Molecular Microbiology (1992) 6(2). 209-219
Formation of active heteroiogous nitrate reductases between nitrate reductases A and Z of Escherichia coli F. Blasco,^* F. Nunzj/ J. Pommier/ R. Brasseur,^ M. Chippaux^ and G. Giordano^ 'Laboratoire de Chimie Bacterienne. CNRS, BP 71, 31 cheminj. Aiguier, 13277 Marseille Cedex 9. France. ^Laboratoire de Chimie-Physique des Macromolecules aux Interfaces, Universite Libre de Bruxelles, CP 206/2. boulevard du Triomphe, 1050 Brussels, Belgium. Summary Two nitrate reductases, NRA and NRZ, are present in Escherichia coli. These isoenzymes have the same aPy. subunits composition and have simitar size and genetic organization. Corresponding subunits of the complexes share at least 75% identity. By subcioning the different genes and expressing them from separate transcriptionai units, we have demonstrated (i) that the translation of the subunits and their assembly are not coupled processes, since subunits produced concomitantiy but independently can meet efficiently and associate to form active enzymes, and (li) that the i>: subunit of a given complex can be replaced by its counterpart from the other isoenzyme to yieid an active membrane-bound heteroiogous enzyme. One such heteroiogous enzyme, a^^z'/z^ has been purified; it is less stable than the native enzymes, more susceptible to thermal denaturation, and shows increased sensitivity to proteolysis. It is also less stably bound to the membrane and, consequently, its activity with physiological electron donors is drastically reduced. The possibility that heteroiogous nitrate reductases could be formed in vivo is discussed with reference to the existence of porin heterotrimers of the outer membrane proteins OmpC, OmpF and PhoE.
Introduction Escherichia coli possesses two respiratory nitrate reductases: NRA and NRZ. Both are membrane-bound complexes composed of three subunits. a. (i and y, encoded respectively, either by the narG. narH and narl genes or Hecsived 17 July, 199i; revised 3 October, 1991- 'For correspondence. Tel. 91 16 40 94; Fax 91 71 89 14.
by the narZ. narYand narV genes (Bonnefoy-Orih et ai. 1981; Blasco et ai, 1989: 1990: 1992. accompanying paper). These genes are in transcription units. narGHJIor narZYWV. of similar size and genetic organization (Blasco et ai, 1989; 1990). The y subunit is the apoprotein of a b-iype cytochrome which is embedded in the membrane and receives electrons from the quinone pool. Electrons are then transferred via the iron-sulphur centres of the p subunit to the molybdenum cofactor on the a subunit. where the reduction of nitrate to nitrite takes place (Blasco et ai, 1989: Chaudhry and MacGregor. 1983: Ruiz-Herrera and DeMoss, 1969). The third gene of each operon. narj or narW, encodes a polypeptide. S, the precise role of which is unclear (Sodergren and DeMoss, 1988: Sodergren etai, 1988: Biasco etai, 1990). During the course of the characterization of NRZ, we obtained a strain which, although fully expressing NRA and overproducing NRZ from a ptasmid. displayed a very low level of nitrate reductase activity (one third of that expected from the narGHJ/operon alone). This discrepancy was not explained by a partial repression of the narGHJ/operon since expression of a narG::lacZ ius\or\ is not affected by the presence and/or expression in trans of the narZYWV operon (Bonnefoy et ai. 1987). The formation of heteroiogous complexes of subunits of both NRZ and NRA offers an alternative explanation. In this paper, we demonstrate that the [i- and ysubunifs originating from one nar operon can associate with the a subunit of the other nar operon. The result of this association is an active heteroiogous nitrate reductase which is much more sensitive to proteolysis than either of the native enzymes. Results All experiments described in this paper have been performed with strain LCB 2048 in which the narGHJI and narZYWV genes have either been deleted or can no longer be expressed (see the Experimental procedures). Consequently, this strain is devoid of both NRA and NRZ activities and does not produce any NRA or NRZ subunits. When plasmid pVA71. which carries the narGHJI operon, is introduced into this strain, the total NRA activity measured in the crude extract is four times that of the parental strain LC8 320. When only the narG or the narHJI genes are expressed, no activity can be detected
210 F.e/ascoet al. Tabfe 1. Nitrate reductase activities in crude extracts and in membranefractionof strains transformed with various riaf plasmids. Benzyl Viologen-Nitrate Heductase Activity Sfrain/Piasmid
Relevant genotype
Expressed subunits
in caide extracts total activity
jn membrane fractions (mg protein)"'
total activity (%}
LCB 320 LCB 2048 LCB 2048 with; pVA71 pFB71,pFN72 pFB80 pFB893. pFNSO pFN90 pFB71,pFN80 pFB893,pFN72
Parental strain AnarGHJI. AnarZYWV
