Download Globins in Nonvertebrate Species: Dispersal by Horizontal Gene

Globins in Nonvertebrate Species: Dispersal by Horizontal Gene
Transfer and Evolution of the Structure-Function
Relationships
Luc Moens,* Jacques VanJEeteren,-f Yves Van de Peer,* Kris Peeters,* Oscar Kapp,$
John Czeluzniak, 5 Morris Goodman, 5 Mark Blaxter,ll and Serge Vinogradofl
*Department of Biochemistry,
University of Antwerp; TDepartment of Morphology, Systematics and Ecology,
University of Ghent; *Department of Radiology and Enrico Fermi Institute, University of Chicago;
SDepartment of Anatomy, Wayne State University School of Medicine; IlWellcome Centre for Parasitic Infections,
Department of Biology, Imperial College of Science, Technology and Medicine; and (#Department of Biochemistry,
Wayne State University School of Medicine
Using a new template based on an alignment of 145 nonvertebrate globins we examined several recently determined
sequences of putative globins and globin-like hemeproteins. We propose that all globins have evolved from a family
of ancestral, approx. 17-kDa hemeproteins, which displayed the globin fold and functioned as redox proteins. Once
atmospheric 0, became available the acquisition of oxygen-binding
properties was initiated, culminating in the
various highly specialized functions known at present. During this evolutionary process, we suggest that (1) high
oxygen affinity may have been acquired repeatedly and (2) the formation of chimeric proteins containing both a
globin and a flavin binding domain was an additional and distinct evolutionary trend. Furthermore, globin-like
hemeproteins
encompass hemeproteins
produced through convergent evolution from nonglobin ancestral proteins
to carry out O,-binding functions as well as hemeproteins whose sequences exhibit the loss of some or all of the
structural determinants of the globin fold. We also propose that there occurred two cases of horizontal globin gene
transfer, one from an ancestor common to the ciliates Paramecium and Tetruhymena and the green alga Chlumydomonus to a cyanobacterium
ancestor and the other, from a eukaryote ancestor of the yeasts Succhuromyces and
Cundidu to a bacterial ancestor of the proteobacterial genera Escherichiu, Alculigenes, and Vitreoscillu.
Introduction
In contrast to vertebrate (V) globins, nonvertebrate
(NV) globins exhibit an extensive variability in all aspects of their structures (Vinogradov
1985; Vinogradov
et al. 1993). They comprise single chain, one-domain
globins, two-domain
and multidomain
globins, which
consist of repeated complete globin units, and chimeric
proteins containing a globin domain linked to a flavinbinding protein (Vinogradov
et al. 1993). In addition,
some of these putative globins particularly from bacteria
(Wakabayashi,
Matsubara, and Webster 1986; Vasudevan et al. 1991; Potts et al. 1992; Cramm, Siddiqui, and
Friedrich 1994), unicellular algae (Couture et al. 1994),
protists (Iwaasa, Takagi, and Shikama 1989; Yamauchi,
Ochiai, and Usuki 1992; Takagi 1993; Takagi et al.
1993), and yeasts (Iwaasa, Takagi, and Shikama 1992;
Zhu and Riggs 1992) may perform functions other than
oxygen binding. We have used the alignment of 145 NV
globins (Kapp et al. 1995) to construct a new NV template and employ it to evaluate several NV putative globins and globin-like
sequences. We also attempt to reconstruct the evolution of the structure-function
relaAbbreviations:
Hmp, hemeprotein;
V, vertebrate; NV, nonvertebrate; Hb, hemoglobin;
Mb, myoglobin; UBP, unit evolutionary period.
Key words: globins,
structure,
evolution,
horizontal
gene transfer.
Address for correspondence
and reprints: Prof. Dr. L. Moens, Department of Biochemistry,
University of Antwerp, Universiteitsplein
1,
B-2610 Wilrijk, Belgium. E-mail: [email protected].
Mol. Biol. Evol. 13(2):324-333.
1996
0 1996 by the Society for Molecular Biology and Evolution.
324
ISSN: 0737-4038
tionship of the globin domain, suggest that all extant
globin proteins can be derived from a monotypic ancestral globin, and propose that two horizontal gene transfers may have occurred between eukaryote and prokaryote ancestors.
Materials and Methods
The accession numbers or references for the sequences studied are listed in table 1.
The NV globin template (table 2) was constructed
as follows. One hundred forty five NV globin amino
acid sequences were aligned using the following guidelines: (1) Related globins with known secondary structure were used as references for matching the helical
segments. When the appropriate crystal structures were
not available, the sperm whale myoglobin (Mb) structure was used. (2) The number of positions assigned to
interhelical regions was minimized (just enough to accommodate the longest sequences). (3) The interhelical
regions were aligned with each other heuristically,
accounting for the preferred conservative
amino acid substitutions. The NV globin template was derived as outlined for construction
of the Bashford, Chothia, and
Lesk (1987) template II with the following modifications. Zero penalty scores were assigned to residues occupying any given position in more than 2% of the sequences. Tyr and Trp were classified as polar residues
and given a score of 0.7 when occupying internal positions at frequences below the 2% cutoff value. Resi-
Globin Evolution
in Nonvertebrate
Species
325
Table 1
Species List of Globin Sequences Used in This Study
Alcaligenes eutrophus flavoHmp
.............
Azorhizobium caulinodans Hmp ..............
A. caulinodans MTRC ......................
Aplysia limacina Mb .......................
Bradyrhizobium japonicum Hmp .............
