Comp. Biochem. Physiol. Vol. 102B.No. 4, pp. 791-793, 1992 Printed in Great Britain
0305-0491/92 $5.00 + 0.00 © 1992Pergamon Press Ltd
HUMAN LIVER GLUTAMIC 3,-SEMIALDEHYDE DEHYDROGENASE: STRUCTURAL RELATIONSHIP TO THE YEAST ENZYME JOHN HEMPEL,* ROLF ECKEY,~" DIANE BERIE,* HANA ROMOVACEK,* DHARAM P. AGARWAL~"and H. WERNER GOEDDE~ *Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, U.S.A. Tel: (412) 648-9552; Fax: (412) 624-1401; and l'Institute of Human Genetics, University of Hamburg, Hamburg, Germany (Received 3 January 1992)
Abstract--The amino acid sequences of nine tryptic peptides (containing altogether 105 amino acids) from human liver glutamic ~,-semialdehyde dehydrogenase (hitherto designated as ALDH4) were found to correspond, at 33~6% identity, to segments from the yeast 1-pyrroline-5-carboxylate (P5C) dehydrogenase encoded by the PUT2 gene.
INTRODUCTION Human aldehyde dehydrogenases (ALDH), catalyzing the oxidation of both aliphatic and aromatic aldehydes, have been classified into two broad groups, "low Kin" and "high Kin" enzymes (Pietruszko et al., 1987). The various A L D H enzymes differ in their electrophoretic properties, catalytic efficiences and subcellular localization (Lindahl and Hempel, 1991). While the "low Km" enzymes (the cytosolic ALDH1 and the mitochondrial ALDH2) have been well-characterized and their primary and genomic structural information is available (J6rnvall et al., 1991), the structural and functional properties of the "high Kin" enzymes are less well known. At least two "high Kin" aldehyde dehydrogenases (ALDH3 or E3, and A L D H 4 or E4) have been identified in the human stomach and liver, respectively (Pietruszko et al., 1987; Agarwal et al., 1989; Eckey et al., 1991). Whereas E1 and E2 enzymes are homotetramers, E3 and E4 are dimers (Agarwal et al., 1989). Human liver contains a high Km, less anodically migrating, A L D H enzyme hitherto designated as E4 or A L D H 4 (Harada et al., 1980). On the basis of substrate specificity, EA has been identified as glutamic ?-semialdehyde dehydrogenase (GSDH, E C 1.5.1.12) or 1-pyrroline-5-carboxylate (P5C) dehydrogenase (Forte-McRobbie and Pietruszko, 1986). GSDH catalyzes the reduction of glutamate to an intermediate, glutamic y-semialdehyde, which is spontaneously converted to the cyclic compound 1-pyrroline-5-carboxylate (P5C). The latter product is an important intermediate in the synthesis of proline and ornithine, and in the interconversion of glutamic acid and proline. Although this enzyme has been purified to homogeneity and characterized with respect to substrate specificity and other physicoehemical, as well as molecular properties, no primary structure data have been reported for the human liver enzyme. 791
In the present work, we have purified to homogeneity the GSDH from autopsy human liver, and compared the amino acid sequences of selected peptides with the known sequences of A L D H isoenzymes from humans and other species. The results indicate a structural relationship between the human liver glutamic 3'-semialdehyde dehydrogenase and 1-pyrroline-5-carboxylate dehydrogenase from the yeast Saccharomyces cerevisiae (Krzywicki and Brandriss, 1984). MATERIALS AND
METHODS
Human autopsy livers were obtained from the Department of Legal Medicine, University of Hamburg. The tissue samples were washed in water and kept frozen at -20°C until used. Extracts were prepared by homogenizing the samples in 30 mM sodium phosphate buffer, pH 6.0 (1 ml buffer/g tissue), containing 1 mM EDTA and i mM dithioerythritol. Enzyme purification Autopsy stomach tissue extract, prepared from about 300-400 g tissue as described above, was centrifuged for 15 min at 27,000 g. The supernatant was dialyzed overnight against a 20-fold volume of extraction buffer and subsequently centrifuged (20min at 27,000g) to remove any denatured proteins. The supernatant was applied to a 5 x 45 cm column of DEAE-Sephadex A50 equilibrated with the extraction buffer. GSDH appeared in the unbound fraction immediately after the void