Isolation and characterization of genes coding for cytochrome b, and ...

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BIOCHEMICAL SOCl ETY TRANSACTIONS

Isolation and characterizationof genes coding for cytochrome b, and cytochrome-b, reductase JACQUELINE S. RIGBY,* PETER C. BULL,* ALAN ASHWORTH,*t ELIZABETH A. SHEPHARD,* INES SANTISTEBAN$ and IAN R. PHILLIPS$ *Department of Biochemistry, University College London, Gower Street, London WCIE 6BT, U.K. and #Department of Biochemistry, St Bartholomew’s Hospital Medical College, University of London, Charterhouse Square, London EClM 6BQ, U.K.

,A a

b

c kb

9 6

10

7.3 3.6

Cytochrome b, and NADH-dependent cytochrome 6,reductase ( E C 1.6.2.2) are components of electron transport 2.7 systems. In the endoplasmic reticulum of liver and many other tissues the proteins are involved in the desaturation of 3.2 fatty acids (Oshino & Sato, 1971) and in the cytochrome I-’150-mediated metabolism of endogenous compounds such as steroids, and of foreign compounds including many drugs, carcinogens and environmental pollutants (Ortiz de Montellano, 1986). In the cytosol of mature erythrocytes, cytochrome b, and cytochrome-b, reductase are present as smaller water-soluble species that are responsible for the reduction of methaemoglobin (Hultquist & Passon, 197 1). The inherited disorder methaemoglobinaemia can be due to a homozygous deficiency of erythrocyte cytochrome-b, reductase (Scott & Griffith, 1959; Hultquist & Passon, 1971) or, in at least one case, to a deficiency of cytochrome b, (Hegesh et al., 1986). In this paper we report the isolation and characterization of rat and human cDNA and rat genomic DNA clones coding for cytochrome b,, and a human cDNA clone coding Fig. 1. Southern blot hybridization analysis of rat (A) and human (B) genomic DNA for cytochrome h,-reductase. Genomic DNA was digested with Hind11 ( A and B, track b), BamHl (B, track a ) or EcoRl ( B , track c), electroCytochrome 6, phoresed through a 0.8% (w/v)agarose gel and transferred to cDNA clones coding for cytochrome b, were isolated by a membrane filter (Hybond-N, Amersham). The membranes screening a rat liver l g t l 1 cDNA expression library with were hybridized with 32P-labelled cDNAs coding for rat antibodies raised against cytochrome b, isolated from rat cytochrome b, ( A ) or human cytochrome-b, reductase ( B ) . liver microsomal membranes. Sequence analysis of the Membranes were washed to a final stringency of 18 mMcloned DNAs showed that cytochrome b, is encoded by two NaCl, 1 mM-sodium phosphate, pH 7.7, 0.1 mM-EDTA, mRNA species of 720 and 820 nucleotides, respectively. 0.1% (w/v)SDS at 65°C. The *As code for identical proteins and differ only in their site of polyadenylation. Analysis of rat genomic DNA by Southern blot hybridization suggested that cytochrome b, is encoded by a large gene (Fig. 1A , track a). To investigate Hind11 (Fig. lB, track b) or EcoRl (Fig. lB, track c ) . No this further we used a cDNA to isolate genomic DNA clones. additional hybridizing fragments were detected under lower Restriction endonuclease mapping, hybridization analysis stringency conditions [final wash at 50°C in 18 mM-NaCl, 1 and DNA sequencing indicate a cytochrome b, gene con- mM-sodium phosphate, pH 7.7, 0.1 mM-EDTA, 0.1% (w/v) taining at least five exons distributed over more than 25 000 SDS] (data not shown). Since the cDNA encodes a sequence of amino acid residues common to both the microsomal base pairs. A rat cytochrome b, cDNA was used as a molecular membrane bound and soluble cytochrome-b, reductases hybridization probe to screen a human liver cDNA library (Ozols et a[., 1984, 1985; Yubusui et al., 1986), it would for cloned sequences coding for the corresponding protein of appear that both forms of the protein are encoded by a single man. Comparison of the sequences of the rat and human gene. cDNAs revealed a high degree of conservation in both their We have used Southern blot hybridization of DNA protein-coding and non-coding regions. isolated from a panel of independent human-rodent somatic cell hybrids to map the cytochrome-b, reductase gene (DIAI locus) to human chromosome 22 (Bull et a[., 1988). This Cytochrome-b, reductase confirms the previous assignment of the locus made on the A synthetic oligonucleotide was used to isolate a cloned basis of starch-gel electrophoretic analysis of cytochrome-b, cDNA coding for human cytochrome-b, reductase. reductase isozymes in human-rodent somatic cell hybrids Northern blot hybridization of human liver RNA showed (Fisher et al., 1977; Junien et a[., 1978). that cytochrome-6, reductase is encoded by an mRNA of approximately 1900 nucleotides. The cDNA hybridized to We thank the M.R.C. for a grant in support of this work. J.S.R. only a single DNA fragment on Southern blots of human and P.C.B. are recipients of M.R.C. post-graduate studentships. genomic DNA digested with either BamHI (Fig. l B , track a), ?Present address: Chester Beatty Laboratories, Institute ol Cancer Research, Fulham Road, London SW3 6JB, U.K.