Caenorhabditis elegans Mb .................
Candida norvegensis flavoHmp ..............
Casuarina glauca 1egHb ....................
Chlamydomonas eugametos Hb L14 10 ........
C. eugametos Hb L1637 ....................
Chironomus thumni Hb III ..................
Escherichia coli colicin A ...................
. coliHmp ..............................
Glycera dibranchiata globin MI1 .............
Glycine maxima IegHb .....................
Hansenula anomala Hmp ...................
Mastigocladus laminosus phycocyanin
OL ......
Nostoc commune Mb .......................
Nostoc sp. Mb ............................
Paramecium caudatum Hb ..................
Parasponia andersonii 1egHb ................
Phaseolus vulgaris 1egHb ...................
Physeter catodon Mb .......................
Rhizobium meliloti Hmp ....................
Saccharomyces cerevisiae flavoHmp ..........
Scapharca inaequivalvis HbI ................
Sesbania rostrata 1egHb II ..................
Sulculus diversicolor “Mb”
.................
Tetrahymena pyriformis Hb .................
Tetrahymena thermophyla Hb ...............
Urechis caupo Hb FI .......................
Vitreoscilla sp. Hb .........................
Proteobacteria
Proteobacteria
Proteobacteria
Mollusca
Proteobacteria
Nematoda
Fungi (Deuteromycota)
Plantae (Spermatophyta)
Plantae (Chlorophyta)
Plantae (Chlorophyta)
Insecta
Proteobacteria
Proteobacteria
Annelida
Plantae (Spermatophyta)
Fungi (Ascomycota)
Cyanobacteria
Cyanobacteria
Cyanobacteria
Protozoa (Ciliophora)
Plantae (Spermatophyta)
Plantae (Spermatophyta)
Mammalia
Proteobacteria
Fungi (Ascomycota)
Mollusca
Plantae (Spermatophyta)
Mollusca
Protozoa (Ciliophora)
Protozoa (Ciliophora)
Echiura
Proteobacteria
dues occurring at surface positions with charge restriction were assigned scores of 0.5 and 0.2 when having
the opposite charge or no charge, respectively.
Scores
of 0.5 and 1.0 were assigned to Val and Trp or Tyr when
they occupied these positions at frequencies below the
cut-off value. At positions with volume restriction, penalty scores were assigned as to internal positions.
Evolutionary
trees based on the neighbor-joining
method (Saitou and Nei 1987) were constructed using
the software package TREECON version 3.0, which was
adapted for the analysis of amino acid sequences, including bootstrapping
(Van de Peer and De Wachter
1993, 1994). Maximum
parsimony
analysis was performed using the PHYLIP software package (version
3.5) of Felsenstein
(1993).
Results and Discussion
Putative Globins and Globin-like
Sequences
The stable folding of a polypeptide
strand around
the heme group is mainly dictated by the hydrophobic
x74334
ACFIXLJ
ACNTRYXA
A0253 1
BJFIXLJ
211115
X68849
SO0560
X72915
X72916
A0255 1
Ml5691
S15992
A02538
A02560
S55723
M75599
M92437
SO5230
A02563
PO2234
PO2185
RHMFIXLJG
A45383
A02535
SO2206
S50084
D13920
D13919
A25537
A02564
Genbank
Genbank
EMBL
PIR
Genbank
Genbank
Genbank
PIR
Genbank
Genbank
Genbank
Genbank
PIR
PIR
PIR
PIR
Genbank
Genbank
(Mulligan
PIR
PIR
PIR
PIR
Genbank
PIR
PIR
PIR
Genbank
Genbank
Genbank
PIR
PIR
1993)
nature of the residues involved in protein-to-heme
and
protein-to-protein
contacts. Nature evolved a number of
protein folds (e.g., cytochrome c, cytochrome b, catalase, etc.) surrounding
the heme group, thereby modulating its functional versatility. As based on a number
of crystal structures, the globin fold consists of seven to
eight helices (A-H) that are connected by short loops
and lock up the heme moiety within a three-on-three
helical sandwich (Pastore and Lesk 1990; Holm and
Sander 1993). There exists considerable
tolerance toward both globin size variation and amino acid substitution, however. Alignment
of several hundreds of V
and NV globins necessitates the use of 182 positions, of
which less than half, 84 positions, are common to all
the globins (Kapp et al. 1995). Amino acid residues that
specify the globin fold are restricted by the hydrophobicity and volume of their side chains. Their restrictions
can be described by templates, and the deviation of the
standard pattern as penalty scores (Bashford, Chothia,
and Lesk 1987). However, the Bashford templates I and
326
Moens et al.
Table 2
Nonvertebrate
Table 2
Continued
Globin Template
PENALTY
0.0
SCORE
0.2
PENALTY
0.5
RKDENQHGASCT
RKDENQHGASCT
ASCTVILM
RKDENQHGASCT
ILMFYW
RKDENQHGASCTP
ASCTVILMF
PV
PV
F
GYW
PV
V
GASCT
V
GYW
Pattern B-C
.. s
.. i
.. i
.. i
.. i
GASCTPDNVE
ASCTVILMF
ILMFYW
VILMFY
ILMF
P
.. i
ASCTVILMFY
.. s
RKDENQHGASCTP
. . req F
. . s RKDENQHGASCTP
Q
V
T
V
G
ST
HLIMK
GYW
GASCT
GASCW
GASCTYW
AQ
GW
V
V
Pattern E
El
E2
E3
E4
E5
E6
E7
E8
El0
El1
El2
El5
El8
El9
E20
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
0.2
0.5
0.7
Pattern H
A4 . . . s
A6 . . . s
A8 . . . i
A10 . . s
Al2 . . i
Al3 . . s
Al5 . . i
B6 .