volume. The eluate containing GSDH activity (substrate: glutamic ~,-semi-aldehyde) was concentrated to 25 ml by ammonium sulfate precipitation (0-55% saturation) and subjected to gel filtration on a 5 x 70 cm column of Sephadex G100. Fractions showing GSDH activity were pooled and applied to a 2.5 x 15 era column of 5'AMP-Sepharose 4B equilibrated with the extraction buffer. The bound enzyme was eluted with a 100 mM sodium phosphate buffer, pH 8.0 containing 1 mM EDTA, 1 mM dithioerythritoland 0.5 mg NADH/ml. The enzyme was further purified by preparative isoelectric focusing on agarose gel in a pH-range of 3.5-9.5 as previously described (Eckey et al., 1988). Remaining impurities were removed by chromatography on a 1 x 50 cm column of Red Sepharose CL-6B equilibrated with the extraction
792
J. HEMPELet al. Fig. I. Comparison of peptide sequences of human liver glutamic ?-semialdehyde dehydrogenase (this work) and yeast 1-pyrroline-5-carboxylate dehydrogenase (Krzywicki and Brandriss, 1984). The upper sequence from each pair is the experimentally determined sequence of a tryptic peptide from the human enzyme, and is designated according to chromatographic isolation. The lower sequences are segments from the yeast enzyme, designated according to their position number in the complete structure. Identical residues are marked with an asterisk. Peptide
Sequence
% Identity
E-23 176 191
Y A V E L E G Q Q P I Y A S D L YAQQ P v
S v L X(Y) s RADG
F-19 204-209
F C Y A D K F V Y A V S
50
G-17 342-350
S A F E Y GGQK G T F E F QGQK
55
G-19 131-139
A A D M L SGPR A A D L I STKY
45
G-23 102-110
A I E A A LAAR V M N A V KAAK
33
G-26 521-537
S T G S I V G Q Q P r G G A(G) C T G A V V S Q Q PWGGA P
60
G-27 125-130
A Q I F L K S A I F L K
66
G-48 252-280
E A G L P P N I E A G L P KGV
J-32 357-368
L Y V P H S L W P Q I K L Y L P E S K S E E F L
I Q F V PADG I N F I LGD
buffer. GSDH was eluted with a linear salt gradient (0-1 M NaC1). Peptide analysis
The purified ALDH4 protein was S-(~4C) carboxymethylated and cleaved with trypsin as described for the human liver ALDHI (Hempel et al., 1984). The tryptic digest was prefractionated by ion exchange HPLC on a 4 x 250 mm poly (2-sulfoethyl aspartamide)-silica column (Nest Group, Southboro, MA) in 5mM potassium phosphate/25% CH3CN, pH 3.0 at 1 ml/min. A gradient of the same solvent containing 0.4 M KCI was applied over 1 hr (Alpert and Andrews, 1988). The eluate was pooled according to u.v. absorbing peaks and selected pools were separately applied to a C-18 reversed-phase HPLC column (Ultrapack, LKB, 4.6 x 250 mm) in 0.1% trifluoroacetic acid and eluted with a linear gradient of acetontrile, with u.v.-monitoring at 214 and 280 nm. Aliquots from u.v.-absorbing peaks were taken for hydrolysis (6N HC1, 0.5% phenol, 110°C in vacuo, 24 hr) and compositional analysis (Beckman 6300 amino acid analyser) to judge peptide purity. Material from selected peaks was submitted to automated Edman degradation in either a Beckman 890M or Porton 2090E sequenator with identification of the derivatives by HPLC.
RESULTS
The preparation was found to be homogeneous by SDS-electrophoresis, and by a lack of cross-reaction with antisera raised against other proteins. Material from 10 different reversed-phase H P L C peaks was submitted to Edman degradation. Two samples were impure, with two peptides each, although it was possible to unambiguously separate two sequences
44
P L FG EXV P VQV TDQV
39 33
from C-terminal part of one of the impure samples after re-digestion with endoprotease Glu-C and separation of the products by rechromatography on the C-18 column. These two sequences and the remaining nine peptides provided 12 sequences of six or more residues, unequivocally covering over 120 residues. O f these, nine sequences are placed relatively unambiguously versus the sequence of the yeast enzyme (Krzywicki and Brandriss, 1984), with % matching residues ranging from 33 to 66% (Fig. 1). The three other sequences (not shown) as yet have no discernable counterpart in the yeast enzyme.
