a mutation at gly314 of the β subunit of the escherichia coli pyridine nucleotide transhydrogenase...

Eur. J. Biochem. 207,733-739 (1992) 0 FEBS 1992

A mutation at Gly314 of the p subunit of the Escherichia coli pyridine nucleotide transhydrogenase abolishes activity and affects the NADP(H)-induced conformational change Suhail AHMAD, Natalie A. GLAVAS and Philip D. BRAGG Department of Biochemistry, University of British Columbia, 2146 Health Sciences Mall, Vancouver BC, Canada

(Received February 6, 1992) - EJB 92 0168

Escherichia coli RH 1 contains a mutation causing complete loss of pyridine nucleotide transhydrogenase activity. A single base change in the chromosomal DNA resulted in the replacement of Gly314 of the f? subunit by a Glu residue. The mutant enzyme was partially purified and its trypsin cleavage products examined. The distinct pattern of polypeptides given by proteolysis of the normal transhydrogenase in the presence of NADP(H) was absent when the mutant enzyme was treated with trypsin. However, the p subunit of the mutant enzyme retained its ability to bind to NAD-agarose. Further substitutions were made at Gly314 converting it to Ala, Val or Cys by the use of site-directed mutagenesis. All substitutions for Gly314 abolished the activity completely. The enzyme containing the Gly314-tAla mutation was studied in detail and behaved exactly as the enzyme containing the Gly314-tGlu mutation. It is concluded that the mutation in the /3 subunit abolished the NADP(H)- induced conformational change in the mutant enzyme. This conformational change, caused by NADP(H) binding, is required to cleave the normal f? subunit at Arg265 by trypsin.

The genes encoding the pyridine nucleotide transhydrogenase were completely resequenced and several corrections have been made to the previously published sequence [Clarke et al. (1986) Eur. J . Biochem. 158,647 - 6531.

Pyridine nucleotide transhydrogenase, found in the cyto- plasmic membrane of Escherichia coli and in the inner membrane of mitochondria, catalyzes the reversible transfer of a hydride ion equivalent between NAD and NADP [l]. The enzyme is of considerable interest since it functions as a proton pump, translocating protons across the membrane according to the equation [Z - 51:

nH2 + NADPH + NAD-nH;, + NADP + NADH.

The mitochondrial and E. coli transhydrogenases show considerable protein sequence similarity although the mitochondrial enzyme is a single polypeptide compared with the two subunits, ci and f?, of the bacterial enzyme [2, 6 - 91. The functional unit of the E. coli enzyme is the c12f?2 tetramer [lo]. We have purified the transhydrogenase and reconstituted its proton pumping activity in artificial phospholipid vesicles [2].

Correspondence to P. D. Bragg, Department of Biochemistry, University of British Columbia, 2146 Health Sciences Mall, Vancouver, BC, Canada V6T 123

Fax: + 1 604 822-5227. Enzymes. Pyridine nucleotide transhydrogenase, NAD(P)+ trans-

hydrogenase (EC 1.6.1 . I ) ; restriction endonucleases (EC 3.1.21.4); exonuclease I11 (EC 3.1.11.2);Sl nuclease(EC 3.1.30.l);calfintestinal alkaline phosphatase (EC 3.1.3.1); DNA polymerase (EC 2.7.7.7); T4 DNA ligase (EC 6.5.1.1); RNase A (EC 3.1.27.5).

Note. The novel nucleotide sequence data published here have been deposited with the EMBL sequence data bank and are available under the accession number X66086. The novel amino acid sequence data have also been deposited with the EMBL sequence data bank.

However, much remains to be learned about the mechanism by which transmembrane proton translocation is coupled to hydride ion transfer between the pyridine nucleotides. An important first stage in this study is to establish the location of the substrate-binding sites.

Our previous nucleotide sequence studies [7] revealed a region in the (x subunit (amino acid residues 170- 189) which showed sequence similarity to the pyridine nucleotide binding fold of some reductases. A similar sequence is present in the mitochondrial transhydrogenase [8]. Using [3Hlp-fluorosulfo- nylbenzoyl-5’-adenosine as a covalent label of nucleotide- binding sites, Wakabayashi and Hatefi Ell] found that there was a NADH-protectable Tyr residue labelled by this reagent. This Tyr residue is equivalent to Tyr226 in the E. coli sequence of the ci subunit. A further Tyr residue, presumed to be the NADP-binding site, was labelled at a site corresponding to Tyr431 of the E. coli f? subunit sequence. The evidence that this sequence contains the NADP-binding site is less firmly established than that for the NAD(H) site on the CI subunit. It has also been reported that binding of NADP(H) increases the susceptibility of the mitochondrial enzyme to tryptic digestion [8] and makes the p subunit of the E. coli enzyme sensitive when otherwise it is resistant to tryptic digestion [12].

In the present paper we have determined the site of a mutation responsible for the complete loss of transhydrogenase activity in the mutant E. coli RH1 [13, 141. This mutation results in the replacement of Glp31J of the p subunit by a Glu residue and renders the p subunit insensitive to tryptic digestion in the presence of NADP(H). Further, substi-

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tutions at Gly314 by Ala, Val or Cys yielded similar results. We conclude that Gly314 is critical for the correct folding of the cytosolic portion of the /3 subunit and that any substitution at this position (alanine being the most conservative replacement) perturbs the conformational change that the normal enzyme undergoes following binding of NADP(H), an event that is essential for the cleavage of the /3 subunit by trypsin.