B9 .
BlO
B13
B14
C2...i
C4 .
C6 .
CD1
CD2
0.0
0.7
Pattern A
SCORE
s
s
s
i
s
s
i
i
.. s
.. i
.. i
.. i
.. i
.. i
.. s
RKDENQHGASCT
RKDENQHGASCTP
RKDENQHGASCTP
GASCTVILMF
RKDENQHGASCTP
RKDENQHGASCTP
PV
V
V
V
V
VL
HQ
GASCTDP
RKDENQHGASCT
ILMF
GASCTVILMFYW
CTVILMF
GASCTVILMFY
ASCTVIL
RKDENQHGASCT
NV
EQHILMKR
PV
V
GASCTYW
S
GAYW
W
GFYW
M
PV
Pattern F
F2 . . . s
RKDENQHGASCT
F3 . . . s
RKDENQHGASCTP
F4 . . . i
TVILMF
F8 . . . req H
FlO . . s
RKDENQHGASCTP
PV
V
C
GASYW
V
Pattern G
FG4 . . i
G5 . . . i
G8 . . . i
GlO . . s
G12 . . i
G15 . . i
G16 . . i
G17 . . s
G18 . . s
TVILM
ILMFYW
ASCTVILMF
RKDENQHGASCTP
VILMF
ASCTVILMFY
VILMF
RKDENQHGASCTP
RKDENQHGASCT
CF
V
GASYW
GASCT
GYW
V
T
GASCYW
GW
GASCYW
T
V
PV
H6
H7
H8
H9
Hll
H12
H13
H15
H17
H19
.
.
.
.
.
.
.
.
.
.
.
.
.
.
..
..
..
..
..
NOTE.-s
s
s
i
s
i
i
s
i
s
i
RKDENQHGASCTP
GAS
ILMFYW
RKDENQHGASCT
GASCTVILMF
GASCTVILMFYW
RKDENQHGASCT
TVILMF
RKDENQHGASCT
GASCTVILMF
= Surface position;
V
CT
V
DPNVEQHLI
GASCT
PV
PV
C
GASYW
PV
i = internal position;
YW
req = essential residue.
II are considerably
biased toward V sequences as the
aligned set of 226 globin sequences comprised only
about 10% NV sequences. Our template is based on 145
NV sequences exclusively
(table 2). This template reflects the high tolerance to amino acid substitution seen
in NV globin. As a result it is generally less restrictive
than the V-based Bashford template II. Some additional
stringency was introduced by giving more weight to volume and charge restriction, classifying Trp and Tyr as
polar instead of hydrophobic
residues, and restricting
zero penalty score assignment to internal residues present in no less than 2% of the sequences. Figure 1 compares the scores obtained by the Bashford, Chothia, and
Lesk (1987) V and our NV templates. Although the V
globins match up well against both templates, the NV
template yields superior results with the NV sequences.
Several putative globins and globin-like
heme proteins
were evaluated by the above criteria. Figure 2 represents
the block of aligned sequences comprising (A) two representative
“true” globins, sperm whale Mb and lupin
hemoglobin
(Hb), (B) several representative
putative
globins
and (C), several globin-like
hemeproteins
(Hmp). We also included the alignments of phycocyanin
C and colicin A, which share the three-on-three
helical
sandwich with globins (Pastore and Lesk 1990; Holm
and Sander 1993).
It can be seen in table 3, that the scores obtained
with the group B sequences, although higher than those
obtained with the “true” globins, exemplified by the Mb
and the plant Hb, are much smaller than those calculated
for the group C globin-like
sequences.
This simply
means that the three-dimensional
structures of the proteins listed in group C definitely deviate from the globin
fold. The penalty scores of the NV globin sequences
(table 3, group B) obtained with the nonvertebrate
template are generally lower than those obtained by applying the Bashford template II, suggesting a better match
Globin Evolution
Non-vertebrate
a
alignment
.
a
I
2
3
4
5
6
7
8
9
10 11 12 13 14
Penalty score
New template
m
Vertebrate
Bashford
iemplate
alignment
120
t
g 100
s
;
80
'
$
60
2
1
40
20
0
0
1
2
3
4
5
New template
6 7 8 9
Penalty score
m
10 11 12 13 14
Bashford template
FIG. 1.-Comparison
of penalty scores of V (Q) and NV (b) globin
sequences against the new NV template and the original Bashford,
Chothia, and Lesk (1987) template II.
of their sequences against the NV template. The relatively greater range in penalty scores obtained with this
template reflects the high range of amino acid substitutions at most positions in NV globin sequences. This
variability
tends to become masked when using the
Bashford template due to its weaker performance
with
NV sequences. As a whole, both templates separate the
B and C groups very well. We do expect, however, that
the NV template will prove a superior tool for aligning
and resolving novel NV globin and globin-like sequences to be detected in the future.
It has been argued that the truncated Hbs of the
protists Paramecium
and Tetrahymena
and the cyanabacterium Nostoc, though similar to each other, show no
homology to other globins and might be evolved from
in Nonvertebrate
Species
327
a different ancestral gene (Takagi 1993; Takagi et al.
