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number AJ316232. The data on the alignment and homology of both nrfA and nrfH have
already been discussed in references 9 and 10.
We used SignalP V2.0 (SignalP-HMM and Signal-NN) [11,12] for the prediction of
the presence and location of signal peptide cleavage sites for gram-negative bacteria and the
program TMHMM 2.0 to predict of transmembrane helices [13,14]. SignalP and TMHMM
are available under the prediction server page of the Center for Biological Sequence
Analysis at www.cbs.dtu.dk/services/tmhmm2.0.html. The signal sequence of lipoproteins
was examined using the program Lipop accessed at PSORT WW Server
(http://psort.nibb.dtu.dk )[15].
The N-terminal sequences of both NrfA and NrfH are the following:
NrfA, 24 XQDVSTELKAPKYKTGIAETETKMSAFKGFPQQYASYMKNNE
NrfH, 1 GTPRNGPWLKWLLGGVAAGVVLMGVLAYAMTTTDQRP
2. Results and Discussion
2.1 Primary Structure
NrfA. As already been described in previous papers [9,10], the deduced amino acid
sequence of NrfA (518 aa) contains four classical c-type heme-binding motifs CXXCH and
a fifth heme-binding site CWXCK, where the proximal histidine residue was replaced by a
lysine. Excluding the cleaved 23 N-terminal aminoacids and the heme prostetic groups, it
has a molecular mass of 56768 Da. The addition of five hemes gives 59848 Da.
The sequence of nrfA encodes for a precursor signal peptide, which shows the
“LA(G/A)|C” consensus motif recognised by signal peptidase II [10].The prediction given
by SignalP, using the HMM version, gives a maximum cleavage site probability between
Gly23 and Cys24 (Fig.1). Interestingly, the NN version, although referred in the literature
to have a better performance in predicting the cleavage site location in gram-negative
bacteria [1], gave slightly different results, being the maximum cleavage site probability
between Ser28 and Thr29. Nevertheless, the results are positive for the presence of a signal
peptide based on S-score (output form signal peptide networks) and mean S-score values
(Fig. 1B). The above mentioned cleavage site was experimentally confirmed by N-terminal
sequencing of the mature protein. Accordingly to the HMM prediction, the N-terminal
sequence of NrfA starts at the 24
th
residue. It shall be stressed out that the chemical
sequencing by Edman degradation doesn't recognise cysteines. Additionally, signal
peptidase II cuts upstream of a cysteine residue to which a gliceride-fatty acid lipid is
attached [16]. For this reason, the signal sequence of lipoproteins, i.e., proteins with a
covalently attached lipid molecule in their mature N-terminus, was examined using the
program Lipop. This program also predicted a lipid attachment to Cys24 with a sequence
consensus motif of CQDV, which gave us an additional evidence for the correct cleavage
site position predicted by the HMM version. Curiously, none of the nrfA from other
organisms published in the literature [see 9 and 10 and references there in] shows this
consensus motive. Thereby, the presence of a lipidic component attached to Cys24 may be
a particular feature of NrfA from D. desulfuricans.
2.2 NrfH
As previously reported [9], the deduced amino acid sequence of NrfH subunit (154 aa)
shows four CXXCH consensus sequences. It has a predicted molecular mass of 16764 Da,
excluding the heme groups. The attachment of four hemes leads to a total molecular mass
of 19228 Da.