MATERIALS AND METHODS

Bacterial strains and plasmids

E. coli strain JM109 [I51 and plasmid pGEM-7Zf (+) were obtained from Promega Corporation. The mutant E. coli strain RH1 (aroD6 arg E3 thi pnt-I), carrying a defect in the pnt genes [13, 141, was kindly provided by Dr. Barbara Bachmann (E. coli Genetic Stock Center). The construction of plasmid pDC 21 carrying the wild-type pnt genes has been described previously [ 161. Plasmid pSA2 was constructed by ligating the HindIII - SmaI fragment, containing the wild- type pnt genes from pDC21, to the HindIII/SmaI-digested pGEM-7Zf( +). The plasmid carrying the 5.0-kb HindIII - HindIII fragment, isolated from the genomic DNA of the mutant E. coli strain RHI, at the HindIII site of pGEM-7Zf( +) was designated as pSA30. Plasmid pSA31 was constructed by ligating the 2.0-kb fragment isolated by digesting plasmid pSA30 DNA with HindIII and BstBI to the 4.0-kb fragment isolated by digesting plasmid pSA2 with HindIII and BstBI. Plasmid pSA32 was constructed by ligating the 3.0-kb fragment isolated by digesting plasmid pSA30 with ApaI and BstEII to the 4.0-kb fragment isolated by digesting plasmid pSA2 with ApaI and BstEII.

Cloning and sequencing of the mutant pnt gene

The genomic DNA from the mutant E. coli strain RH1 was prepared by the procedure described by Wilson 1171. The genomic DNA was digested overnight with the restriction enzyme HindIII and the 5-kb HindIII -Hind111 fragment containing the pnt genes was subcloned into the plasmid pGEM- 7Zf( +). E. coli JM109 was transformed with the recombinant plasmids and the positive clones were identified by the unique restriction digestion pattern. Plasmid DNA was prepared by the alkaline lysis procedure as described by Maniatis et al. [I81 with the following modifications. The cells were pelleted in an Eppendorf centrifuge and the cell pellet was suspended in 0.2 ml glucose solution (25 mM Tris/HCl pH 8.0 containing 50 mM glucose and 10 mM EDTA). After 5 min at room temperature, 0.4 ml freshly prepared alkaline solution (1 YO SDS in 0.1 M NaOH) was added, the contents were mixed by inverting the tube a few times, and the tube was transferred to ice. After 5 min, 0.3 ml 7.5 M ammonium acetate was added, the contents were mixed by inverting the tube a few times and the tubes were left on ice. After 10 min, the tubes were centrifuged in an Eppendorf centrifuge for 10 min at 4 C ; 0.6 ml of the supernatant was transferred to another tube and was recentrifuged for 5 min at room temperature; 0.48 ml of this supernatant was transferred to a fresh tube and the DNA was precipitated by the addition of 0.34ml isopropanol. After 10 min, the tubes were centrifuged for 10 min. The pellet was washed with 1 ml7oY0 (by vol.) ethanol and dried at room temperature. The DNA pellet was dissolved in 0.1 mi 10 mM Tris/HCl pH 8.0 containing 1 mM EDTA and 1 &ml RNase A. This procedure yielded very high qual- ity plasmid DNA (> 80% closed circular form) that was used

for the generation of the nested sets of deletions and for DNA sequencing.

Nested sets of deletions were prepared by exonuclease III/ S1 nuclease digestion as described by Heinrich [19]. DNA sequencing was performed by the dideoxy-chain-termination method [20] with the Sequenase kit. Primer binding sites on the plasmid or synthetic oligodeoxyribonucleotide primers were used. Restriction endonuclease digestion, ligation and transformation were performed by standard methods [I 81.

Site-directed mutagenesis

Plasmid pSA2 carrying the wild-type pnt genes was used to isolate single-stranded phagemid DNA. Site-directed mutagenesis was performed to convert Gly314 to Ah, Val or Cys by the method of Taylor et al. [21] using the reagents and protocol as outlined in the Amersham mutagenesis kit except that competent E. coli JM 109 cells were used for transformation. The plasmid DNA was prepared from individual colonies and the mutants were identified by double-stranded DNA sequencing. Finally, the entire coding region of the pnt genes from each mutant was completely sequenced to eliminate the possibility of unwanted changes in the DNA sequence, using overlapping synthetic primers.

Solubilization and purification of transhydrogenase

The method of Clarke and Bragg [2] was used with modifications. E. coli JM109 pSA30 cells (2 g) were suspended at 1 g in 5 ml buffer A (50 mM Tris/H2S04, 1 mM EDTA, 1 mM dithiothreitol, pH 7.8) and disrupted by passage through a French press at 1400 kg/cm2. Unbroken cells were removed by low-speed centrifugation and the membrane vesicles sedi- mented by centrifuging the supernatant for 2 h at 50000 rpm in a Beckman 60 Ti rotor. The membrane vesicles (1 .0 g) were suspended at 1 g/5 ml buffer A to which was then added 1 M KCl, 30 mM sodium cholate and 30 mM sodium deoxy- cholate. The mixture was stirred at 0°C for 20 min. The supernatant obtained following centrifugation of the mixture at 50000 rpm (Beckman 60Ti rotor) was dialyzed against buffer A containing Brij 35 (1 mgiml). The dialyzed solution was loaded on a DEAE-BioGel A column (1.5 x 16 cm) equilibrat- ed in buffer A (+ Brij 35). A 200-ml linear gradient of 0 - 200 mM NaCl in buffer A was applied; 5-ml fractions were collected. The enzyme was located by SDS/PAGE. Fractions containing the transhydrogenase (40 ml) were desalted and the buffer exchanged to buffer B (10 mM sodium phosphate, pH 7, containing 1 mM dithiothreitol, 1 mM EDTA and 0.5 mg/ml Brij 35) by ultrafiltration through an Amicon PMIO filter. The normal transhydrogenase of JM83 pDC21 was purified further through an NAD-Agarose column as described previously 121.

Alternatively, the inner membrane containing enriched levels of transhydrogenase was isolated through the use of sucrose density gradient centrifugation and detergent washing as described by Tong et al. [12].

