Plant Physiol. (1992) 99, 693-699 Received for publication September 20, 1991 0032-0889/92/99/0693/07/$01 .00/0 Accepted December 11, 1991 Expression in Escherichia coli of Cytochrome c Reductase Activity from a Maize NADH:Nitrate Reductase Complementary DNA' Wilbur H. Campbell Phytotechnology Research Center and Department of Biological Sciences, Michigan Technological University, Houghton, Michigan 49931-1295 ABSTRACT the deduced amino acid sequences of NR to related mam- A cDNA clone was isolated from a maize (Zea mays L. cv malian protein sequences because of its similarity to the Mo- W64AxW183E) scutellum Xgtl 1 library using maize leaf pterin domain of sulfite oxidase, and the C-terminal region NADH:nitrate reductase Zmnrl cDNA clone as a hybridization of NR has sequence homology to NADH:Cyt b5 reductase, probe; it was designated Zmnr1S. ZmnrlS was shown to be an which is a FAD-containing enzyme (3, 22). In between the NADH:nitrate reductase clone by nucleotide sequencing and com- Mo-pterin and FAD domains, NR has similarity to mam- parison of its deduced amino acid sequence to Zmnrl. Zmnr1S, malian Cyt b5. This central Cyt b domain of NR is bridged to which is 1.8 kilobases in length and contains the code for both the other two domains on either side of it by highly variable the cytochrome b and flavin adenine dinucleotide domains of sequence regions that appear to be hinges (3). These hinge nitrate reductase, was cloned into the EcoRI site of the Esche- regions are probably exposed in native NR and susceptible to richia coli expression vector pET5b and expressed. The cell attack by proteolytic enzymes (15, 22). Hence, native NR can lysate contained NADH:cytochrome c reductase activity, which is cleaved by into stable fragments that a characteristic partial activity of NADH:nitrate reductase depend- readily be proteinases ent on the cytochrome b and flavin adenine dinucleotide domains. retain partial catalytic activities of holo-NR, such as methyl Recombinant cytochrome c reductase was purified by immunoaf- viologen NR, NADH:Cyt c reductase, or NADH:ferricyanide finity chromatography on monoclonal antibody Zm2(69) Sepha- reductase (15, 22). The stable fragments appear to be the rose. The purified cytochrome c reductase, which had a major domains of the native NR or combinations of two domains; size of 43 kilodaltons, was inhibited by polyclonal antibodies for for instance, the fragment with methyl viologen NR activity maize leaf NADH:nitrate reductase and bound these antibodies is composed of the Mo-pterin and Cyt b domains, the Cyt c when blotted to nitrocellulose. Ultraviolet and visible spectra of reductase fragment contains the Cyt b and FAD domains, oxidized and NADH-reduced recombinant cytochrome c reduc- and the ferricyanide reductase fragment contains only the tase were nearly identical with those of maize leaf NADH:nitrate FAD domain (3, 15, 22). These catalytically active and stable reductase. These two enzyme forms also had very similar kinetic NR in out some of the properties with respect to NADH-dependent cytochrome c and fragments of have been useful sorting ferricyanide reduction. complexities of the biochemistry of NR (2, 3, 22). However, it is difficult to produce these fragments in sufficient amounts for many studies, especially those involving physical and chemical measurements. An alternative approach is to produce the NR domains and their catalytically active combinations as recombinant pro- Higher plant NADH:NR2 (EC 1.6.6.1) catalyzes the first teins in coli cDNA clones for NR. The a of Escherichia using step in nitrate assimilation and is key site for regulation FAD domain of maize leaf NADH:NR was overexpressed in this pathway (22). The enzyme is a homodimer composed of E. coli using part of the Zmnrl cDNA (9) and purified to two approximately 100-kD polypeptide chains with each con- homogeneity by NADH elution from blue Sepharose (11). taining one equivalent of FAD, heme-Fe, and Mo-pterin (2, The recombinant FAD domain of NR has an Mr of 30,000, 3, 17). Each of these electron-carrying cofactors appears to be NADH:ferricyanide reductase activity, and a UV-visible spec- bound into a specific region of the enzyme's polypeptide, trum essentially identical with mammalian NADH:Cyt b5 which folds into distinct domains. The N-terminal region of reductase, which demonstrated that FAD is bound to the NR can be recognized as the Mo-pterin domain by comparing recombinant protein in a functional manner (1 1). The recom- binant FAD domain has now been crystallized, and analysis This research was supported by National Science Foundation of its three-dimensional structure is underway (G. Lu, W.H. grant DCB-9020773 and U.S. Department ofAgriculture Competitive Campbell, Y. Lindqvist, and G. Schneider, unpublished re- Research grant 90-37280-5474. 