1993). However, these proteins fit all the essential determinants of the globin fold, and their V and NV templates scores are still comparable to the scores for other
NV globins (table 3). Furthermore, the monomeric globin sequences from the bacterium Vitreoscilla, the green
alga Chlamydomonas,
and the N-terminal
globin domains of the chimeric proteins from the yeasts Saccharomyces and Candida and the Proteobacteria Escherichia and Alcaligenes also appear to be “true” globins by
the above criteria.
In contrast, none of the group C sequences listed
in table 3 meets these criteria. The globin-like domains
of the chimeric proteins from the mollusc SuZcuZus, the
yeast Hansenula, and the Rhizobium spp. can be aligned
to have CD1 Phe and F8 His, only at the expense of a
considerable
increase in gap length. Furthermore,
their
penalty scores are considerably
higher when compared
to those of “true” globins (table 3 groups A and B).
Phycocyanin
C and colicin A share the three-on-three
a-helical sandwich with the globins (Pastore and Lesk
1990; Holm and Sander 1993). However, they lack the
CD1 and F8 His residues and have high scores with both
templates (table 3), in agreement with the earlier suggestion (Holm and Sander 1993) that their three-dimensional structures converged toward a stable three-onthree a-helical sandwich similar to the globin fold. Such
convergence
is not surprising in view of the increased
evidence for a limited number of supersecondary
structures (Dorit, Schoenbach,
and Gilbert 1990; Chothia
1992; Blundell and Johnson 1993; Orengo et al. 1993).
The recent detection of a globin-like fold in diphtheria
toxin (Orengo and Taylor 1993) is probably yet another
example.
Origin
and Early Evolution
of Globin
We hypothesize that redox Hmps existed during the
early Proterozoic, approx. 2,000 Mya, well before dioxygen accumulated
in the atmosphere
(Jenkins 1991).
Oxygen-reactive
globins likely evolved as an offshoot
with the advent of atmospheric
oxygen (Keilin 1966,
p.95-104; Riggs 1991). Structural similarities with phycocyanin, colicin, and diphteria toxin, but also with hemocyanin, hemerythrin (Volbeda and Ho1 1982), and the
cytochromes
b2 and b5 (Runnegar
1984) may reflect
very remote common ancestry as well as convergence.
The origin of globin is rather hypothetical, but stronger
evidence exists for the hypothesis that ancestral globin
functioned as an oxygen-utilizing
enzyme. The reactivity with O2 and its binding are determined by the nature
of the amino acid residues surrounding the heme (Perutz
1989). Many V and NV Hbs and Mbs show monooxygenase activity and their ferric derivatives
can be reduced by NADH (Ferraiolo and Mieyal 1982; Ferraiolo,
328
Moens et al.
6
7
5
3
4
8
9
1
2
xx12345678901234567-89012345678901234-567890--12345678901-2345678-------------9012345678901234-56789012345678901234567890
10
15
20
15
10
15 1
1
1
5
1
5
1
5
10
15
5
Max xx___-----____~_AAAAAAAAAAAA--________
BBBBB--BBBBBBBBBBB-CCCCCCC-------------CCCC-----DDDDDDD-EEEEEEEEEEEEEEEEEEEEEEEEEE
D
Mbfld------------NN~-~~a~~--------BBB~--BBBbbB_~BB-CCCcC~C-------------C~CCCC-CCDDDDDDD-EEEeE~e~e~eeE~ee~-----AA
DDDDDD-DD
(a)
Phc
GIY
Ure
Sea
Apl
Chi
Gli
LUP
1
0
------------VLSEGEW-QLVLHVWAKVEA-------DVAGH--GQDILI~S-HPETLEK-------------FDRFKH-LKTEAEMKA-SEDLKKHGVTVLTALGH
------------GLSAAQR-QVIAATWKDIAGAD--------NGAGV--GKDCLI~SA-HP~V-------------FG-FSG--------AS-DP
GVAALGAKVLAQIGVAVSHL--GD
------------GLTTAQI-KAIaDHWFLNIKGC-_------LQ~--ADSIFFK~TA-YPGDLAF-------------FHKFSS-V-PLYGLRS-NPAY~~L~~D~~G------PSVYDAAAQLTADVK-KDLRDSWKVIGS---------DKKGN--GV~T~AD-NQETIGY-------------FK~-GNV-S~-M-A-ND~RGHSIT~~QNFIDQL---D
------------SLSAAEA-DLAGKSWAPVFA---------NKN~--GLD~V~EK-FPDS~F-------------FADFKGK--SVADI~-SP~R~S~~E~---~