Treatment of solubilized transhydrogenase with trypsin

Membrane-bound or purified, solubilized transhydrogenase in buffer B (0.5 - 0.6 mg/ml) was treated with di- phenylcarbamoyl-chloride-treated trypsin, in the presence or absence of pyridine nucleotides (0.4 mM), at a trypsin/transhydrogenase protein mass ratio of 1 : 100. After 15 min, the reaction was stopped by addition of soya bean trypsin inhibi-

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PLASMID

PGEM

ACTIVITY

H N L - - L - 0.19

pSA3l

94 l k b

Fig. 1. Restriction endonuclease maps of plasmids containing the normal and mutant pnt genes and transhydrogenase activities of everted membrane vesicles prepared from cells harbouring each of the plasmids. Plasmids were constructed and transformed into E. coli JM109. The solid lines are DNA inserted into pGEM (broken line). DNA from pSA2 is shown as a thin solid line. The wider solid line is DNA derived from pSA30. CI, b indicate regions of DNA of pSA2 coding for the CI

and 0 subunits of the transhydrogenase. Symbols: A, ApaI; BB, BstBI; BE, BstEII; E, EcoRI; H, HindIII; N, NsiI; S, Smal. Enzyme activity is expressed as pnol 3-acetylpyridine - adenine dinucleotide reduced min- ' (mg membrane protein)-'.

tor (trypsin/inhibitor = 1 : 2, by mass). Samples were then examined by SDS/PAGE.

Transhydrogenase assay The assay was performed as described by Clarke and Bragg

[ l a

Electrophoresis

[22]. Gels were stained with Coomassie blue [23]. SDSjPAGE was performed by the method of Laemmli

Materials Restriction endonucleases, calf intestinal alkaline phos-

phatase, Klenow fragment of E. coli DNA polymerase, T4 DNA ligase, exonuclease I11 and S1 nuclease from mung bean were obtained from Pharmacia Fine Chemicals and were used according to the manufacturers instructions. Geneclean kit was purchased from Bio-101 . 5-Bromo-4-chloro-3-indolyl B-d-galactopyranoside and isopropyl P-d-thiogalactoside were obtained from Bethesda Research Laboratories. Sequenase version 2.0 DNA sequencing kit was purchased from U. S. Biochemicals Corporation. Site-directed mutagenesis kit and [cI-~'P]~ATP were obtained from Amershdm Corporation. Pyridine nucleotides, bovine pancreas di- phenylcarbamoyl-chloride-treated trypsin, soya bean trypsin inhibitor, and N'-linked NAD-Agarose were supplied by Sigma Chemical Company. Electrophoresis reagents were obtained from Bio-Rad. The oligodeoxyribonucleotides were synthesized at the DNA synthesis facility of the University of British Columbia. They were purified before use.

RESULTS Cloning and sequencing of the mutant pnt genes

The pnt genes of E. coli are encompassed within a 5.0-kb HindIII - HindIII fragment [16]. Therefore, in order to clone

67

43

30

Fig. 2. SDS/PAGE of membrane vesicles from E. coli JM109 transformed with the plasmids indicated. Molecular mass markers (values in kDa) are shown in the left track. The CI and subunits of the transhydrogenase are marked.

thepnt genes from the mutant E. coli strain RHI [13,14], the genomic DNA was isolated from the mutant cells and was subjected to Hind111 digestion. The digested DNA was run on an agarose gel. The region corresponding to the 5.0-kb fragment was excised and the DNA was eluted using the Geneclean kit. The eluted DNA was ligated to the plasmid pGEM-7Zf( +) that had already been digested with Hind111 and dephosphorylated using the calf intestinal alkaline phosphatase. The ligated mixture was used to transform competent E. coli JM109 cells and the cells were plated onto LB agar plates containing ampicillin as well as isopropyl P-d-thiogalactoside and 5-bromo-4-chloro-3-indolyl P-d-galactopyranoside for colour screening. A total of 43 white (recombinant) colonies were picked for mini-plasmid preparations and the positive clones were identified by the unique EcoRI/XhoI restriction digestion patterns (data not shown). A total of three positive clones (two in one orientation and the third in the opposite orientation) were obtained. The clone that was tran- scribed in the same direction as that of the lacZ gene on the plasmid (designated as pSA 30) was used for further studies.

Membranes were prepared from E. coli JM109 cells containing the non-recombinant plasmid pGEM-7Zf( + ) (control cells), as well as from the JM109 cells containing the wild-type pnt genes (clone pSA2) and the pnt genes from the mutant E. coli strain (clone pSA30) on the plasmid. The membranes were assayed for transhydrogenase activity and the results are presented in Fig. 1. Membranes prepared from cells containing pSA2 exhibited enhanced transhydrogeiiase activity compared to the control cells without the recombinant plasmids. Membranes prepared from cells containing the cloned mutant pnt genes (clone pSA30) exhibited the basal activity of the control cells. The membranes were analyzed by PAGE (Fig. 2). Membranes prepared from cells containing pSA2 or pSA30 exhibited over-expression of both CI and p subunits of the transhydrogenase as compared to the control cells (pGEM);

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A pntA-,

247 ATG CGA ATT GGC ATA CCA AGA GAA CGG I T A ACC AAT GAA ACC CGT GTT GCA GCA ACG CCA i b w t A r p 11. Wy 11s P r o A r g W u A r g Leu Thr A r n W u Thr A r g Val A l a A I s Thr Pro

301 RRA ACA GTG GAA UIG CTG CTG RRA CTG GGT ACC GTC GCG GTA GAG AGC GGC GCG GGT 21bLys Thr V a l Wu O n Leu Leu L y s Leu Wy me Thr Val A l a V a l W u S e r G l y A l a Wy

