- Email: [email protected]
Expression and purification of mammalian 5-aminolevulinate synthase
336
HEME
[381
of porphobilinogen deaminase from human or animal erythrocytes or from animal liver and the stability of the enzyme, together with the commercial availability of porphobilinogen, mean that preuroporphyrinogen may be prepared in any biochemical or medical laboratory. More recently, the expression of the hemC gene specifying the deaminase from recombinant bacterial strains has aided further the isolation of the enzyme. The ability to assay the activity of uroporphyrinogen III synthase relies on the fact that the enzyme-catalyzed transformation of the unstable 1-hydroxymethylbilane, prueroporphyrinogen, into uroporphyrinogen III is much faster than the spontaneous nonenzymatic cyclization to form uroporphyrinogen I. However, the involvement of a substrate with a halflife of less than 5 min under the assay conditions means that there will always be a background formation of uroporphyrinogen I not only during the preuroporphyrinogen generation stage but also during the uroporphyrinogen III synthesis stage. The unavoidable formation of uroporphyrinogen I is the main difficulty in the assay of the synthase, because at low levels of synthase there will be a proportional increase in the preuroporphyrinogen available for chemical cyclization to give uroporphyrinogen I. In contrast, at highlevels of the synthase there will be less uroporphyrinogen I formed because preuroporphyrinogen will be utilized by the synthase. This will lead to an underestimate of the synthase activity at high levels of the synthase enzyme. The comparison of the direct assay readings and predicted ratios of isomer III to isomer I with the ratios determined by HPLC provides an important validation of the rapid assay (Fig. 4). Acknowledgment We are grateful to the BBSRC for financial support.
[38] E x p r e s s i o n a n d P u r i f i c a t i o n o f M a m m a l i a n 5-Aminolevulinate Synthase By H A R R Y A . DAILEY a n d TAMARA A . DAILEY
The first step in the biosynthesis of any tetrapyrrole is the synthesis of the compound 5-aminolevulinate (ALA). In nature two ways have evolved to produce this compound. 1 In one the starting material is glutamate in 1p. M. Jordan, in "Biosynthesis of Heme and Chlorophylls" (H. A. Dailey, ed.), p. 55. McGraw-Hill, New York, 1990.
METHODS IN ENZYMOLOGY, VOL. 281
Copyright © 1997 by Academic Press All rights of reproduction in any form reserved. 0076-6879/97 $25
[38]
MAMMALIAN 5-AMINOLEVULINATE SYNTHASE
337
the form of glutamyl-tRNA. Two enzymes, glutamyl-tRNA reductase and glutamate-l-semialdehyde aminotransferase, catalyze the formation of ALA from glutamyl-tRNA. This pathway is referred to as the C5 pathway and is found in plants, archaebacteria, and many eubacteria. 2 In contrast, nonplant eukaryotes and a few eubacteria utilize a single enzymatic step to form ALA from succinyl-CoA and glycine.1 This enzyme is named ALA synthase (ALAS) (EC 2.3.1.37) and synthesis of ALA by ALAS is referred to as the C4 pathway. In chickens and mammals two ALAS enzymes a r e f o u n d . 3'4 These two enzymes, which are highly homologous in structure and function, are encoded by separate genes and are subject to different forms of regulation. ALAS-l, or the housekeeping form of ALAS, is present in all nonerythroid cell types. It has a short half-life and is subject to regulation by the end product of the pathway, protoheme. Details of the regulation of ALAS can be found in reviews by May et al. 5 and Dierks. 3 A second form of the enzyme is ALAS-2, or the erythroid-specific form. This enzyme, which is encoded on the human and mouse X chromosome,6 is induced only during the period of increased heme synthesis in developing erythroid cells. Unlike ALAS-l, it does not appear to be regulated by heme, but is subject to regulation by iron, due to the presence of a 5' ironresponsive element (IRE), as well as a variety of erythroid-specific factors] It does not appear, however, that ALAS-2 alone is responsible for regulation of heme synthesis in these cells. 