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Biochemical Society Transactions ( 1 996) 24
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Amino acid sequence alignment of a ‘small’ citrate
synthase from Aeudomonas aeruginosa PAC 514, with
other citrate synthase sequences.
COLIN G MITCHELL and SEAN CK ANDERSON
Biomedicine and Biotechnology Research Group,
Department of Biological Sciences, Napier University,
Edinburgh, EHlO SDT, U.K.
A complex diversity relating to the Krebs cycle enzyme
citrate synthase (CS), has been shown to exist regarding
the nature and molecular interactions of its subunits [I].
Gram-negative bacteria contain a hexameric ’large’ (L)
enzyme (Mr 240000) which is subject to allosteric control
by NADH. Eukaryotes and Gram-positive bacteria contain
a dimeric ‘small’ (S) enzyme (Mr IOOOOO) which is subject
to isosteric control by ATP. Both types of CS have a single
subunit type, suggesting that the quaternary structure
determines regulatory properties. A strain of Pseudomonas
aeruginosa was shown to possess both the L- and S-forms
of CS This was an unusual finding and further studies of
the Pseudomonas group revealed a more complex
diversity, where species possessed all L-form, all S-form or
varying proportions of both CS types [2]. In those
Pseudomonads which possessed both isoenzymes, several
questions arise: (a) are the amino acid sequences of the CS
isoenzymes similar? (b) are there distinct CS structural
genes? (c) what is the sequence identity with other CS
sequences, particularly from the archaebacteria?
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1 D K K A O L I I E B S A P V E L P V L S G T
Figure 1. Alignment of
N-terminal amino acid
sequences of citrate synthases.
The CS’s were from the following sources: a. porcine heart
(‘small’); b. Bacillus sp C4 (‘small’); c. E.coli K12 (wildtype, ‘large’); d. Rickettsia prowarakii (‘small’); e.
Thermoplasma acidophilum (‘small’); f E.coli (mutant of
K12, ‘small’); g. Ps. aeruginosa PAC514 (‘small’ ); h. Ps.
aeruginosa 13474(‘large’). The molecular size of each CS
from which the amino acid sequence data was derived, is
given after the source. The hyphens (-) represent gaps
made to maximise alignment and the ‘?’ are undetermined
residues.
Although the existence of two CS genes in Saccharomyces
cerevisiae is well documented [3], evidence has now been
provided suggesting the presence of two CS genes in
Escherichia coli [4] and Bacillus subtilis [S], organisms
considered for many years to contain a single molecular
form of CS. There is also evidence that CS isoenzymes
may have different metabolic roles [6].
Citrate synthase isoenzymes have recently been purified to
homogeneity, from Pseudomonas aeruginosa PAC514 [7].
We have determined the N-terminal 23 amino acids from
the ‘small’ CS isoenzyme (the sequence for the ‘large’ CS
has not yet been determined) and compared this to other
CS’s of known sequence. The multiple alignment of these
sequences have shown high percentage identities within the
Eukaryotes (up to 92%), and the Gram-negative bacteria
(up to 75%). However, identities between the Eukaryotes,
eubacteria and the archaebacteria are low (less than 26%).
Figure 1 shows the alignment of these CS’s and several
features are worth noting: (a) there is significant homology
(36%) with the ‘small’ CS from E.coli, and Bacillus (27%)
but there is <5% homology with a ’large’ CS from Ps.
aeruginosa UM74; (b) although the homology with the
archaebacterium T.acidophilurn is low ( 18%). there are
sufficient similarities between these CS’s and the ‘small’
E.colr CS to suggest that further sequencing may reveal a
more significant homology; (c) the ‘small’ CS from
Pseudomonas aeruginosa PAC514 appears to be about 40
residues shorter at the N-terminus than the porcine CS and
other eubacterial CS’s as is the case for Bacillus sp CJ,
Tacidophilum and the ‘small’ CS from E.coli.
Our previous work [7] has shown that the CS isoenzymes
from Pseudomonas aerugrnosa PAC514 are distinct and
suggest that they are derived from different structural
genes. The low homology of the ‘small’ CS with the
sequence from a ‘large’ CS from Ps. aeruginosa UM74
and the significant homology with the ‘small’ CS from
E.coli , which has been shown to possess distinct CS genes
[4], supports this view.
We would like to thank Napier University Research and
Consultancy Committee for a Research Studentship to
S.C.K.A. and research support to C.G.M.
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