167 CRA CTG GCA AGT TTl' GAC GAT bAA GCG m GTG CAA GCG GGC GCT GAA ATT GTA GAA GCC 4 1 ) ~ " leu A I ~ sr me L ~ S A I ~ me vai GI" A I ~ w y A I ~ wu I IS vai GI" w y

427 AAT AGC GTC TGG CAG TCA GAG ATC ATT CTG AAG GTC ART GCG CCG TTA GAT GAT GAA ATT 61bArn S e r Val T r p G l n S e r Wu I l e I I B Leu Lys Val A s n A l a Pro Leu A l p Asp Wu I I B

487 GCG TTA CTG AAT CCT GGG ACA ACG CTG GTG AGT m A T TGG CCT GCG CAG AAT CCG GAA 8 1 ) A l a Leu Leu A r n Pro Wy Thr Thr Leu Val S e r me I I B T r p P r o A l a W n A r n Pro Wu

547 TTA ATG CAA WA CTT GCG GAA CGT AAC GTG ACC GTG ATG GCG ATG GAC TCT GTG CCG CGT 1OlPLei l W l W n Lya Lsu A l a Glu A i g A r n Val Thr Val Msl A l a Msl A s p Ser Val P r o A r g

607 ATC TCA CGC G.3 CAA TCG CTG GAC GCA CTA AGC TCG ATG GCG AAC ATC GCC GGT TAT CGC i z 1 P I l e S e r Acg A l a W n S e r Leu A s p A l a Leu Ser Ser Msl A l a A s n I l e A l a Wy T y r A r g

667 GCC ATT GTT GAA GCG GCA CAT GAA lTl' GGG CGC TTC TIT ACC GGG CAA ATT ACT GCG GCC I 4 i P A l a I I e Val G l u A l a A l a Hxs G l u Phe G l y A r g Phe Phe Thr G l y G l n I l e Thl A l a A I a

1 2 1 GGG AAA GTG CCA CCG GCA AAA GTG ATG GTG AIT GGT GCG GGT GTT GCA GGT CTG GCC GCC 161)Wy L y s Val P i 0 Pro A l a Lys Val WI Val I I e Gly A I a G l y Va l A l a G l y Leu A l a A I a

781 ATT GGC GCA GCA PAC AGT CTC GGC GCG ATT GTG CGT GCA TTC GAC ACC CGC CCG GAA GTG 1 8 1 ) l l e GIy A I a A l a Asn S e r Leu Gly A12 I I e Val A r q A l a Phe A s p Thr A r g Pro W u Val

847 AAA GAA CAA GTT CAA AGT ATG GGC GCG GAA TTC CTC GAG CTG GAT m AAA GAG GAA GCT ZOlbLys Glu G l n Va l G l n S e r Wl G l y A I a G l u Phe Leu G l u Leo A s p Phe L y s G l u G l u A l a

907 GGC AGC GGC GAT GGC TAT GCC M A GTG ATG TCG GAC GCG TTC ATC AAA GCG GAA ATG GAA 2 2 1 P W y Ser G l y A s p G l y T y r A l a Lys Val Msl Ser A s p A I a Phe I I e L y s A I a GI" Msl W u 961 CTC TIT GCC GCC CRG GCR AAA GAG GTC GAT ATC ATT GTC ACC ACC GCG CTT ATT CCA GGC 241DLeu Phe A l a A l a Gln A I a L y r G l u Val Asp I I e I I e Val Thr Thr A l a Leu I I e Pro Gly

1021 AAA CCA GCG CCG AAG CTA ATT ACC CGT GAA ATG GTT GAC TCC ATG AAG GCG GGC AGT GTG 261bLyr P r o A l e P r o L y s Leu I I e Thr A r q G l u W l Val A s p Ser Msl Lys A I a Gly Ser Val

1087 A n GTC GAC CTG GCA GCC CAA RAC GGC GGC RAC TGT GAA TAG ACC GTG CCG GGT GAA ATC 2 8 1 ) l l e Va l A s p Leu A l a A I a G l n A r n Gly G l y As" Cys G l u T y r Thr Val Pro Gly GI" l i e

1141 TTC ACT ACG GAA AAT GGT GTC L-L"? GTG ATT GGT TAT ACC GkT CTT CCG GGC CGT CTG CCG 301)phs Thr Thr Glu A r n Gly Val L y s Val I I e Gly T y r Thr A s p Leu Pro Gly A r g Leu P r o

1201 ACG CAA TCC TCA CAG CTT TAC GGC ACA AAC CTC G I T RAT CTG CTG AAA CTG TTG TGC AAA 321)Thr Gin S e r S e r G l n Leu T y r Gly Thr A m Leu Va l AS" Leu Leu Lys Leu Leu Cyr L y s

1267 GAG bAA GAC GGC AAT ATC ACT GTT GAT m GAT GAT GTG GTG ATT CGC GGC GTG ACC GTG 341)Glu L y s A s p Gly A r n I I e Thr Val Asp Phe A l p A s p Val Val I l e A r g Gly V a l Thr Val

1327 ATC CGT GCG u;C GAA ATT ACC TGG CCG GCA CCG CCG ATT CAG GTA TCA GCT CAG CCG CAG 3 6 1 b I l e A r p A l a G l y G l u Ile Thr T r p Pro A l a Pro P r o I I e G l n Val S e r A l a G l n Pro G l n

1381 GCG GCA 0.A WA GCG GCA CCG GAA GTG AAA ACT GAG GAA AAA TGT ACC TGC TCA CCG TGG 3 8 l b A l s A l a Wn L y r A l a A l a P r o G l u Val Lys Thr G l u G l u L y s Cys Thc Cya Ser P r o T r p