8 Deficiency of activity of ALAS-2 has been demonstrated to be the cause of X-linked sideroblastic anemia (see Ref. 9). A number of genetic mutations have been identified in individuals with pyridoxine-responsive X-linked sideroblastic anemia, and some have been demonstrated to result in decreased enzyme activity.1°-12 2 y. j. Avissar, J. G. Ormerod, and S. I. Beale, Arch. Microbiol. 151, 513 (1989). 3 p. Dierks, in "Biosynthesis of Heine and Chlorophylls" (H. A. Dailey, ed.), p. 201. McGrawHill, New York, 1990. a R. D. Ridable, M. Yamamoto, and J. D. Engel, Proc. Natl. Acad. Sci. U.S.A. 86, 792 (1989). 5 B. K. May, C. R. Bhasker, M. J. Bawden, and T. C. Cox, Mol. Biol. Med. 7, 405 (1990). 6 p. D. Cotter, H. F. Willard, J. L. Gorski, and D. F. Bishop, Genomics 13, 211 (1992). 7 T. C. Cox, M. J. Bawden, M. Alison, A. Martin, and B. K. May, E M B O J. 10, 1891 (1991). 8 H. Lake-Bullock and H. A. Dailey, Mol. Cell. Biol. 13, 7122 (1993). 9 S. S. Bottomly, in "Wintrobe's Clinical Hematology" (G. R. Lee, T. C. Bithell, J. Forester, J. W. Athens, and J. N. Lakens, eds.), 9th Ed., Vol. 1. Lea & Febiger, Philadelphia, 1993. 10E. Prades, C. Chambon, T. A. Dailey, H. A. Dailey, J. Bri6re, and B. Grandchamp, Hum. Genet. 95, 424 (1995). 11 p. D. Cotter, M. Baumann, and D. F. Bishop, Proc. Natl. Acad. Sci. U.S.A. 89, 4028 (1992). 12T. C. Cox, S. S, Bottomley, J. S. Wiley, M. J. Bacoden, C. S. Mathews, and B. K. May, N. Engl. J. Med. 330, 675 (1994).
338
rtEME
[38]
All forms of ALAS appear to be homodimers with pyridoxal 5'-phosphate as a cofactor. In eukaryotes the enzyme is nuclear encoded, cytoplasmicaUy synthesized as a precursor with a mitochondrial targeting sequence, and then translocated into the mitochondrial matrix where it is processed into its mature form (see Ref. 3 for review). Expression and Purification of Murine 5-Aminolevulinate Synthase 2 The cDNA for mouse ALAS-2 is supplied by P. Curtis (Wistar Institute, Philadelphia, PA) and is in two fragments (pMS-6 and pMS-1213). These fragments have been combined to give a full-length ALAS-2 cDNA in plasmid pHD2J 4 Using this plasmid as starting material two different expression vectors have been constructed in our laboratory. One, pGF23, encodes the production of a mature-length ALAS-2 under the control of an alkaline phosphatase promoter TM and a second, pTAD-ALAS2, encodes the production of a mature-length ALAS-2 under the control of a Tac promoterJ ° Both plasmids when expressed in Escherichia coli JM109 and grown under appropriate conditions give high yields of protein. The deciding factor in selection of vector has only to do with choice or ease of induction technique. Following growth and induction of protein synthesis, ceils are harvested by centrifugation at 6000 g for 15 min at 4°. The cell pellet is suspended in buffer A [20 mM potassium phosphate (pH 7.2), 1 mM EDTA, 5 mM mercaptoethanol, 10% (v/v) glycerol, and 20 /zM pyridoxal phosphate] that contains 100/.~M phenylmethylsulfonyl fluoride (PMSF) and the cells disrupted by sonication at maximum power (twice for 30 sec). The disrupted cells are centrifuged at 15,000 g for 20 min at 4° to remove cellular debris and the supernatant is carefully decanted. Saturated ammonium sulfate solution, pH 8.0, is slowly added to yield 40% (w/v) saturation and after 30 min at 4° this solution is centrifuged (15,000 g for 20 rain at 4°). The resulting pellet, which contains ALAS-2, is resuspended in a minimal volume of buffer A with PMSF and applied to an Ultrogel AcA 44 column (1.5 x 30 cm) equilibrated with buffer A. Fractions from this column that contain ALAS activity are pooled and applied to a DEAE-Sephacel column (2.5 x 12 cm) that is equilibrated with buffer A. The column is successively washed with 100 ml of buffer A and 100 ml of buffer A with 40 mM KC1 before the protein is eluted with buffer A with 100 mM KC1. Fractions containing ALAS activity are pooled and stored at - 2 0 °. From 1 liter of culture one obtains approximately 50 mg of enzyme. 13D. S. Schoenhautand P. J. Curtis, Gene (Amsterdam) 48, 55 (1986). 14G. C. Ferreira and H. A. Dailey,J. Biol. Chem. 268, 584 (1993).
