The periplasmic nitrate reductase from Escherichia coli :a heterodimeric molybdoprotein with a double-arginine signal

sequence and an unusual leader peptide cleavage site

Gavin Thomas 1, Laura Potter, Je¡ A. Cole *School of Biochemistry, University of Birmingham, Birmingham B15 2TT, UK

Received 4 November 1998; received in revised form 19 November 1998; accepted 20 November 1998

Abstract

The periplasmic nitrate reductase, NapA, from Escherichia coli was identified as a 90 kDa molybdoprotein which co-migrated during polyacrylamide gel electrophoresis with the di-haem c-type cytochrome, NapB. The DNA sequence of the5P end of the napA gene and the N-terminal amino acid sequences of both NapA and NapB were determined. The 36 residueleader peptide for NapA includes the double-arginine motif typical of proteins to which complex redox cofactors are attachedin the cytoplasm prior to targeting to the periplasm. The pre-NapA leader sequence is both unexpectedly long and, unless twosuccessive proteolysis steps are involved, is cleaved at the unprecedented sequence G-Q-Q-. Nap activity was suppressed duringgrowth in the presence of tungstate and was absent from a mutant unable to synthesise the molybdopterin cofactor. z 1999Published by Elsevier Science B.V. All rights reserved.

Keywords: Nitrate reduction; Periplasmic nitrate reductase; Twin-arginine; Leader peptide

1. Introduction

Periplasmic nitrate reductases (Nap) were ¢rstcharacterised biochemically as water-soluble, hetero-dimeric complexes of a 90 kDa molybdoprotein anda 16 kDa di-haem cytochrome c [1^3]. Subsequentgenome sequencing data have revealed the presenceof genes for similar enzymes in all of the majorgroups of prokaryotes, though only in a few caseshave the enzymes been studied [4,5].

All of the periplasmic nitrate reductases so farfound are encoded by operons which include fourcommon genes located in the order napDABC[6^8]. At least ¢ve other genes also occur in di¡erentcombinations in di¡erent organisms [7,8]. Resultsfrom the Escherichia coli Genome Sequencing Proj-ect (GenBank Entry U00008 [9]) revealed that the 47min region of the chromosome includes seven geneswhich as we recently demonstrated encode a func-tional Nap activity [10]. Based upon similarities ordi¡erences to nap genes in other bacteria, we desig-nated these genes napFDAGHBC. However, due tosevere G-C compressions which caused sequencingerrors, translation of the initially published DNAsequence of the nap operon results in a NapA poly-

0378-1097 / 99 / $20.00 ß 1999 Published by Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 1 2 1 - 4

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* Corresponding author. Tel. : +44 (121) 414 5440;Fax: +44 (121) 414 3982; E-mail: j.a.cole@bham.ac.uk

1 Present address: Nitrogen Fixation Laboratory, John InnesCentre, Norwich, UK.

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peptide with no recognisable signal sequence to di-rect its export into the periplasm. Furthermore, ac-cording to SWISSPROT Database entry P33937,NapB is a c-type cytochrome with a short, acidicN-terminus beginning with the sequence MEFGSE:if this is correct, it is unlikely that NapB would beexported, and the napB ORF overlaps that of napH.Such sequence data clearly con£ict with our demon-stration that E. coli exports a soluble nitrate reduc-tase and NapB into the periplasm [10]. The aims ofthe work to be described were therefore to identifythe constituent polypeptides in Nap from the peri-plasm of E. coli, determine their N-terminal aminoacid sequences and hence deduce the structures ofthe leader peptides and the sequence of the cleavagesites. The DNA sequence of the 5P end of napA wasalso redetermined to correct any errors in the data-bases. The results con¢rm the current expectationthat Nap in E. coli is a heterodimer of a small, c-type cytochrome with a conventional leader peptideand a molybdoprotein exported by the recently de-scribed `double-arginine' targeting pathway [11^14].

2. Materials and methods

2.1. Bacterial strains and growth conditions

Derivatives of E. coli K-12 were used. StrainJCB7120 is a parental strain used previously to iden-tify Nap activity [10]. Strain JCB355 is a hemN de-rivative of JCB387 and is defective in the anaerobicsynthesis of all c-type cytochromes [15,16]. The roleof the mobAB locus in Nap activity was investigatedusing strain TP1000 [16]. Controls with the isogenicparental strain were included in all experiments withthese mutants.