1441 CGT RRA TAC GCG TTG ATG GCG CTG GCA ATC ATT CTT TTI GGC TGG ATG GCA AGC GTT GCG 4 O l b A r g L y s T y r A l a Leu Wl A l a Leu A l a I I e I l e Leu Phe Gly T i p wl A l a Set Val A I a

1507 CCG bAA GAA TTC CTT GGG CAC TTC ACC GTT TTC GCG CTG GCC TGC GTT GTC GGT TAT TAC 421bPro L y s Wu Phs L e u G l y His Phe Thr Va l Phs A I a Leu A l a Cys V a l Val Gly T y r T y r

1561 GTG GTG TGG AAT GTA TCG CAC GCG CTG CAT ACA CCG TTG ATG TCG GTC ACC AAC GCG A?? 4 4 l b V a l Val T l p ASn Val Ser H19 A l a Leu Hts Thr Pro Leu Mat S e r V a l Thr Asn A l a I I e

1627 TUI GGG ATT ATT G n GTC twI GCA CTG TK CAG ATT GGC CAG GGC GGC TGG GTT RGC TTC 461)Ser Wy I I e I l e Val V a l GIy A l a Leu Leu Gln I I e G l y G l n G l y G l y Trp V a l Ser Phe

1687 CTT AGT m ATC GCG GTG CTT RTA GCC AX ATT AAT ATT TTC GGT u;c TTC ACC GTG ACT 481)Leu S e r Phe I I B A I a Val L e u 11s A l a Ser l i e A s n 118 Phe G l y G l y m e Thr Va l Th i

1141 CAG CGC ATG CTG bAA ATG TTC CGC AAA AAT TAA SOl*Gln A r g W I Leu Lys MI me A r g L y s A r n . . .

pntB+ B 1790 ATG TCT GGA GGI\ TTA GTT ACA GCT GCA TAC ATT GTT GCC GCG ATC CTG TIT ATC TTC AGT

lbwi Ser Gly Gly Leu Val Thr A l a A l a T y r I I e Val A1a A I a I I e Leu Phe I I e Phe S e r

1850 CTG GCC GGT CTT TCG AAA CAT GAA ACG TCT CGC CAG GGT AAC AAC TTC GGT ATC GCC GGG 21bLsu A I a Wy Leu S e r L y s Hlr G l u Thr Ser A r g Gln Gly Asn Asn Phe Gly I I e A1a GIy

1910 ATG GCG A n GCG P A ATC GCA ACC ATT TIT twI CCG GAT ACG GGT AAT GTT GGC TGG ATC 41)Msl A 1 a I l e A l a Leu IIe A l a Thr I l e h e G l y P r o A s p Thr G l y As" V a l Gly T i p I I B

1970 TTTG CTG GCG ATG GTC ATT GGT GGG GCA ATI GGT ATC CGT CTG GCG bAG P&A GTT GAA ATG 61)Leu Leu A l a Wl Val I I e Gly G l y A l a I I e G l y 11.3 A r q Leu A I s Lys L y r Va l G l u Wl

2030 ACC GAA ATG CCA GAA CTG GTG GCG ATC CTG CAT AGC TK GTG GGT CTG GCG GCA GTG CTG 81)Thr G l u Ma1 P r o Wu Leu Val A l a 11s Leu H I S Ser me Val G l y Leu A l a A l a Val Leu

2090 GTT GGC TTl' AAC AGC TAT CTG CAT CAT GAC GCG U;ii ATG GCA CCG A n ' CTG GTC AAT A n 10i)vai G I ~ he AS^ ~ e r ~ y i ~ e u h i t s n,s A S ~ A I ~ G I ~ mi A I ~ P ~ O I I ~ i e u v a ~ A S " i l e

2150 CAC CTG ACG GAA GTG TTC CTC GGT ATC TTC ATC GGG GCG GTA ACG TTC ACG GGT TCG GTG 1 2 1 P H t s Leu Thr G l u V a l Phe Leu G l y I I e Phe I I e Gly A l a Val Thr Phs Thr Gly Ser Val

2210 GTG GCG TTC GGC AAA CTG TGT GGC AAG ATT TCG TCT AAA CCA TTG ATG CTG CCA AAC CGT 1 4 1 ) V a l A l a h e Gly L y r Leu Cys G l y Lys I I B Ser S e r L y s Pro Leu & I Leu Pro Asn A l p

2210 CAC kAA ATG AAC CTG GCG GCT CTG GK GTT TCC TIC CTG CTG CTG ATT GTA T T I GTT CGC 161)His L y s Ma1 A s n Leu A l a A I a Leu Val Val S e r Phe Leu Leu Leu I I B Val Phe Val A r q

2330 ACG GAC AGC GTC GGC CTG CAA GTG CTG GcI\ TTG CTG RTA ATG ACC GCA ATT GCG CTG GTA I 8 l b T h r A s p Ser Val Gly Leu Gln Val Leu A l a Leu Leu I l e Msl Thr A l a I l e A l a Leu Val

2390 TK GGC TGG CAT TTA GTC GCC TCC ATC GGT GGT G.3 GAT ATG CCA GTG GTG GTG X G ATG 201bRe Gly T r p H l s Leu Val A I a Ser 11s Gly G l y A l a A s p Msl P r o Val Val Val Ser Msl

2450 CTG AAC TCG TAC TCC GGC TGG GCG GCT GCG GCT GCG GGC m ATG CTC AGC AAC GAC CTG 221bLsu A s 0 S e r T y r Ser Gly T r p A l a A l a A l a A l a A l a Gly Phe Msl Leu Ser A m A s p Leu

2510 CTG ATT GTG ACC GGT GCG CTG GTC GGT TCT TCG GGG GCT A X CTT TCT TAC ATT ATG TGT 2 4 1 B L e ~ I I e Val Thr G l y A l a Leu Va l Gly Ser Ser Gly A l a l i e Leu S e r T y r I I e W l Cys