[381
MAMMALIAN 5-AMINOLEVULINATE SYNTHASE
339
5-Aminolevulinate S y n t h a s e Assay A variety of A L A S assays are available and choice of assay will be dependent on enzyme activity and level of purity of the sample. A radiochemical assay described by B r o o k e r et aL 15 is effective and sensitive, although costly. We have used this procedure only on crude tissue or cell extracts. The most common assay procedure is the one based on use of modified Ehrlich's reagent. 16When proper precautions are taken this procedure is accurate and inexpensive. There is also a coupled continuous spectrophotornetric assay that should prove useful in assaying purified ALAS. 17 Properties of R e c o m b i n a n t M u r i n e 5-Aminolevulinate S y n t h a s e 2 ALAS-2 as prepared herein has a molecular weight of approximately 56,000 as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. This along with the obsery.ation that gel filtration of the purified protein on Sephadex G-200 yields a molecular weight of just over 100,000 supports previous reports that the enzyme is a homodimer. 1 The enzyme contains a noncovalently bound pyridoxal 5'-phosphate, which may be removed by dialysis. Functional holoenzyme is restored by incubation with pyridoxal 5'-phosphate. Although it has been shown that Lys-313 of the enzyme will form a covalent adduct with the cofactor, TM site-directed mutagenesis studies indicate that this residue is not required for binding of cofaetor, but may be involved in catalysis. 19 Determination of the actual residues involved awaits X-ray crystallographic structure determination. The kinetic constants for recombinant murine ALAS-2 are similar to those determined for the enzyme from mammalian tissues. The Km for glycine is 51 m M and for succinyl-CoA is 55/xM. The kca is approximately 0.5 rain -1.14 Summary We have described a procedure for production and purification of recombinant, mature-length mouse ALAS-2. The fact that E. coli utilizes the C5 path for A L A production means that there is no problem with contamination of the recombinant ALAS-2 by host cell enzyme, such as one may have with a yeast expression system. While the detailed procedure ~5j. D. Brooker, G. Srivastava, B. K. May, and W. H. Elliott, Enzyme 28, 109 (1982). 16L.-F. Lien and D. S. Beattie, Enzyme 28, 120 (1982). 17G. A. Hunter and G. C. Ferreira, Anal Biochem. 226, 221 (1995). 18G. C. Ferreira, P. J. Neame, and H. A. Dailey, Protein Sci. 2, 1959 (1993). 19G. C. Ferreira, U. Vaiapey, O. Hafey, G. A. Hunter, and M. J. Barber, Protein Sci. 4, 1001 (1995).
340
HEME
[391
produces enzyme in good yield with relatively common protein purification techniques, future expression systems may be developed to take advantage of the rapid purification achieved by the use of a 6-histidine (His6) aminoterminal tag and metal chelate chromatography. Such approaches in this laboratory with protoporphyrinogen oxidase, 2°'21 coproporphyrinogen oxidase, 22 and uroporphyrinogen decarboxylase have resulted in the production and purification of enzymes whose kinetic and physical parameters are essentially identical to those of proteins lacking the His6 tag.
Acknowledgment This work was supported by NIH Grants DK32303 and DK35898 (to H.A.D.).