Bull, P. C., Shephard, E. A,, Povey, S., Santisteban, I. & Phillips, I. R. (1988)Ann. Hum. Gene[. 52,263-268 Fisher, R. A,, Povey, S., Bobrow, M., Solomon, E., Boyd, Y. & Carritt,B.(1977)Ann. Hum. Genet. 41, 151-155

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627th MEETING, NOTTINGHAM Hegesh, E., Hegesh, J. & Kaftory, A. (1986) N.Engl. J. Med. 314, 757-76 1 Hultquist, D. E. & Passon, P. G. ( 197 I ) Notiire (London) New Biol. 229,252-254 Junien, C., Vibert, M., Weil, D., Van-Cong, N. & Kaplan, J. C. ( 1 978) Hum. Genet. 42,233-239 Ortiz de Montellano, P. R. (ed.)( 1986) Cyiochrome 1'-450: Structure, . Mec/innism, and Biochemistry, Plenum, New York Oshino. N. & Sato, R. ( 1 97 I ) J . Biochem. 69. 169- 1 80 Ozols. J.. Carr, S. A. & Strittmatter. P. (1984) J . Biol. C'hem. 259. 13349-13354

Ozols, J., Korza, G., Heinemann, F. S., Hediger, M. A. & Strittmatter, P. ( 1985) J. Biol. Chem. 260, 1 1 953- I 1 96 1 Scott, E. M. & Griffith, 1. V. (1959) Biochim. Biophys. Acto 134, 584-586 Yubusui, T., Miyata, T., Iwanga, S., Tamura, M. & Takeshita, M. ( 1086)J. Hiochem. 99,407-422

Received 20 June I988

Site-directed mutagenesis of the aromatic amino acid aminotransferase of Escherichiu coli MARTIN J. CARTLAND,* MICHAEL G. HUNTER,? IAN G. FOTHERINGHAM,t GEOFFREY C. ROWLAND* and ROBERT E. GLASS* *Biochemistty Department, Nottirigham Uriiver.yityMedical School, Clifoti Boulevard, Nottingharn N G 7 ZUH, U.K. and ? The Nirtrasweet Company, 601 East Kerisirigton Road, Moirtit Prospect, IL 60065, U.S.A.

Tahle I , Thertnolahility of varinnts of the E. coli nromntic amino iic,irl ~rri~iriorrcor.~er~ictsc creaiecl by .suppression of'cr t~otisetiscrniiiution crt position I I5 Half-life of activity (min) Mutant pozition