2570 bAG GCG ATG AAC CGT TCC TTl' ATC AGC GTT ATT GCG GGT GGT TTC GGC ACC GAC GGC TCT 261bLys A l a &I A r n A r q S e r Phe I l e S e r Val I I e A I a Gly G l y Phe Gly Thr A s p Gly Ser

2630 TCT ACT GGC GAT GAT CAG GAA GTG GGT GAG CAC CGC GAA ATC ACC GCA GAA GAG ACA GCG 281)Ser Thr Wy Asp A s p G l n G l u Val Gly G l u H I S A i g Glu I l e Thr A l a G l u G l u Thr * l a

2690 GAA CTG CTG AAA AAC TCC CAT TCA GTG A X ATT ACT CCG G& TAC GGC ATG GCA GTC GCG 3 0 1 1 W u Leu L e u L y s A s n S e r H l s Ser Val I I e I I e Thf Pro Gly Tyr Gly Msl A I a Val A l a

2150 CAG GCG CAA TAT CCT GTC GCT GAA AfT ACT GAG AAA TTG CGC GCT CGT GGT ATT AAT GTG 3 2 i ) G l n A l a G l n T y r P r o Val A l a GI" l i e Thi G l u L y s Leu A r q A l a A r g Gly 11-3 A r n Val

2810 CGT rrC GGT ATC CAC CCG GTC GCG GGG CGT TTG CCT GGA CAT ATG AAC GTA TTG CTG GCT 3 4 1 ) A r g Phe Gly I I e H i s P r o Val A l a Gly A r q Leu P r o Gly H I S Msl A r n Val Leu Leu A l a

361)Glu A l a L y s Va l P r o T y r Asp I I e Val leu G l u Msl A s p G l u l i e A r n Asp Asp me A l a

2930 GAT ACC GAT ACC GTA CTG GTG ATT GGT GCT AAC GAT ACG G T T AAC CCG GCG GCG CAG GAT 381bArp Thr Asp Thr V a l Leu Val I I e G l y A l a Asn A s p Thr Val A r n P i 0 A I a A l a Gin A s p

2990 GAT CCG PAC AGT CCG ATT GCT GGT ATG CCT GTG CTG GRR GTG TGG AAA GCG CRG AAC GTG 4 O 1 ) A r p P r o L y l S e r P r o I I e A l a Gly lvel P r o Val Leu G l u Va l T r p L y r A l a G l n Asn Val

421) I I B V a l Phe LyS A r g S e r hiel A s o Thr Gly T y r A l a Gly V a l Gln Asn Pro Leu W e Phe

4 4 1 ) L y r G l u A s n Thr H i s Msl Leu Phe Gly A s p A l a L y r A l a S e r Val A s p A I a 118 Leu L ~ L 3170 GCT CTG TAA

4 6 1 ) A l a Leu . . .

2870 GAA GCA AAA GTA CCG TAT GAC ATC GTG CTG GAA ATG CAC GAG AX AAT GAT GAC m GCT

3n50 ATT GTC m AAA CGT TCG ATG ARC ACT u;c TAT GCT GGT GTG CAA AAC CCG CTG TTC TTC

1110 AAG GAA AAC ACC CAC ATG CTG m GGT GAC rcc MA GCC AGC GTG CAT GCA ATC CTG asa

Fig. 3. Nucleotide sequence of (A) the pntA and (B) the pntB genes. Transcription and translation are from left to right. The nucleotide which is altered in pSA30 is indicated by an asterisk.

only membranes from cells containing pSA2 exhibited enhanced transhydrogenase activity.

In order to determine which of the subunits was defective in the mutant strain, the region corresponding to the CI or the f l subunit of the pnt genes in clone pSA2 was replaced by the corresponding region from clone pSA30 and the resulting clones, designated as pSA31 and pSA32 respectively, were constructed as outlined in Fig. 1 and Materials and Methods. Membranes prepared from cells containing clone pSA31 or pSA32 were assayed for transhydrogenase activity. Only the membranes from cells containing the clone pSA31 exhibited elevated levels of activity (Fig. l), even though both CI and f l subunits of the transhydrogenase were over-expressed in cells containing clone pSA31 or pSA32 (Fig.2). These data suggested that the defect was in the f r subunit of the transhydrogenase.

The pnt genes from clone pSA30 were then completely sequenced by generating nested sets of deletions by digestion with exonuclease 111 and S1 nuclease. The DNA sequence was found to be altered at several places in both the CI and the f l subunits of the transhydrogenase compared with the published sequence [7]. Some of the alterations involved frame shifts. In order to determine which changes constituted errors in the published sequence and to establish the change(s) that

was the result of the mutation in RH1, the corresponding regions were sequenced from the wild-type genes (from clone pSA2). This was achieved by constructing deletion mutants or by the use of synthetic oligonucleotide primers. There was complete agreement between the DNA sequences obtained from the wild-type and the mutant genes (except for the single base change constituting the mutation). This analysis resulted in the following changes in the published wild-type pnt gene sequence [7].

a) An extra G at position 365 results in a frame shift in the a subunit; however, an extra A at position 402 and an extra C at position 410 (numbers corresponding to the new sequence; Fig. 3) restored the reading frame and resulted in an extra amino acid compared to the published sequence. The translated protein sequence from nucleotide position 364 - 411 is now different. The revised sequence shows greater similarity with the corresponding region of the pnt gene from mitochondria [S] than the previously published sequence.

b) A C in place of G at position 1232 (base number 1229 in the published sequence) results in the conversion of an Arg to a Thr residue.

c) An extra C at position 1756 (after base number 1753 in the published sequence) results in the continuation of the protein sequence for an additional seven amino acids at the

737

A C G T A C G T

Normal Mutant Fig. 4. Sequencing of gels ofpntB DNA showing the region in which the alteration G+A in the mutant results in replacement of a Gly by a Glu residue in the amino acid sequence.