(up6Y)A.(ap&y)z None
NO
1.85
Formation of active heteroiogous nitrate reductases between nitrate reductases A and Z of Escherichia coli F. Blasco,^* F. Nunzj/ J. Pommier/ R. Brasseur,^ M. Chippaux^ and G. Giordano^ 'Laboratoire de Chimie Bacterienne. CNRS, BP 71, 31 cheminj. Aiguier, 13277 Marseille Cedex 9. France. ^Laboratoire de Chimie-Physique des Macromolecules aux Interfaces, Universite Libre de Bruxelles, CP 206/2. boulevard du Triomphe, 1050 Brussels, Belgium. Summary Two nitrate reductases, NRA and NRZ, are present in Escherichia coli. These isoenzymes have the same aPy. subunits composition and have simitar size and genetic organization. Corresponding subunits of the complexes share at least 75% identity. By subcioning the different genes and expressing them from separate transcriptionai units, we have demonstrated (i) that the translation of the subunits and their assembly are not coupled processes, since subunits produced concomitantiy but independently can meet efficiently and associate to form active enzymes, and (li) that the i>: subunit of a given complex can be replaced by its counterpart from the other isoenzyme to yieid an active membrane-bound heteroiogous enzyme. One such heteroiogous enzyme, a^^z'/z^ has been purified; it is less stable than the native enzymes, more susceptible to thermal denaturation, and shows increased sensitivity to proteolysis. It is also less stably bound to the membrane and, consequently, its activity with physiological electron donors is drastically reduced. The possibility that heteroiogous nitrate reductases could be formed in vivo is discussed with reference to the existence of porin heterotrimers of the outer membrane proteins OmpC, OmpF and PhoE.
Introduction Escherichia coli possesses two respiratory nitrate reductases: NRA and NRZ. Both are membrane-bound complexes composed of three subunits. a. (i and y, encoded respectively, either by the narG. narH and narl genes or Hecsived 17 July, 199i; revised 3 October, 1991- 'For correspondence. Tel. 91 16 40 94; Fax 91 71 89 14.
by the narZ. narYand narV genes (Bonnefoy-Orih et ai. 1981; Blasco et ai, 1989: 1990: 1992. accompanying paper). These genes are in transcription units. narGHJIor narZYWV. of similar size and genetic organization (Blasco et ai, 1989; 1990). The y subunit is the apoprotein of a b-iype cytochrome which is embedded in the membrane and receives electrons from the quinone pool. Electrons are then transferred via the iron-sulphur centres of the p subunit to the molybdenum cofactor on the a subunit. where the reduction of nitrate to nitrite takes place (Blasco et ai, 1989: Chaudhry and MacGregor. 1983: Ruiz-Herrera and DeMoss, 1969). The third gene of each operon. narj or narW, encodes a polypeptide. S, the precise role of which is unclear (Sodergren and DeMoss, 1988: Sodergren etai, 1988: Biasco etai, 1990). During the course of the characterization of NRZ, we obtained a strain which, although fully expressing NRA and overproducing NRZ from a ptasmid. displayed a very low level of nitrate reductase activity (one third of that expected from the narGHJ/operon alone). This discrepancy was not explained by a partial repression of the narGHJ/operon since expression of a narG::lacZ ius\or\ is not affected by the presence and/or expression in trans of the narZYWV operon (Bonnefoy et ai. 1987). The formation of heteroiogous complexes of subunits of both NRZ and NRA offers an alternative explanation. In this paper, we demonstrate that the [i- and ysubunifs originating from one nar operon can associate with the a subunit of the other nar operon. The result of this association is an active heteroiogous nitrate reductase which is much more sensitive to proteolysis than either of the native enzymes. Results All experiments described in this paper have been performed with strain LCB 2048 in which the narGHJI and narZYWV genes have either been deleted or can no longer be expressed (see the Experimental procedures). Consequently, this strain is devoid of both NRA and NRZ activities and does not produce any NRA or NRZ subunits. When plasmid pVA71. which carries the narGHJI operon, is introduced into this strain, the total NRA activity measured in the crude extract is four times that of the parental strain LC8 320. When only the narG or the narHJI genes are expressed, no activity can be detected
210 F.e/ascoet al. Tabfe 1. Nitrate reductase activities in crude extracts and in membranefractionof strains transformed with various riaf plasmids. Benzyl Viologen-Nitrate Heductase Activity Sfrain/Piasmid
Relevant genotype
Expressed subunits
in caide extracts total activity
jn membrane fractions (mg protein)"'
total activity (%}
LCB 320 LCB 2048 LCB 2048 with; pVA71 pFB71,pFN72 pFB80 pFB893. pFNSO pFN90 pFB71,pFN80 pFB893,pFN72
Parental strain AnarGHJI. AnarZYWV
(up6Y)A.(ap&y)z None
NO
1.85
