or only hypothetical noncatalytic fun ction).
So we can conclude that the evolution of this class
probably occurred t hrough the death-and-birth process.
WhetherthisisalsotrueforclassIIandclassIIIremainsto
be proved. It is clear that this mode of evolution in heme
proteins can create novel reaction specificities, thus repre-
senting significant evolutionary advantage in both prokary-
otic and eukaryotic genomes.
Conclusion

5. Welinder, K.G. (1992) Superfamily of plant, fungal and bacterial
peroxid ase s. Curr. Opin. Struct. Biol. 2, 388–393.
6. Za
´
mocky´ ,M.,Regelsberger,G.,Jakopitsch,C.&Obinger,C.
(2001) The molecular peculiarities of catalase-peroxidases. FEBS
Lett. 492, 177–182.
7. Finzel, B.C., Poulos, T.L. & Kraut, J. (1984) Crystal structure of
yeast cytochrome c peroxidase refined at 1.7-A
˚
resolution. J. Biol.
Chem. 259, 13027–13036.
8. Patterson, W.R. & Poulos, T .L. (1995) Crystal structure of
recombinant pea cytosolic ascorbate peroxidase. Biochemistry 34,
4331–4341.
9. Sharp, K.H., Mewies, M., M oody, P.C. & Rave n, E.L. (2003)
Crystal structure of the ascorbate-peroxidase ascorbate complex.
Nat. Struct. Biol. 10, 303–307.
10. Wada,K.,Tada,T.,Nakamura,Y.,Kinoshita,T.,Tamoi,M.,
Sigeoka, S. & Nishimura, K. (2002) Crystallization and pre-
liminary X-ray diffraction studies of catalase-peroxidase from
Synechococcus PCC 7942. Acta Crystallogr. D 58, 157–159.
11. Ca rp ena, X., Gua rn e, A., Ferre r, J.C., Alz ari, P.M., F ita , I. &
Loewen, P.C. (2002) Crystallization and preliminary X-ray ana-
lysis of t he h ydroperoxidase I C-terminal domain fro m Escherichia
coli. Acta Crystallogr. D 58, 853–855.
12. Yamada, Y., Fujiwara, T., Sato, T., Igarashi, N. & Tanaka, N.
(2002) The 2.0A
˚
crystal structure of catalase-peroxidase from

NatlAcad.Sci.USA97, 10866–10871.
20. Nei, M., Gu, X. & Sitnikova, T. (1997) Evolution by the birth-
and-death process in multigene families of the vertebrate i mm une
system. Proc.NatlAcad.Sci.USA94, 7799–7806.
21. Jeanmougin, F., Thompson,J.D.,Gouy,M.,Higgins,M.&
Gibson, T.J. (1998) Multiple sequence alignment with ClustalX.
Trends Biochem. Sci. 23, 403–405.
22. Nicholas, K.B. & Nicholas, H.B. Jr (1997) GeneDoc: a tool for
editing and annotating multip le sequence alignments. (Distrib uted
by the author.)
23. Kumar, S., Tamura, K., Jakobsen, I.B. & N ei, M . ( 2001) MEGA
2: molecular evolutionary genetics analysis software. Bioinfor-
matics 17, 1244–1245.
24. Felsenstein, J. (1996) Inferring phylogenies from protein sequenc es
by parsimony, distance, and likelihood method. Methods Enzy-
mol. 266, 418–427.
25. Page, R.D.M. (1996) Treeview: an application to display phylo-
genetic trees on personal computers. Comput. Appl. Biosci. 12,
357–358.
26. Strimmer, K. & von Haeseler, A. (1996) Quartet puzzling: a
quartet maximum likelihood method for reconstructing tree
topologies. Mol. Biol. Evol. 13, 964–969.
27. Whelan, S. & Goldman, N. (2001) A general empirical model of
protein evolution derived from multiple protein families using a
maximum-likelihood approach. Mol. Biol. Evol. 18, 691–699.
28. Laskowski, R.A. (2001) PDBsum: summaries and analyses of
PDB structures. Nucleic Acids Res. 29, 221–222.
3308 M. Za
´
mocky´ (Eur. J. Biochem. 271) Ó FEBS 2004

¨
ller, P.G., R u
¨
ker, F. & Obinger, C. (2003) The catalytic
role of the distal asparagine-histidine couple in cat alase-peroxi-
dases. Eur. J. Biochem. 270, 1006–1013.
36. Jespersen, H.M., Kjaersgard, I.V.H., Ostergaard, L. & Welinder,
K.G. (1997) From sequence analysis of three novel ascorbate
peroxid ase s f rom Arabidopsis thaliana to structu re, function and
evolution of seven types of a scorbate peroxidase. Biochem. J. 326,
305–310.
37. Sivaraja, M., Goodin, D.B., Smith, M. & Hoffman, B.M. (1989)
Identification by endor of Trp191 as the free-radical site in cyto-
chrome c pe roxidase in co mpound ES. Science 245, 738–740.
38. Jakopitsch, C., Regelsberger, G., Furtmu
¨
ller, P.G., Ru
¨
ker, F.,
Peschek, G.A. & Obinger, C. (2002) Engineering the proximal
heme cavity of catalase-peroxidases. J. Inorg. Bioc hem. 91, 78–86.
39. Wengenack, N.L., Uhl, J .R., St Amand, A.L., T omlison, A.J.,
Benson,L.M.,Naylor,S.,Kline,B.C.,Cockerill,F.R.&3rd&
Rusnak, F. (1997) Recombinant Mycobacterium tuberculosis KatG
(S315T) is a competent catalase-peroxidase with reduced ac tivity
towards isoniazid. J. Infect. Dis. 176, 7 22–727.
40. Zamocky, M. & Koller, F. (1999) Understanding the structure and
function of catalases: clues from m olecular evolution and in vitro
mutagenesis. Prog. Biophys. Mol. Biol. 72, 19–66.
41. Ragan, M.A. (2001) Detection of lateral gene transfer among