C-terminal before terminating at bases 1777 - 1779. Again, the added sequence shows similarity with the sequence of the corresponding region of the mitochondrial pnt gene [8].

d) The wild-type (as well as the mutant) sequence corresponding to the b subunit showed the omission of a single base (G after position 2449; base number 2446 in the published sequence) and the addition of a single base (C at position 2510; after base number 2506 in the published sequence) resulted in a frame shift between bases 2450-2512 from the published DNA sequence. The revised protein sequence exhibited con- siderably greater similarity to the corresponding region of the mitochondrial transhydrogenase. The complete revised DNA and the amino acid sequences of the a and the p subunits of the E. colipnt genes are presented in Fig. 3.

In contrast to the above changes, the DNA sequence from the mutant E. coli strain was found to differ in a single base (A in place of G at position 2730; base 2726 in the older sequence) that resulted in the conversion of the triplet for Gly314 to that for Glu in the p subunit of thepnt genes. The position of mutation is marked by an asterisk in Fig. 3. Fig. 4 shows the appropriate region of the sequencing gels showing wild-type and mutant sequences.

Binding of substrates to the mutant transhydrogenase

The DNA sequencing data showed that the mutant differed from the wild-type enzyme in a single amino acid within the /3 subunit. Since the mutation lies within the p subunit that corresponds to the C-terminal portion of the mitochondrial transhydrogenase, the latter being proposed to contain the NADP(H)-binding site [l 11, the possibility that NADP(H) binding was affected in the mutant protein was explored.

Yamaguchi et al. [24] have shown that the presence of NADP(H) during tryptic digestion of the mitochondrial transhydrogenase results in cleavage at additional sites, that are otherwise resistant, probably due to a conformational change of the C-terminal brought about by NADP(H) binding. We have also recently shown 1121 that the p subunit of the E. coli transhydrogenase is resistant to proteolytic digestion by trypsin but becomes sensitive in the presence of NADP or NADPH. Further, the fi subunit binds to an NAD-Agarose column, presumably through the interaction with the NADP(H) binding site. Similar experiments were performed with the mutant enzyme to determine the effect of mutation on NADP(H) binding. This was accomplished by solubilizing and partially purifying the defective transhydrogenase from membrane vesicles of JM109 pSA30. The purified enzyme showed CI and p subunits migrating as expected on SDSjPAGE (Fig. 5) . The normal enzyme isolated as described before [2] from JM83 pDC21 was used as a control.

A B

1 2 3 4 5 1 2 3 4 5

Fig.5. Effect of substrates on trypsin digestion of (B) normal and (A) mutant purified transhydrogenases. The transhydrogenases were digested for 15 min with trypsin at a ratio of trypsin/transhydrogenase of 1 : 100 (by mass) in the absence (lane 1) or presence of 0.4 mM NAD (lane 2), NADH (lane 3), NADP (lane 4) or NADPH (lane 5). SDSjPAGE was carried out on the reaction mixtures. The positions of migration of the subunits and of the resultant polypeptides (molecular mass in kDa) are indicated. The undigested purified soluble transhydrogenase of the mutant is shown in the extreme left-hand lane.

Trypsin treatment of the soluble normal enzyme of JM83pDC21 in the absence of substrates, or in the presence of 0.4mM NAD or NADH, resulted in cleavage of the a subunit only yielding 43-kDa, 29-kDa and 16-kDa polypeptides as expected 1121. However, cleavage of the p subunit to 30-kDa and 25-kDa fragments occurred when NADP or NADPH was present (Fig. 5B). By contrast with the normal enzyme, trypsin treatment of the transhydrogenase from JM109pSA30 in the presence of NADPH or NADP did not lead to cleavage of the /3 subunit. In the presence of these substrates (or of NAD or NADH), cleavage of the a subunit gave the same cleavage fragments as found in the absence of substrate (Fig. 5A).

The mutant and normal enzymes were treated with trypsin and the digestion products applied to an NAD-Agarose column. Unbound materials were removed by washing with buffer containing 20 mM NaCl and bound fragments then eluted with 10 mM NADH (Fig. 6A). Although salt eluted some p subunit from the normal enzyme, most was eluted by NADH, indicating that it had bound through a pyridine- nucleotide-binding site to the immobilized NAD of the gel matrix. A nearly identical pattern was obtained with the mutant enzyme (Fig. 6B).

The binding of the mutant enzyme to the NAD-Agarose column showed that NADP(H) binding was not affected but rather the enzyme has become resistant to conformational change. Since the mutation converts Gly314 to a Glu residue, which is not a good replacement, we utilized site-directed mutagenesis to selectively convert Gly314 to Ala, its most closely related amino acid, as well as to Val or Cys. Membranes were prepared from cells carrying these substitutions and were analyzed for enzyme activity and membrane assembly of the transhydrogenase. The data are presented in Table 1. Substi- tution of Gly314 by Ala, as well as by Val and Cys, resulted in complete loss of activity even though both CI and p subunits were assembled in the membrane and constituted major peptides in gels. The enzyme carrying the Gly 314-Ala mutation was studied in more detail.

Tryptic digestion was carried out in the absence or presence of NAD(H) or NADP(H). The results are shown in Fig. 7. Again, the p subunit was not digested by trypsin even in the presence of NADP(H), a result similar to that obtained with the enzyme carrying the Gly314+Glu mutation.

738

A 1 2 3 4 5 6 7 8 910

45:

29.

16.