DISCUSSION
A structure has been known for an enzyme from yeast (Krzywicki and Brandriss, 1984), which catalyzes the same reaction as the present G S D H or A L D H 4 from human liver. Thus it was of immediate interest to compare the sequences obtained to the yeast sequence. The ability to readily place several peptides vs the yeast P5C dehydrogenase deduced sequence suggests a c o m m o n ancestor for the enzymes from these two sources. The range in per cent matching residues, and the presence of three sequences not yet alignable with the yeast enzyme suggests that some regions are substantially more conserved than others. Some of these sequences are being used as the basis for the design of oligonucleotide probes for P C R amplification of the G S D H ( A L D H 4 ) gene (Cooper and Isola, 1990). When the entire sequences become known, it is anticipated that
Human A1DH4 peptides the three sequences for which no yeast enzyme counterpart was found will be incorporated into the entire sequence. REFERENCES
Agarwal D. P., Eckey R., Rudnay A.-C., Volkens T. and Goedde H. W. (1989) High Km aldehyde dehydrogenase isozymes in human tissues. In Enzymology and Molecular Biology of Carbonyl Metabolism 2 (Edited by Weiner H. and Flynn T. G.), pp. 119-131. Alan R. Liss, New York. Alpert A. I. and Andrews P. C, (1988) Cation exchange chromatography of peptides on poly(2-sulfoethyl aspartamide)-silica. J. Chromat. 443, 85-96. Cooper D. L. and Isola N. (1990) Full-length cDNA cloning utilizing the polymerase chain reaction. Biotech. 9, 60-65. Eckey R., Agarwal D. P., Volkens T. and Goedde H. W. (1988) In Alcohol Toxicity and Free Radicals Mechanisms (Edited by Nordmann R., Ribiere C. and Rouach H.) pp. 171-176. Pergamon Press, Oxford. Eckey R., Timmann R., Hempel J., Agarwal D. P. and Goedde H. W. (1991) Biochemical, immunological and molecular characterization of a "high Kin" aldehyde dehydrogenase. In Enzymology and Molecular Biology of Carbonyl Metabolism 3 (Edited by Weiner H., Wermuth B. and Crabb D. W.), pp. 43-52. Plenum Press, New York.
793
Forte-McRobbie C. M. and Pietruszko R. (1986) Characterization of human liver "high Kin" aldehyde dehydrogenase as glutamic v-semialdehyde dehydrogenase. J. biol. Chem. 261, 2154-2163. Harada S., Agarwal D. P. and Goedde H. W. (1980) Electrophoretic and biochemical studies of human aldehyde dehydrogenase. Life Sci. 26, 1771-1780. Hempel J., von Bahr-Lindstr6m H. and J6rnvall H. (1984) Aldehyde dehydrogenase from human liver: primary structure of the cytosolic enzyme. Eur. J. Biochem. 141, 21-35. J6rnvall H., Persson B., Krook M. and Hempel J. (1991) Alcohol and aldehyde dehydrogenases. In The Molecular Pathology of Alcoholism (Edited by Palmer N.), pp. 130-156. Oxford University Press, Oxford. Krzywicki K. A. and Brandriss M. C. (1984) Primary structures of the nuclear PUT2 gene involved in the mitochondrial pathway for proline utilization in S. cerevisiae. Mol. Cell. Biol. 4, 2837-2842. Lindahl R. and Hempel J. (1991) Aldehyde dehydrogenases: what can be learned from a baker's dozen sequences. In Enzymology and Molecular Biology or Carbonyl Metabolism 3 (Edited by Weiner H., Wermuth B. and Crabb D. W.), pp. 1-8. Plenum Press, New York. Pietruszko R., Ryzlak M. T. and Forte-McRobbie C. M. (1987) Multiplicity and identity of human aldehyde dehydrogenases. Alcohol Alcoholism. Suppl. 1, 175-179.
0305-0491/92 $5.00 + 0.00 © 1992Pergamon Press Ltd
HUMAN LIVER GLUTAMIC 3,-SEMIALDEHYDE DEHYDROGENASE: STRUCTURAL RELATIONSHIP TO THE YEAST ENZYME JOHN HEMPEL,* ROLF ECKEY,~" DIANE BERIE,* HANA ROMOVACEK,* DHARAM P. AGARWAL~"and H. WERNER GOEDDE~ *Department of Molecular Genetics and Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, PA 15261, U.S.A. Tel: (412) 648-9552; Fax: (412) 624-1401; and l'Institute of Human Genetics, University of Hamburg, Hamburg, Germany (Received 3 January 1992)
Abstract--The amino acid sequences of nine tryptic peptides (containing altogether 105 amino acids) from human liver glutamic ~,-semialdehyde dehydrogenase (hitherto designated as ALDH4) were found to correspond, at 33~6% identity, to segments from the yeast 1-pyrroline-5-carboxylate (P5C) dehydrogenase encoded by the PUT2 gene.