-------------LSADQI-STvQASFDKvKG-----------D--PVGILYAVFKA_DSKIIGEL--------------VAFTEKQD-ALVSSSFEAFKA---------NIPQY--S~TSILEK-~~DL-------------FSFL----~GVDP-T-NP~TG~~~~~QL~SG
-----------GALTESQA-ALVKSSWEEFNA----------NIPKH--TH~IL~EI-APAAKDL-------------FSFLKG--TSEV-PQN-NPELQ~~~~~QLE~G
(b)
Cd
Pha
SeS
Cas
Pan
Par
TeP
Tet
CYP
CYM
Ch4
Ch6
Vit
Hmp
Ale
Sac
Can
-----------SMNRQEIS-DLCVKSLEGRMVGTEA----QNIEN--GNA~Y~TN-FPDLRVY-------------~G-AEKYT-ADDVKK-SE~DK~~L~HL~-V--YT
-----------GAFTEKQE-ALVNSBWEAE'K G-------NIPQY--SVVFYTSILEK-APAAKNL-------------FSFL----ANGVDP-T-NPKLTAS~a~~QLRANG
------------GFTDKQE-ALVNASYEK-------NLPGH--SVLFYSFILEK-EPAAKGL-------------FSFLKD--SDGV-PQN-NPSLQAHAEKVFC;LVHDAG
CC)
PW
CoA
Sul
Han
Rhm
Acf
Azo
Bjf
1
1
1
1
1
1
1
1
1
6
7
3
4
5
8
1
2
0
12345678--------9-012345678901234-5--67890123456789012345---67890-12345678901234567890123456789012xxxxx
15
20
25
1
5 PlO
1
5
10
15
1
5
10
FFFFFFFF--------F-FFFFFFFFFFF--------GGGGGGGGGGGGGG~GGGGG--------HHHHHHHHH~HHHHHHHHHHHHHHHH-----xxxxx
P
----------------F-FFffFFFFFFX--FF_F--FF-F--~~G~~~GgGGgg~GX------- ---HHHHHHHhHHhhHHhHHHhXHHHHHHHHHH--------G---GG-G
GGGG
(a)
Phc
Gly
Ure
Sea
Apl
Chi
Gli
LUP
EAE-------------L-KPLAQSHATKH--K--I--PIKYLEFISEAIIHVLHSR----HPGDF-GADAQGAMNKALELFRKDIAAKYKELGYQG-------EGKMVAQ--------M-KAVCVRHKGYG-NKH-I--KAQISGLQS-------------GNAGAL--------M-KSHDA--MG---I--TP~~QLLKL~~QEEF-SA------DPTTV~~DMG~V~----------------NPDDLVCV--------V-EKHIT----RK-I-_SAA-----------------NAGKMSAM--------L-SQFAKEHVGFG-----V--GSAQFENVRSMFPGFVAS-VAAPP------AGALIIDALKAAGK--------------PNIEAD--------V-NTFVASHKPRG-----V-_THD-------------TW----A--------D-AALGSVHAQKA-------TDPQ---------------VWSDAT--------L-KNLGSVHVSKG-----V--AD~FP~EAILKTIKEWGA----KW-SEELNSA~IAYDE~IVIKKEMDD~----------
(b)
Gel
Pha
SaS
Cas
Pan
Par
Tap
Tet
CYP
CYM
Ch4
Ch6
Vit
MP
Ale
Sac
Can
NEEVFKGY--------V-RETINRIIRI--Y-K--M--DPALWMAFFTVFTGnESNLPHV-----AV----VA--------D-ASIHSQKG-----V--NDSQFL~~LK~K~VGD----~-TDELST~E~D~IK~YA------------WV---LA--------D-ASLSVBVQKG----V-_TDPHS------------HAWYDNNT--------L-KRLCSIHLKN---K--I--TDPHFEVMKGALLGTIKEIKE----NW-SDEMGQA~EAYNQLVATIKAEMKE------------KVTVKESD--------L-KRIGAIHFKTG-----V--~E~~RFALLPSST--------NA--WT----------G-RNLICEVHAN--MG---V--SNAQFPTVIGHLRSAGTGA-GV---AAA-LVEQTVAVAET-----------------EHT?mGK_--_____------NH--YK----------G-K~~KG--M-N--L--QNLHFDAIIE~TLKE-LGV-------TDAVIN~
NH__YK_-----_-_-G-Km
KG--M-N--L--QNSHFDAIIENLAAl'LKE-LGV-------SDQIIGEAAKVI EHTRKDCLGK---_________--KQ--YG----------G-RPKTHAG--L-N--L--QQPILNK--------------KQ--YG----------G-RPKTHAG--L-N--L--QQPILNK--------------AE--WK----------G-KCMRTAHKD--LVPH-L--TDV~QAW~LSD~E-L---GVT---PGDIAD~~TKTE~~PR~G~SNR-----SE--WK----------G-KCMRTARKD--LVPH-L--SDVHFQA~SD~TE-L---GVP---PEDITD~~STRTE~~P~------------LPAILPA---------V-KKIAVKRCQ-----AG-V--~YPI~Q~L~KEVL---GD~--TDDILDA~~GVI~~IQVEADLYA~VE----LPALLPA---------V-EK~KHTS--F--Q-I--KPEQ~I~E~LA~DEMF----SP---GQEVLDA~~G~
INREAF,IYNENAS----PNSLMAV---------L-KNIANKIIAS--L--G-V--KPEQYPIVGEHLL~KEVL---GN~--TDDIISA~~G~D~GMESELYE~~----LSVLMDH---------V-KQIGHKHRA--L--L--Q-I--KPEHYPIVGE~L~~VL---GD~--TPEIINAWGEAYQAIADIFITVEKKMYEEAL------LTPISGF---------V-NQIVLKHCG--LG---I--KPDQYPWGESLVQAPKQTLIDAEASVYKTLA------
(c)
Phy
CoA
SUl
Han
Rhm
Acf
Azo
Bjf
FIG. 2.-Alignment
known crystal structure:
of some NV globins and globin-like sequences with globins of known three-dimensional
structure. (a) Globins with
Physeter catodon (Phc), Glyceru dibrunchiutu MI1 (Gly), Urechis cuupo (Ure), Scuphurcu inequivulvis I (Sea), Aplysiu
Globin Evolution
Table 3
Penalty Scores Against the V Template of Bashford,
Chothia, and Lesk (1987) and the New NV Template
Group
A .....