Amino acid substituted

37°C

42°C

In Escherichia coli, the aspartate and the aromatic amino Serine h 2 Leu- I I5 acid aminotransferases are related enzymes (Fotheringham et Glutaniinc al.. 1986) with overlapping, but different, substrate speciTyrosine 47 6 ficities. Both enzymes can synthesize tyrosine aspartate and Leucine 65 39 Glutarninc/tryptophan phenylalanine, while the aromatic form can also synthesize 70 35 lcucine (Gelfand & Steinberg, 1977). In addition, there is Wild-type evidence that the aromatic form is more thermolabile (I. G. Fotheringham, personal communication). With the availability of a detailed crystal structure for aspartate amino- plasmid-borne tyrB (Leu-1 15) nonsense mutation was transtransferase from pig (Ford el al.. 1980),and the knowledge of formed into a transaminase-deficient strain carrying a parthe close homology of both prokaryotic and cukaryotic ticular suppressor and the amino acid auxotrophic aminotransferases, the aromatic amino acid aminotransfer- requirements were tested at different temperatures. ase provides an ideal opportunity to investigate the above Substitution of glutamine at position 1 15 (Leul 15Gln) properties. produced a variant incapable of growth at 37 and 42°C in the Recent site-directed mutagenesis studies on aspartate absence of aspartate. Substitution of tyrosine (Leul 15Tyr) aminotransferase have concentrated on residues known to be gave a similar result, albeit only at 42°C. It is possible, thereinvolved in the catalytic process. In particular, the critical fore, that Leu- 115 may be in a region of the enzyme which active-site lysine (Malcolm & Kirsch, 1985) and the arginine effects the specificity for the precursor of aspartate, oxaloresidue involved in substrate specificity (Cronin ef al., 1987) acetate. have been altered and the properties of the resultant mutant The thermostability of the variant has also been studied in enzymes investigated. cell extracts. For this purpose, a simple linked assay was In this work, an alternative approach was adopted. Non- adopted: aspartate was used as the amino donor to asense mutations were introduced into the structural gene for ketoglutarate, the oxaloacetate produced being converted to the aromatic amino acid aminotransferase (fyrB) to give an malate via malate dehydrogenase. The drop in NADH levels amber codon approximately every 50 residues. By using can then easily be monitored spectrophotometrically. By nonsense suppression to insert known amino acids at the using this system, it is also possible to get an indication of the mutant sites, a number of protein variants have been gener- aspartate/oxaloacetate specificity of the mutant enzymes. ated (see below). The results of these experiments are shown in Table 1. During the process of selecting the sites for mutation, no In the Leul 15Gln mutant, no activity was observed in any consideration was given to their position in the 3-dimen- of the extracts prepared. This supports the results of the sional structure in order to prevent bias towards residues of phenotypic tests that suggested ;1 change in the specificity for known importance. This 'semi-random' approach may oxaloacetate/aspartate. expose important regions of the enzyme which are otherwise In short, protein engineering using informational suppression of in vitro generated nonsense mutations in fyrB overlooked by the more conventional methods. Site-directed mutagenesis (in M13mpl8 with subsequent provides a powerful means for elucidation of residues subcloning into a pATl53-derived vector) has been used to involved in enzyme function. insert the sequence CTAG at six different sites (TAG being the stop codon). This allowed monitoring of the mutation at Cronin, C. N., Malcolm, B. A. & Kirsch, J. F. ( I 987) J. Am. Chem. SOC.109,2222-2223 all stages as CTAG is the restriction target of Mae1 as well as the core sequence of a number of other enzymes such as Ford, G. C., Eichele, G. & Jansonius, J. N. (1980) /'roc. Nntl. Accirl. Sci. U.S.A. 11,2559-2563 Nhel, Spel and Xbal. We have available ten different suppressors, permitting the creation of potentially 60 different Fotheringham, I. G., Dacey, S. A,, Taylor, P. T., Smith, T. J., Hunter, M. G., Finlay, M. E., Primrose, S. B., Parker, D. M. & Edwards, enzyme variants. The preliminary results for one such R. M. ( 1 986) Hiochem. J. 234,593-604 position, Leu-115, making use of six amino acid substitu- Gelfand, D. H. & Steinberg, R. A. ( 1977)J . Bmeriol. 130,429-440 tions are presented here. Malcolm, B. A. & Kirsch, J. F. ( I 985) Biochem. Biophys. Kes. The continuing ability of the different Leu-1 15 mutants to Commun. 132,915-921 synthesize tyrosine, phenylalanine, aspartate and leucine in vivo has been investigated using a simple plate study. The Received 16 June 1988 Vol 17