2o H. A. Dailey and T. A. Dailey, J. Biol. Chem. 271, 8714 (1996). 21 T. A. Dailey and H. A. Dailey, Protein Sci. 5, 98 (1996). 2z A. Medlock and H. A. Dailey, J. Biol. Chem. 2719 32507 (1996).
[39] E x p r e s s i o n , P u r i f i c a t i o n , a n d C h a r a c t e r i s t i c s o f Mammalian Protoporphyrinogen Oxidase
By
T A M A R A A . DAILEY a n d H A R R Y A . DAILEY
The penultimate enzyme of the heme biosynthetic pathway, protoporphyrinogen oxidase (PPO) (EC 1.3.3.4), catalyzes the six-electron oxidation of protoporphyrinogen IX to form protoporphyrin IX (Scheme 1). 1-3 The enzyme has been purified from mouse4 and bovines liver, and from yeast.6 More recently, the enzyme has been cloned from Bacillus subtilis, 7-9 Myxo-
1 R. J. Porra and J. E. Falk, Biochem. Z 90, 69 (1964). 2 R. Poulson and W. J. Polglase, J. Biol. Chem. 250, 1269 (1975). 3 H. A. Dailey, in "Biosynthesis of Heme and Chlorophylls" (H. A. Dailey, ed.), p. 123. McGraw-Hill, New York, 1990. 4 H. A. Dailey and S. W. Karr, Biochemistry 26, 2697 (1987). 5 L. J. Siepker, M. Ford, R. de Kock, and S. Kramer, Biochim. Biophys. Acta 913, 349 (1987). 6 j. M. Camadro, F. Thome, N. Brouillet, and P. Labbe, J. Biol. Chem. 269, 32085 (1994). 7 M. Hansson and L. Hederstedt, J. Bacteriol. 174, 8081 (1992). 8 M. Hansson and L. Hederstedt, J. Bacteriol. 176, 5962 (1994). 9 T. A. Dailey, P. N. Meissner, and H. A. Dailey, J. Biol. Chem. 269, 813 (1994).
METHODS IN ENZYMOLOGY, VOL. 281
Copyright © 1997 by Academic Press All rights of reproduction in any form reserved. 0076-6879/97 $25
HEME
[381
of porphobilinogen deaminase from human or animal erythrocytes or from animal liver and the stability of the enzyme, together with the commercial availability of porphobilinogen, mean that preuroporphyrinogen may be prepared in any biochemical or medical laboratory. More recently, the expression of the hemC gene specifying the deaminase from recombinant bacterial strains has aided further the isolation of the enzyme. The ability to assay the activity of uroporphyrinogen III synthase relies on the fact that the enzyme-catalyzed transformation of the unstable 1-hydroxymethylbilane, prueroporphyrinogen, into uroporphyrinogen III is much faster than the spontaneous nonenzymatic cyclization to form uroporphyrinogen I. However, the involvement of a substrate with a halflife of less than 5 min under the assay conditions means that there will always be a background formation of uroporphyrinogen I not only during the preuroporphyrinogen generation stage but also during the uroporphyrinogen III synthesis stage. The unavoidable formation of uroporphyrinogen I is the main difficulty in the assay of the synthase, because at low levels of synthase there will be a proportional increase in the preuroporphyrinogen available for chemical cyclization to give uroporphyrinogen I. In contrast, at highlevels of the synthase there will be less uroporphyrinogen I formed because preuroporphyrinogen will be utilized by the synthase. This will lead to an underestimate of the synthase activity at high levels of the synthase enzyme. The comparison of the direct assay readings and predicted ratios of isomer III to isomer I with the ratios determined by HPLC provides an important validation of the rapid assay (Fig. 4). Acknowledgment We are grateful to the BBSRC for financial support.
[38] E x p r e s s i o n a n d P u r i f i c a t i o n o f M a m m a l i a n 5-Aminolevulinate Synthase By H A R R Y A . DAILEY a n d TAMARA A . DAILEY
The first step in the biosynthesis of any tetrapyrrole is the synthesis of the compound 5-aminolevulinate (ALA). In nature two ways have evolved to produce this compound. 1 In one the starting material is glutamate in 1p. M. Jordan, in "Biosynthesis of Heme and Chlorophylls" (H. A. Dailey, ed.), p. 55. McGraw-Hill, New York, 1990.