Organism/protein
NV
Template
V
Template
Physeter Mb
Lupinus Hb
0.2
2.2
0.5
2.0
8.8
4.4
5.9
7.3
8.3
0.9
3.9
1.2
0.7
3.1
12.8
8.4
10.9
13.1
12.3
6.5
9.8
7.8
7.7
7.8
14.1
18.2
25.0
25.0
20.4
27.0
22.2
22.9
18.7
22.9
29.1
27.6
20.7
31.2
21.9
22.0
B
.. ...
Paramecium Hb
Tetrahymena pyriformis Hb
Tetrahymena thermophyla Hb
Chlamydomonas 4
Nostoc commune
Saccharomyces flavoHb
Candida flavoHb
Vitreoscilla Hb
Escherichia coli HMP
Alcaligenes Hmp
C
.....
Sulculus Mb
Hansenula Hmp
Rhizobium Hmp
Azorhizobium (Acf) Hmp
Azorhizobium (MTRC) Hmp
Bradirhizobium Hmp
Phycocyanin
C
E. coli colicin A
Onady, and Mieyal 1984; Bakan et al. 1991). Structurally engineered
cytochrome
c in which Met 80 is replaced by Ala, evinces spectral characteristics
resembling those of Mb and is capable of reversible O2 binding to the Fe 2+ heme (Wallace and Clark-Levis
1992;
Lu et al. 1993). The purple sulfur bacterium Chromatium vinosum has a covalently bound heme able to bind
O2 and CO, exhibiting spectroscopic
properties similar
to ferric Mb (Gaul, Gray, and Knaff 1983). A number
of bacterial oxidases and mixed function oxidases, collectively called cytochromes
o, are Hmps able to bind
CO, CN, and O2 (Jurtshuk and Yang 1980; Poole 1984).
The creation of a reversibly oxygen-binding
cytochrome c mentioned previously illustrates that a functional shift can arise from minor structural changes in
the vicinity of the heme group without any alteration in
the protein fold. Thus functional shifts are more readily
in Nonvertebrate
Species
329
achieved than fundamental
alterations
of the protein
fold. Hence, we assume that globin sequences that exhibit all the determinants
of the globin fold (A and B,
but not C groups of fig. 1 and table 3) are derived from
a direct common monomeric globin ancestor.
Evolution
of Oxygen
Affinity
As the concentration
of atmospheric O2 increased
after the radiation of the photosynthetic
bacteria, about
2,000 Mya (Jenkins 1991), specialized proteins evolved
to protect cells against oxidative damage, e.g., superoxide dismutase and the various peroxidases,
some of
which are Hmps (Hassan and Schiavone
1991). Conceivably, ancestral globin was also recruited for oxygen
binding and scavenging. This would imply a selective
pressure toward enhanced
O2 affinity. In the plant
1egHbs and globin from the yeast Candida (Oshino et
al. 1973) and the trematode flatworm Dicrocoelium (Di
Iorio et al. 1985; Smit et al. 1986) this is achieved by
a very high “on” rate. In contrast, globin from the insect
Gastrophilus (Phelps et al. 1972) and, particularly,
the
pseudocoelic globin from the nematode Ascaris (Gibson
et al. 1993) have high oxygen avidity resulting from a
very low “off” rate. The clam Lucina has two globin
species: type I being characterized by a high “on” rate,
type II having a slow “off” rate (Kraus and Wittenberg
1990). The simplest conclusion is that high oxygen affinity may have evolved repeatedly in the past. The molecular basis of high oxygen avidity is generally unknown, but in Ascaris pseudocoelic globin it appears to
be due to the formation of a second hydrogen bond between O2 and the hydroxyl group of B 10 Tyr, in addition
to the usual one formed with E7 Gln (His) (De Baere
et al. 1994; Kloek et al. 1994). Because these residues
occur in Ascaris muscle globin (Blaxter et al. 1994) and
several other NV globins, such as plant Hbs and the
chimeric Hbs of the yeasts Saccharomyces and Candida
(Oshino et al. 1973; Gibson et al. 1989; Iwaasa, Takagi,
and Shikama 1992; Zhu and Riggs 1992), which have
much higher “off” rates, other residues are probably
needed to position the B helix properly with respect to
the heme.
c
limacina (Apl), Chironomus tumni III (Chi), Glycine maxima (Gli), Lupinus luteus II (Lup). (b) Monomeric globin from Caenorhabditis elegans
(Cel), Phaseolus vulgaris (Pha), Sesbania rostrata II (Ses), Casuarina glauca I (Cas), and Parasponia andersonii (Pan), Nostoc commune (CyP)
and Nostoc sp. (CyM), and the globin domains of the chimeric flavoHmps from Saccharomyces cerevisiae (Sac), Candida norvegensis (Can),
Escherichia coli (HMP), and Alcaligenes eutorophus (Ale). (c) Phycocyanin
(Ychain from Mastigocladus laminosus (Phy), the carboxy-terminal
fragment of colicine A (CoA) from Escherichia coli, and the globin-like domain in Hmps from Sulculus diversicolor (Sul), Hansenula anomala
(Han), Rhizobium meliloti (Rhm), Azorhizobium caulonidans (Acf and Azo, the MTRC gene), and Bradyrhizobium japonicum (Bjf). The
characteristics
of the globin fold are indicated on top of the block of aligned sequences. The maximum length (182 positions) of the helical
segments is indicated as Max. The myoglobin fold (Mb fold) displays the template I of Bashford, Chothia, and Lesk (1987), which consists of
71 positions including 34 interior positions with low, medium, and severe restrictions in size (indicated by lowercase, lowercase boldface, and
capital boldface letters, respectively),
32 positions occupied by surface residues (underlined), and 5 positions where any residue is acceptable
(boldface X). The distal and proximal residues are indicated by D and I?