4B

B 1 2 3 4 5 6 7 8 9 1 0

4% ‘B

Fig. 6. Binding of trypsin-digestion fragments to NAD-Agarose. (A) Purified transhydrogenase of JM83pDC21 was digested with trypsin for 10 min. The products wereloaded on an N6-linked NAD-Agarose column (1 x 6 cm) and eluted with the indicated buffers. SDS/ polyacrylamide gels were run on the eluted fractions. Lanes 1-4, buffer B (10 mi); lanes 5 and 6 , 20 mM NaCl in buffer B (10 ml); lanes 7- 10,10 mM NADH in buffer B (5 ml). (B) Digestion products of partially purified G314E mutant transhydrogenase of JM109pSA30 were loaded on an NAD-Agarose column. Lanes 1 - 3, column eluted with buffer B (10 ml); lanes 4 and 5, 20 mM NaCl in buffer B (10 ml); lanes 6-10, 10 mM NADH in buffer B (5 ml). The fi subunit and digestion fragments of the SI subunit are indicated by their molecular masses (in kDa).

Table 1. Membrane assembly and enzyme activity in everted membrane vesicles of mutant pyridine nucleotide transhydrogenases in which GIy314 of the f i subunit has been replaced by other amino acids.

Plasmid Amino acid at Extent of en- Specific position 314 of zyme assembly activity the p subunit in the membrane

pmol min-’ mg protein- ’

~ pGEM-7Zf( +) ~ 0.29 pSA2 GIY +++ 11.30

pSA71(3) Ala + + + 0.41 pSA70(9) Val + + 0.07 pSA70(11) CYS + + + 0.07

pSA30 Glu + + + 0.11

DISCUSSION

The pyridine nucleotide transhydrogenase is an integral inner membrane protein that catalyzes the transhydrogen- ation of NADP by NADH with concomitant translocation of protons across the membrane [2, 51. Although the genes encoding the enzyme from both E. coli and bovine mitochondria have been cloned and sequenced, much of the information on the substrate binding sites and proton translocating region remains largely unresolved. For the mitochondrial enzyme, the identity of an amino acid involved at the NAD(H) binding site has been well established by employing analogs of the

a. P’

A B 1 2 3 4 5 6 7 1 2 3 4 5 6 7

.p ;: ‘43

4 29

4 6 4 1 6

Fig. 7. Effect of substrates on trypsin digestion of transhydrogenases in (B) normal and (A) Ala314 mutant purified membranes. The membranes were digested for 15 min with trypsin at a trypsin/membrane protein ratio of 1 : 100 (by mass) in the absence (lane 3) or presence of 0.4 mM NAD (lane 4), NADH (lane 5), NADP (lane 6 ) or NADPH (lane 7). SDSjPAGE was carried out on the reaction mixtures. The positions of migration of the c1 and p subunits and of the resultant polypeptides (molecular mass in kDa) are indicated. The undigested purified membranes are shown in lane 1 ; lane 2, trypsin and trypsin inhibitor.

substrate [l I]. The radiolabelled analog was bound to the enzyme followed by tryptic digestion. The peptides were separ- ated and the labelled peptide was subjected to N-terminal sequencing to identify the labelled amino acid. However, the information on the identity of other amino acids involved in NAD(H) binding as well as the amino acid(s) involved in NADP(H) binding is either lacking or less certain [Ill.

Even such preliminary studies have not been reported for the E. coli enzyme, and the probable NAD(H) and NADP(H) binding sites can only be extrapolated based on sequence alignments of the bacterial and the mitochondrial pnt genes and the deduced amino acid sequences [7, 81. However, the bacterial system offers the unique advantage of isolating mutants defective in the enzyme. One such E. coli mutant that lacks the transhydrogenase activity completely has been described [14]. Thus, it was of considerable interest to determine the amino acid residue(s) altered in the mutant protein as they might be involved in substrate binding sites. The pnt genes were, therefore, cloned and sequenced from the mutant E. coli strain. The DNA sequence of the mutant pnt genes differed in a single base (base number 2730 involving the P subunit) from the wild-type sequence. This single base change in the pntB gene of E. coli RH1 results in a change of Gly314 of the f l subunit of the transhydrogenase to a Glu residue. This is associated with complete loss of pyridine nucleotide transhydrogenase activity and loss of trypsin cleavage of a susceptible bond in the p subunit in the presence of NADP(H). However, the P subunit of the mutant enzyme retained the ability to bind to NAD-Agarose. Since the Psubunit binds to NAD- Agarose, presumably through the interaction of the NADP(H) binding site, the binding of NADP(H) per se to the /3 subunit is probably not affected in the mutant enzyme. However, this binding of NADP(H) to the P subunit causes a conformational change that makes an otherwise buried Arg residue (Arg265) sensitive to tryptic digestion [12]. It seems probable that this conformational change brought about by NADP(H) is abolished in the mutant enzyme. It is possible that Gly314 is located at a critical turn within the P subunit polypeptide and that replacement of this residue by Glu yields a conformationally different form of the enzyme that is unable to undergo the conformational change which occurs following binding of NADP(H). Thus, there is no cleavage of the mutant P subunit at Arg265 by trypsin.

739

Support for the above hypothesis comes from studies of the enzyme in which Gly314 has been changed to other amino acids by site-directed mutagenesis. Replacement of Gly314 by Ala produced a non-functional enzyme which was also resistant to the a-subunit cleavage by trypsin even in the presence of NADP(H). Thus, the Gly-tAla change also pro- duces an enzyme that is unable to undergo conformational changes following NADP(H) binding. The results presented in this paper highlight the importance of proteolytic enzymes in elucidating subtle conformational changes in complex pro- teins brought about by substrate binding.

This work was supported by a grant from the Medical Research Council of Canada (MRC) and by an MRC studentship to N. Glavas.

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a mutation at gly314 of the β subunit of the escherichia coli pyridine nucleotide transhydrogenase...

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