Bacteria were grown anaerobically in 2 l of mini-mal salts (MS) medium supplemented with fullstrength nutrient broth (NB), 0.4% glycerol, 40mM fumarate and 20 mM nitrate, harvested andwashed in 50 mM potassium phosphate bu¡er, pH7.4. Periplasmic proteins were released as describedby McEwan et al. [1] except that the concentration ofEDTA was 5 mM. The periplasmic proteins wereconcentrated by freeze drying to a volume of 2 to3 ml.

To investigate the role of molybdate in Nap activ-

ity, similar cultures were grown in the absence of 10WM sodium molybdate or with 10 WM sodium tung-state.

2.2. Polyacrylamide gel electrophoresis and stainingmethods

Concentrated periplasmic proteins were separatedon native 7.5% polyacrylamide gels. After electro-phoresis, gels were placed in a solution of dithion-ite-reduced methyl viologen containing 20 mM ni-trate. Nitrate reductase activity was detected as acolourless band against a dark purple background[10]. The band of activity was excised and soakedin SDS-PAGE sample bu¡er. The proteins releasedwere denatured by heating to 90³C for 10 min andseparated by SDS-PAGE.

Both native and denaturing gels were stained ¢rstto detect haem peroxidase activity associated with c-type cytochromes [17] and then with Coomassie Bril-liant Blue to detect total protein content.

2.3. DNA sequencing

Plasmid pJG600, which contains the complete nap-ccm region [10], was transformed into E. coli strainJM109 and RNA-free single stranded DNA was pre-pared [18]. Primers designed to amplify 200 bparound the 5P end of napA and the PharmaciaLKB T7 DNA sequencing kit were used to deter-mine the nucleotide sequence of this fragment bythe dideoxy chain termination method [19]. Sequenc-ing was repeated with de-aza NTPs (Pharmacia)after initial sequencing revealed severe G-C compres-sions within the coding region of napA.

3. Results and discussion

Periplasmic proteins from E. coli strain JCB7120were separated by non-denaturing PAGE andstained for nitrate reductase activity [10]. The samegel was also stained for peroxidase activity associ-ated with the covalently bound haem of c-type cyto-chromes. A single band of nitrate reductase activitywas found which coincided with the weakest of thethree cytochrome c bands (Fig. 1, tracks 1 and 3).The lower and fainter band of activity visible in lane

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1 corresponds to the most abundant c-type cyto-chrome in the periplasmic fraction, cytochrome c552

(track 3). Activity is due to the reduction of nitriteformed as the product of NapA activity by this peri-plasmic nitrite reductase [20].

Periplasmic proteins were also isolated from ahemN mutant, strain JCB355, which is unable tosynthesise cytochromes during anaerobic growth.Nap activity was again detected, but associatedwith a band of decreased electrophoretic mobilitywhich lacks c-type cytochrome, implying thatNapA is active with the arti¢cial electron donor,methyl viologen, in the absence of NapB (Fig. 1,tracks 2 and 4).

Polypeptides associated with the band of Nap ac-tivity from strain JCB7120 were electroeluted andseparated by SDS-PAGE. More than 10 di¡erentcomponents were detectable by Coomassie staining,only one of which was a c-type cytochrome detect-able by staining for covalently bound haem. Thiswas the 16 kDa NapB. The N-terminal sequencesof the most abundant polypeptides, which migratewith apparent masses of 16 and 90 kDa, were deter-mined by automated Edman degradation. The N-ter-minal sequence of the 90 kDa band was Q/E-A-I-K-W-D-K, but the 16 kDa component was clearly a

mixture of polypeptides. Cytochrome c550 (NapB)was therefore puri¢ed as described previously [21],shown to co-migrate with the c-type cytochrome as-sociated with Nap activity, and the N-terminal se-quence was determined to be A-N-G-V-D-F-S-.This sequence was used to search the predictedORFs from the complete E. coli genome sequence(release M52). Only a single match was found whichcorresponded to residues 35 to 42 of the predictedNapB sequence listed in SWISSPROT entry P33937.Note, however, that there is an alternative possibletranslation start which would result in a pre-apocy-tochrome with the N-terminal sequence M-K-S-H-D-L-K-K-A-L-. The sequence of the ¢rst 26 residueswould then correspond to a conventional Sec-de-pendent leader peptide with a cleavage site, V-W-A, which conforms to the 33; 31 rule for cleavageby the leader peptidase [22]. We therefore concludethat the second methionine in SWISSPROT entryP33937 is the correct translation start for NapB.