2 Abbreviations: NR, nitrate reductase; anti-FD, rabbit polyclonal sults). The Cyt b domain of Chlorella NADH:NR has also antibody for SDS-denatured recombinant FAD domain of NR; anti- been expressed in E. coli, and although this domain has no NR, rabbit polyclonal antibody for nitrate reductase; FAD, flavin catalytic activity by itself, the recombinant protein has a adenine dinucleotide; IgG, immunoglobulin G; IPTG, isopropyl 3-D- visible spectrum very similar to native NR (5). thiogalactopyranoside. Reported here is the expression in E. coli of a fragment of 693 694 CAMPBELL Plant Physiol. Vol. 99, 1992 NADH:NR with NADH:Cyt c reductase activity. The recom- the chromosome of the DE3 cell strain and is under the binant protein was purified using monoclonal antibody-based control of the inducible lac UV5 promoter (24). Cells were immunoaffinity chromatography, which was developed for grown to 0.6 A600nm before induction with IPTG. After 2.5 to purification ofholo-NR from maize leafextracts (12). Recom- 3 h of induction with IPTG, the cells were harvested by binant Cyt c reductase has immunochemical, kinetic, and centrifugation. spectral properties virtually identical with NADH:NR purified from maize leaves by the same method. Purification of Recombinant Cyt c Reductase MATERIALS AND METHODS E. coli cells harvested from two 1-L cultures were suspended in 1 L of 0.1 M KPO4 (pH 7.5) and 1 mM EDTA and frozen Isolation of Maize Scutellum cDNA Clones for NR at -70°C for at least 12 h before thawing. No additives were Total RNA was isolated from nitrate-induced scutella of required to lyse the cells because they were expressing T7 maize (Zea mays L. cv W64AXW183E) seedlings and shown lysozyme from the pLysS plasmid (23). After thawing, cell to contain a transcript that hybridized to the Zmnrl cDNA debris was removed by centrifugation for 30 min at 20,000g, clone, as previously described (10). cDNA was synthesized and the supernatant fluid was mixed with 7 to 10 mL of from polyadenylated RNA isolated on mAP paper (Amer- Zm2(69) Sepharose (monoclonal antibody Zm2(69) coupled sham, Chicago, IL) and cloned into EcoRI-cut Xgtl 1 arms to cyanogen bromide-activated Sepharose 4B [Pharmacia] at (Stratagene, San Diego, CA) after EcoRI-NotI linker-adaptor 1 mg of IgG/mL gel) at 4°C for 45 min (12). The gel was (GAATTCGCGGCCGC) ligation (Librarian cDNA cloning recovered by vacuum filtration and washed with a total of 2 kit; Invitrogen, San Diego, CA). The maize scutellum cDNA L of suspension buffer using three washes with the gel col- library was screened by hybridization with radioactive Zmnrl lected by filtration in between each wash. The washed gel was insert DNA, and four positive clones were isolated and plaque mixed with a minimum volume of suspension buffer and purified (9, 18). Insert DNA was excised with EcoRI and packed into a column (0.9 x 15 cm). The column was eluted subcloned into the EcoRI site of Bluescript II vector (Strata- with 10 mm glycine (pH 11), and 1. 1-mL fractions were gene). Nucleotide sequencing was carried out by the dide- collected into tubes containing 15 ML of 2 M NaPO4, pH 7 oxynucleotide method on the double-stranded DNA of these (12). Crude extract, the first filtrate obtained immediately subclones using T7, T3, SK, and KS primers (Stratagene), as after the binding step, and purified fractions eluted from the well as six custom primers derived from the nucleotide se- immunoaffinity column were assayed for Cyt c reductase quence of Zmnrl (9). One of these clones, designated activity (21), protein (16), and A413nm (purified fractions only). ZmnrlS, is very similar to Zmnr1 in nucleotide sequence and contains 1804 base pairs, which makes it 300 base pairs Purification of Other Enzyme Forms and Miscellaneous shorter than Zmnrl (9). The other three clones were either Methods smaller or contained some nucleotide sequence regions not related to Zmnrl. Previously described methods were used to purify maize leafNADH:NR (12) and the recombinant FAD domain (1 1). Construction of the Expression Vector Spectra ofpurified Cyt c reductase and maize leafNADH:NR were analyzed with a Shimadzu UV- 1201 UV/visible spectro- ZmnrlS insert was excised with EcoRI and cloned into the photometer at 25°C. These proteins were reduced with solid EcoRI site of the E. coli expression vector pET5b (Novagen, NADH to generate their reduced spectra. Kinetic analyses Madison, WI). After the clone was transformed into E. coli were done at 25°C with a Gilford 2400-2 UV/visible spectro- strain DH5a- competent cells, three clones were selected photometer measuring the change in absorbance with time containing the ZmnrlS insert, and the orientation was deter- for Cyt c reduction at 550 nm and for ferricyanide reduction mined by nucleotide sequencing using the T7 primer, which at 340 nm (NADH), which were converted to micromoles per binds to the T7 promoter in the pET vector series (24).