330
Moens et al.
Evolution
of Chime&
A
Globins
A different evolutionary
trend is highlighted by the
chimeric flavoHbs of bacteria and yeasts (Vasudevan et
al. 1991; Iwaasa, Takagi, and Shikama 1992; Zhu and
Riggs 1992; Cramm, Siddiqui,
and Friedrich
1994),
which are part of group B (table 3). The globin and the
C-terminal
FAD-binding
domains
show considerable
homology to corresponding
domains in other members
of the group. The flavin-binding
domain appears to be
homologous
to other flavoprotein
reductases: NADPH
sulfite reductase (Ostrowski
et al. 1989), toluate 1,2
dioxygenase
(Neidle et al. 1991), cytochrome P450 reductase (Karplus, Daniels, and Herriott 1991), and nitric
oxide synthase (Bredt et al. 1991). The HMP protein of
E. coli acts as a dihydropteridine
and ferrisiderophore
reductase (Vasudevan et al. 1991; Andrews et al. 1992).
Because O2 binding by the heme moiety limits FAD
reduction, HMP could serve as an oxygen sensor, which
might regulate the activity of transcriptional
regulator
proteins (Poole, Ioannidis, and Or-ii 1994), such as the
Fnr protein (Spiro and Guest 1991). Vitreoscilla Hb
lacks the second domain but interacts with a separate
NADPH reductase (Dikshit et al. 1989). Reduced AZcaligenes Hmp binds O2 reversibly and mediates reduction of various dyes and cytochrome c (Cramm, Siddiqui, and Friedrich 1994), which is suggestive of superoxide anion production.
Possibly, this Hmp utilizes O2
as a catalyst in the reduction of nitrate or nitrite to nitrous oxide and nitrogen. Vitreoscilla Hb supports aerobic growth in E. coli lacking functional terminal oxidases (Dikshit et al. 1991), and expression of the globin
from Saccharomyces
is considerably enhanced after disruption of the electron transport system. Thus these proteins can act as alternative oxidases, a function also performed in plants by an unrelated flavoHmp (Kumar and
Sol1 1992).
The Hmps from the abalone molluscs SuZcuZus diversicolor (Suzuki and Takagi 1992) and Nordotis madaka (Suzuki 1994) function as a Mb but have sequences more similar to human indoleamine
2,3-dioxygenase.
Alternatively,
linkage of a globin domain to a different
protein may lead to progressive alteration of the structural determinants
of the globin fold during evolution,
as in the case of Hansenula and rhizobial Hmps.
Horizontal
Gene Transfers
in Globin
Evolution
The evolutionary
tree depicted in fig. 3a clearly
resolves three separate clusters, one comprising
metazoan species and plants, a second comprising
ciliates,
Chlamydomonas,
and Nostoc, and a third combining the
two yeasts along with the three Proteobacteria.
Essentially the same picture is obtained using a maximum
parsimony (PROTPARS)
method (fig. 3b). On the contrary, a phylogenetic
tree based on SSU t-RNA sequenc-
Distance
0.1
H
ekgans
Candida
norvegensis
Escherichia coli
Aicaligenes eutrophus
Saccharomyces cerevestae
Vitreoscllla
zs4588~
-i-_
Gly-zera dibranchiata
I
-
Distance
I
Urechis caupo
Chmxwnus thumml
Scaphsrca insquwalvis
Caenorhatditis elegans
Aplysla limacma
Physeter catodon
Pararponla andersonii
Casuanna glaucs
Lupinw luteus
Sesbania rosbats
PhaseOlUSwgans
Giycine max
Tetrahymena tennoph~ia
TefIahymeM pyllfon?llS
Nostoccommure M
Noetoccmmune P
Pararnecitm~caudaium
Chlamydcmonas rhetnatdii 6
Chlamydornonas rheinardii4
saccharomycss csrs”Ieiae
Viiscdla
Eschenchta coli
Alcaligeros eutmphus
Candida “ON~S”SS
0.1
I
FIG. 3.-(u)
Unrooted neighbor-joining
tree constructed from globin sequences including those aligned in fig. 2. The evolutionary distance separating any pair of species is given by summing the lengths
of the connecting branches along the horizontal axis, using the scale
on top, which represents 10 amino acid replacements per 100 amino
acids, corrected for superimposed events. Deletions/insertions
were ignored. Bootstrap values higher than 50% are indicated at each node.
(b) Consensus tree (PROTPARS) with bootstrap values using the same
sequences as in a. One most parsimonious
tree (unrooted) needed a
total of 2,795 substitutions. The trees were constructed using the Philip
3.5 software package of Felsenstein (1993). (c) Neighbor-joining
tree
constructed from 16/18S rRNA sequences from the species (or closely
related species) also listed in a, as well as a few informative additional
data. Interpretation of the tree as in a.