Having determined the N-terminal sequence ofNapA, we then redetermined the sequence of the 5Pend of the napA gene which includes a long G-C-richregion encoding alanine residues (Fig. 2). In agree-ment with the revised sequence in the current E. coligenome database and the partial sequence proposedby Berks et al. [7], ¢ve di¡erences from the sequencepublished by GenBank entry U00008 were found.This resulted in two changes in the reading frameand the insertion of an extra alanine codon. TheN-terminal sequence of the pre-NapA therefore in-cludes a consensus twin-arginine transfer sequencewhich directs the export of proteins binding molyb-dopterin and other redox cofactors into the peri-plasm by a recently described Sec-independent path-

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Fig. 2. The DNA sequence of the 5P end of the napA gene. Thepredicted amino acid sequence of NapA is shown. The sequenceof the signal peptide is in bold. The predicted ribosome bindingsite and start codon are underlined.

Fig. 1. Nitrate reductase activity stain and haem stain of peri-plasmic proteins from E. coli. Tracks 1 and 2 contain periplasmicproteins from strains JCB7120 and JCB355 stained for nitrate re-ductase activity after separation by non-denaturing polyacryl-amide gel electrophoresis. Tracks 3 and 4: the same gel was sub-sequently stained for haem-dependent peroxidase activity.

G. Thomas et al. / FEMS Microbiology Letters 174 (1999) 167^171 169

way [12^14]. Unexpectedly, however, pre-NapA iscleaved not at the G-V-A (positions 27 to 29) orA-R-A (positions 29 to 31), but after the G-Q-Q atpositions 34 to 36. Not only is the leader peptidelonger than expected, but also the leader peptidasecleavage site for pre-NapA is, as far as we are aware,unprecedented. The N-terminal amino acid sequencedata for mature NapA were unequivocal: neverthe-less, the unlikely possibility cannot be excluded thatNapA is cleaved twice, once by the leader peptidaseduring export, and then by a second, periplasmicprotease activity.

Nap activity was lost rapidly during the separationof NapA from NapB by anion exchange chromatog-raphy. Stability studies with periplasmic proteinsfrom strain JCB7120 revealed that all activity waslost within a few hours at 30³C. About 90% of theactivity had been lost after 48 h at 4³C and no ac-tivity remained after 4 days at 4³C. There was asimilar rate of loss of Nap activity from periplasmicproteins from the hemN mutant, implying that Napactivity is unstable both in the presence and absenceof NapB.

No Nap activity was detected in a mobAB deletion

mutant. More activity was detected when strainJCB7120 was grown in the presence of 10 WM mo-lybdate than in its absence (Fig. 3, tracks 1 and 2),and virtually no activity was detectable in the peri-plasm of bacteria which had been grown in the pres-ence of tungstate but without added molybdate (Fig.3, track 3). These experiments con¢rm that Nap is amolybdoprotein. The low level of Nap activity stillpresent in the culture grown with tungstate was mostlikely due to e¤cient scavenging of traces of molyb-date present in the other medium components.

Acknowledgments

We are grateful to Duncan Wisbey for help withthe puri¢cation of NapB; to the BBSRC for theaward of a CASE Studentship, supported by BritishNuclear Fuels plc, to G.T.; and to the UK MedicalResearch Council for a Research Studentship forL.P. This research was funded by BBSRC ProjectGrant C07661 to J.A.C.

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Fig. 3. The e¡ect of loss of molybdate and the presence of tung-state on Nap activity. Periplasmic proteins of strain JCB7120grown with molybdate (lane 1), without molybdate (lane 2) orwith tungstate instead of molybdate (lane 3). The gel was stained¢rst for nitrate reductase activity and then for nitrite reductaseactivity. Protein loading was identical for lanes 1 and 2; lane 3contained 70% of the protein in the other lanes.

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