METHODS IN ENZYMOLOGY, VOL. 281
Copyright © 1997 by Academic Press All rights of reproduction in any form reserved. 0076-6879/97 $25
[38]
MAMMALIAN 5-AMINOLEVULINATE SYNTHASE
337
the form of glutamyl-tRNA. Two enzymes, glutamyl-tRNA reductase and glutamate-l-semialdehyde aminotransferase, catalyze the formation of ALA from glutamyl-tRNA. This pathway is referred to as the C5 pathway and is found in plants, archaebacteria, and many eubacteria. 2 In contrast, nonplant eukaryotes and a few eubacteria utilize a single enzymatic step to form ALA from succinyl-CoA and glycine.1 This enzyme is named ALA synthase (ALAS) (EC 2.3.1.37) and synthesis of ALA by ALAS is referred to as the C4 pathway. In chickens and mammals two ALAS enzymes a r e f o u n d . 3'4 These two enzymes, which are highly homologous in structure and function, are encoded by separate genes and are subject to different forms of regulation. ALAS-l, or the housekeeping form of ALAS, is present in all nonerythroid cell types. It has a short half-life and is subject to regulation by the end product of the pathway, protoheme. Details of the regulation of ALAS can be found in reviews by May et al. 5 and Dierks. 3 A second form of the enzyme is ALAS-2, or the erythroid-specific form. This enzyme, which is encoded on the human and mouse X chromosome,6 is induced only during the period of increased heme synthesis in developing erythroid cells. Unlike ALAS-l, it does not appear to be regulated by heme, but is subject to regulation by iron, due to the presence of a 5' ironresponsive element (IRE), as well as a variety of erythroid-specific factors] It does not appear, however, that ALAS-2 alone is responsible for regulation of heme synthesis in these cells. 8 Deficiency of activity of ALAS-2 has been demonstrated to be the cause of X-linked sideroblastic anemia (see Ref. 9). A number of genetic mutations have been identified in individuals with pyridoxine-responsive X-linked sideroblastic anemia, and some have been demonstrated to result in decreased enzyme activity.1°-12 2 y. j. Avissar, J. G. Ormerod, and S. I. Beale, Arch. Microbiol. 151, 513 (1989). 3 p. Dierks, in "Biosynthesis of Heine and Chlorophylls" (H. A. Dailey, ed.), p. 201. McGrawHill, New York, 1990. a R. D. Ridable, M. Yamamoto, and J. D. Engel, Proc. Natl. Acad. Sci. U.S.A. 86, 792 (1989). 5 B. K. May, C. R. Bhasker, M. J. Bawden, and T. C. Cox, Mol. Biol. Med. 7, 405 (1990). 6 p. D. Cotter, H. F. Willard, J. L. Gorski, and D. F. Bishop, Genomics 13, 211 (1992). 7 T. C. Cox, M. J. Bawden, M. Alison, A. Martin, and B. K. May, E M B O J. 10, 1891 (1991). 8 H. Lake-Bullock and H. A. Dailey, Mol. Cell. Biol. 13, 7122 (1993). 9 S. S. Bottomly, in "Wintrobe's Clinical Hematology" (G. R. Lee, T. C. Bithell, J. Forester, J. W. Athens, and J. N. Lakens, eds.), 9th Ed., Vol. 1. Lea & Febiger, Philadelphia, 1993. 10E. Prades, C. Chambon, T. A. Dailey, H. A. Dailey, J. Bri6re, and B. Grandchamp, Hum. Genet. 95, 424 (1995). 11 p. D. Cotter, M. Baumann, and D. F. Bishop, Proc. Natl. Acad. Sci. U.S.A. 89, 4028 (1992). 12T. C. Cox, S. S, Bottomley, J. S. Wiley, M. J. Bacoden, C. S. Mathews, and B. K. May, N. Engl. J. Med. 330, 675 (1994).