Globin Evolution
in Nonvertebrate
Species
331
A
FAD:pyrophos
<--_--_-_-_-_-_-_-_>
Ale
Hmp
Sac
Can
Wos
FAD:isoallx
<----_-_-_-_-_--_>
lQPGGWKGWRTFVIRKKRPESDVITSFILKPAXGPY
~PQ_PpcYVS_-----_
----VNFEPGQYTSVAIDV--PALGLQQIRQYSLSDKPNGRTYRI
*KAGGWKGTRDFRIVAKTPRSALITSFE
LEPVDGGAV----AEYRPGQY
LGVWIXP--SGFPHQKIRQYSLTRKPDGKGYRIAVKREEGGQ------VS------VKHEMRKNFPAGLVS------l----WPGWKP PEITAKKYVASDIVKFTVKPKFGSGIKLESLPITPGQYITVNTHPIRQKNQYDALRHYSLCSASTKNGLRFA
*----WEEPKDERVTKLVKED~~L~~--FKLK--PIIPGEYISFRWDIIHNPDITDIQPREYSISQDVKENEYRISVR--------DIGIVS------lQ~FASLATNEKEKQ~~KG~EYEEWRWCIMPTIVS
YHTRDGKGPVHHGVCS
NADPIi:rilmse
<--_-_-_----_--_--->
Ale
Hmp
Sac
Can
No8
VHAmRDRLRmAKTYKNLDLFVFY
NLLRDRVRVGDQVKLAAP
YGSFHIDVD----AKTPIVLISGGVGLTPMVSMLK-VALQAPPRQWFVHGARNSA
NWLHNHAMlCDVVKLVAPAGDFFUAVA----DDTPVTLISAGVGQTPMLANLDTUKAGHTAQWWFHAA=GDVHAFAD
KVKKLGQSLPRFTAHTWY
KYLHKDAKVGDKIKLSAPAGDFAINKELIIiQNKVPLVLLSSGVGVTPLLAKLEKQVKCNPNRPIYWIQSSYD~
ELLAKCANVDKII-EHALKDGKDVKLYYSNRSYQ--SKPFRKFFSNLKKKNNGKFKLN-DYINKKLQVGDI VPVHAPVG--THKYDSISKKG-KVAVLAGGIGITPMIPII
SwLNR-IQ~DwPCpV-RGApS--FHLpRNpQvpcILVG~~I~~~~RQ~IQ~~PC~~SKIDHI~~G~
NADPH:adenine
<----_-_--_---_-_>
Ale
_P
Sac
Can
Nos
B
----WPLPEDVQGRDYDYP-CLMVRQIEKSILLPDADYYICGPIP~QHD~~IH~I~~PD~~----------------RQPSKADRAKGQFDSE-GLKDLS
KLEGAFSDPTHQFYLCGPVGFMQFTAKQLVDLGVKQKNIHYE---------------------------VHTDTE-PLID-AAPLKEKSPA1UDW------------------------NYISAE-~INPDEYD~~~A~KF~YLVGKGVSD--~EF~~DP--------------KL~AYSREPDRPKR~QD~~~~~~HI~~DV-~D~IQRI~~SK~A~IS~D~Y~IFG
Distance
0.1
H
HMP
ALC
S
NOS
FOG. 4.-(u)
Alignment of the flavin-binding
domain of the flavoHmps from Succharomyces cerevisiue, Cundidu norvegensis, Escherichiu
coli, and Alculigenes eutrophus with human nitric oxide synthase (Bredt et al. 1991). The putative NADPH- and FAD-binding sites are indicated.
(b) Neighbor-joining
tree constructed from these sequences, with the nitric oxide synthase sequence taken as outgroup. Interpretation of the tree
as in fig. 3.
es of the same or closely related species clearly assigns
all bacteria to a single cluster, well separated from the
eukaryotes (fig. 3~). This agrees well with the generally
accepted
idea that eubacteria
and eukaryotes
have
evolved separately for at least 1,800 million years (Doolittle et al. 1986) and possibly much longer. These conflicting lines of evidence can be reconciled by assuming
that the Nostoc commune ancestor acquired the globin
gene from a common ancestor to Paramecium,
Tetruhymena, and Chlamydomonas.
If the transferred globin
has evolved at the same rate as observed in animals
(UEP approx. 5 Myr) the postulated gene transfer occurred approx. 600 Mya.
The evidence for globin gene transfer in the yeastProteobacteria
cluster (fig. 3~) is even more compelling.
A common ancestor to E. coli, A. eutrophus, and Vitreoscilla acquired a copy of the chimeric flavoHmp
gene from an ancestor of S. cerevisiae not long after it
separated from its earlier common ancestor with C.
norvegensis,
less than 400 Mya. The flavoprotein
domain was subsequently
removed in the line leading to
the Vitreoscilla. This scenario is compatible
with the
evolutionary
tree based on the flavoprotein domains (fig.
4). The branching order of E. coli, A. eutrophus, Vitreoscilia, S. cerevisiae, and C. norvegensis is not informa-
tive as the branches are supported by bootstrapping
values that are too low. Lateral gene transfers between an
E. coli and a eukaryote ancestor were also reported for
cytosolic
glyceraldehyde-3-phosphate
dehydrogenase
(from eukaryote
to prokaryote)
and aldolase type II
(from prokaryote to eukaryote)
(Doolittle et al. 1990;
Smith, Feng, and Doolittle 1992). In both instances the
E. coli sequence clusters with the yeast sequences.
Acknowledgments
This work was supported by grant no 2.0023.94
from the Belgian Fund for Joint Basic Research. J.R.V.
is a Research Director with the National Fund for Scientific Research. S.N.V. is supported in part by grants
from the National Institutes of Health (DK 38674 and
DK 30382). O.H.K. is supported by a grant from the
Department of Energy no. DE-FG02-86-ER60437.
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MANOLO GOUY, reviewing
Accepted
October
2, 1995
editor