338
rtEME
[38]
All forms of ALAS appear to be homodimers with pyridoxal 5'-phosphate as a cofactor. In eukaryotes the enzyme is nuclear encoded, cytoplasmicaUy synthesized as a precursor with a mitochondrial targeting sequence, and then translocated into the mitochondrial matrix where it is processed into its mature form (see Ref. 3 for review). Expression and Purification of Murine 5-Aminolevulinate Synthase 2 The cDNA for mouse ALAS-2 is supplied by P. Curtis (Wistar Institute, Philadelphia, PA) and is in two fragments (pMS-6 and pMS-1213). These fragments have been combined to give a full-length ALAS-2 cDNA in plasmid pHD2J 4 Using this plasmid as starting material two different expression vectors have been constructed in our laboratory. One, pGF23, encodes the production of a mature-length ALAS-2 under the control of an alkaline phosphatase promoter TM and a second, pTAD-ALAS2, encodes the production of a mature-length ALAS-2 under the control of a Tac promoterJ ° Both plasmids when expressed in Escherichia coli JM109 and grown under appropriate conditions give high yields of protein. The deciding factor in selection of vector has only to do with choice or ease of induction technique. Following growth and induction of protein synthesis, ceils are harvested by centrifugation at 6000 g for 15 min at 4°. The cell pellet is suspended in buffer A [20 mM potassium phosphate (pH 7.2), 1 mM EDTA, 5 mM mercaptoethanol, 10% (v/v) glycerol, and 20 /zM pyridoxal phosphate] that contains 100/.~M phenylmethylsulfonyl fluoride (PMSF) and the cells disrupted by sonication at maximum power (twice for 30 sec). The disrupted cells are centrifuged at 15,000 g for 20 min at 4° to remove cellular debris and the supernatant is carefully decanted. Saturated ammonium sulfate solution, pH 8.0, is slowly added to yield 40% (w/v) saturation and after 30 min at 4° this solution is centrifuged (15,000 g for 20 rain at 4°). The resulting pellet, which contains ALAS-2, is resuspended in a minimal volume of buffer A with PMSF and applied to an Ultrogel AcA 44 column (1.5 x 30 cm) equilibrated with buffer A. Fractions from this column that contain ALAS activity are pooled and applied to a DEAE-Sephacel column (2.5 x 12 cm) that is equilibrated with buffer A. The column is successively washed with 100 ml of buffer A and 100 ml of buffer A with 40 mM KC1 before the protein is eluted with buffer A with 100 mM KC1. Fractions containing ALAS activity are pooled and stored at - 2 0 °. From 1 liter of culture one obtains approximately 50 mg of enzyme. 13D. S. Schoenhautand P. J. Curtis, Gene (Amsterdam) 48, 55 (1986). 14G. C. Ferreira and H. A. Dailey,J. Biol. Chem. 268, 584 (1993).
[381
MAMMALIAN 5-AMINOLEVULINATE SYNTHASE
339
5-Aminolevulinate S y n t h a s e Assay A variety of A L A S assays are available and choice of assay will be dependent on enzyme activity and level of purity of the sample. A radiochemical assay described by B r o o k e r et aL 15 is effective and sensitive, although costly. We have used this procedure only on crude tissue or cell extracts. The most common assay procedure is the one based on use of modified Ehrlich's reagent. 16When proper precautions are taken this procedure is accurate and inexpensive. There is also a coupled continuous spectrophotornetric assay that should prove useful in assaying purified ALAS. 17 Properties of R e c o m b i n a n t M u r i n e 5-Aminolevulinate S y n t h a s e 2 ALAS-2 as prepared herein has a molecular weight of approximately 56,000 as determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. This along with the obsery.ation that gel filtration of the purified protein on Sephadex G-200 yields a molecular weight of just over 100,000 supports previous reports that the enzyme is a homodimer. 1 The enzyme contains a noncovalently bound pyridoxal 5'-phosphate, which may be removed by dialysis. Functional holoenzyme is restored by incubation with pyridoxal 5'-phosphate. Although it has been shown that Lys-313 of the enzyme will form a covalent adduct with the cofactor, TM site-directed mutagenesis studies indicate that this residue is not required for binding of cofaetor, but may be involved in catalysis. 19 Determination of the actual residues involved awaits X-ray crystallographic structure determination. The kinetic constants for recombinant murine ALAS-2 are similar to those determined for the enzyme from mammalian tissues. The Km for glycine is 51 m M and for succinyl-CoA is 55/xM. The kca is approximately 0.5 rain -1.14 Summary We have described a procedure for production and purification of recombinant, mature-length mouse ALAS-2. The fact that E. coli utilizes the C5 path for A L A production means that there is no problem with contamination of the recombinant ALAS-2 by host cell enzyme, such as one may have with a yeast expression system. While the detailed procedure ~5j. D. Brooker, G. Srivastava, B. K. May, and W. H. Elliott, Enzyme 28, 109 (1982). 16L.-F. Lien and D. S. Beattie, Enzyme 28, 120 (1982). 17G. A. Hunter and G. C. Ferreira, Anal Biochem. 226, 221 (1995). 18G. C. Ferreira, P. J. Neame, and H. A. Dailey, Protein Sci. 2, 1959 (1993). 19G. C. Ferreira, U. Vaiapey, O. Hafey, G. A. Hunter, and M. J. Barber, Protein Sci. 4, 1001 (1995).
340
HEME
[391
produces enzyme in good yield with relatively common protein purification techniques, future expression systems may be developed to take advantage of the rapid purification achieved by the use of a 6-histidine (His6) aminoterminal tag and metal chelate chromatography. Such approaches in this laboratory with protoporphyrinogen oxidase, 2°'21 coproporphyrinogen oxidase, 22 and uroporphyrinogen decarboxylase have resulted in the production and purification of enzymes whose kinetic and physical parameters are essentially identical to those of proteins lacking the His6 tag.
Acknowledgment This work was supported by NIH Grants DK32303 and DK35898 (to H.A.D.).
2o H. A. Dailey and T. A. Dailey, J. Biol. Chem. 271, 8714 (1996). 21 T. A. Dailey and H. A. Dailey, Protein Sci. 5, 98 (1996). 2z A. Medlock and H. A. Dailey, J. Biol. Chem. 2719 32507 (1996).
[39] E x p r e s s i o n , P u r i f i c a t i o n , a n d C h a r a c t e r i s t i c s o f Mammalian Protoporphyrinogen Oxidase
By
T A M A R A A . DAILEY a n d H A R R Y A . DAILEY
The penultimate enzyme of the heme biosynthetic pathway, protoporphyrinogen oxidase (PPO) (EC 1.3.3.4), catalyzes the six-electron oxidation of protoporphyrinogen IX to form protoporphyrin IX (Scheme 1). 1-3 The enzyme has been purified from mouse4 and bovines liver, and from yeast.6 More recently, the enzyme has been cloned from Bacillus subtilis, 7-9 Myxo-
1 R. J. Porra and J. E. Falk, Biochem. Z 90, 69 (1964). 2 R. Poulson and W. J. Polglase, J. Biol. Chem. 250, 1269 (1975). 3 H. A. Dailey, in "Biosynthesis of Heme and Chlorophylls" (H. A. Dailey, ed.), p. 123. McGraw-Hill, New York, 1990. 4 H. A. Dailey and S. W. Karr, Biochemistry 26, 2697 (1987). 5 L. J. Siepker, M. Ford, R. de Kock, and S. Kramer, Biochim. Biophys. Acta 913, 349 (1987). 6 j. M. Camadro, F. Thome, N. Brouillet, and P. Labbe, J. Biol. Chem. 269, 32085 (1994). 7 M. Hansson and L. Hederstedt, J. Bacteriol. 174, 8081 (1992). 8 M. Hansson and L. Hederstedt, J. Bacteriol. 176, 5962 (1994). 9 T. A. Dailey, P. N. Meissner, and H. A. Dailey, J. Biol. Chem. 269, 813 (1994).
METHODS IN ENZYMOLOGY, VOL. 281
Copyright © 1997 by Academic Press All rights of reproduction in any form reserved. 0076-6879/97 $25