INTRODUCTION Human aldehyde dehydrogenases (ALDH), catalyzing the oxidation of both aliphatic and aromatic aldehydes, have been classified into two broad groups, "low Kin" and "high Kin" enzymes (Pietruszko et al., 1987). The various A L D H enzymes differ in their electrophoretic properties, catalytic efficiences and subcellular localization (Lindahl and Hempel, 1991). While the "low Km" enzymes (the cytosolic ALDH1 and the mitochondrial ALDH2) have been well-characterized and their primary and genomic structural information is available (J6rnvall et al., 1991), the structural and functional properties of the "high Kin" enzymes are less well known. At least two "high Kin" aldehyde dehydrogenases (ALDH3 or E3, and A L D H 4 or E4) have been identified in the human stomach and liver, respectively (Pietruszko et al., 1987; Agarwal et al., 1989; Eckey et al., 1991). Whereas E1 and E2 enzymes are homotetramers, E3 and E4 are dimers (Agarwal et al., 1989). Human liver contains a high Km, less anodically migrating, A L D H enzyme hitherto designated as E4 or A L D H 4 (Harada et al., 1980). On the basis of substrate specificity, EA has been identified as glutamic ?-semialdehyde dehydrogenase (GSDH, E C 1.5.1.12) or 1-pyrroline-5-carboxylate (P5C) dehydrogenase (Forte-McRobbie and Pietruszko, 1986). GSDH catalyzes the reduction of glutamate to an intermediate, glutamic y-semialdehyde, which is spontaneously converted to the cyclic compound 1-pyrroline-5-carboxylate (P5C). The latter product is an important intermediate in the synthesis of proline and ornithine, and in the interconversion of glutamic acid and proline. Although this enzyme has been purified to homogeneity and characterized with respect to substrate specificity and other physicoehemical, as well as molecular properties, no primary structure data have been reported for the human liver enzyme. 791
In the present work, we have purified to homogeneity the GSDH from autopsy human liver, and compared the amino acid sequences of selected peptides with the known sequences of A L D H isoenzymes from humans and other species. The results indicate a structural relationship between the human liver glutamic 3'-semialdehyde dehydrogenase and 1-pyrroline-5-carboxylate dehydrogenase from the yeast Saccharomyces cerevisiae (Krzywicki and Brandriss, 1984). MATERIALS AND
METHODS
Human autopsy livers were obtained from the Department of Legal Medicine, University of Hamburg. The tissue samples were washed in water and kept frozen at -20°C until used. Extracts were prepared by homogenizing the samples in 30 mM sodium phosphate buffer, pH 6.0 (1 ml buffer/g tissue), containing 1 mM EDTA and i mM dithioerythritol. Enzyme purification Autopsy stomach tissue extract, prepared from about 300-400 g tissue as described above, was centrifuged for 15 min at 27,000 g. The supernatant was dialyzed overnight against a 20-fold volume of extraction buffer and subsequently centrifuged (20min at 27,000g) to remove any denatured proteins. The supernatant was applied to a 5 x 45 cm column of DEAE-Sephadex A50 equilibrated with the extraction buffer. GSDH appeared in the unbound fraction immediately after the void volume. The eluate containing GSDH activity (substrate: glutamic ~,-semi-aldehyde) was concentrated to 25 ml by ammonium sulfate precipitation (0-55% saturation) and subjected to gel filtration on a 5 x 70 cm column of Sephadex G100. Fractions showing GSDH activity were pooled and applied to a 2.5 x 15 era column of 5'AMP-Sepharose 4B equilibrated with the extraction buffer. The bound enzyme was eluted with a 100 mM sodium phosphate buffer, pH 8.0 containing 1 mM EDTA, 1 mM dithioerythritoland 0.5 mg NADH/ml. The enzyme was further purified by preparative isoelectric focusing on agarose gel in a pH-range of 3.5-9.5 as previously described (Eckey et al., 1988). Remaining impurities were removed by chromatography on a 1 x 50 cm column of Red Sepharose CL-6B equilibrated with the extraction
792
J. HEMPELet al. Fig. I. Comparison of peptide sequences of human liver glutamic ?-semialdehyde dehydrogenase (this work) and yeast 1-pyrroline-5-carboxylate dehydrogenase (Krzywicki and Brandriss, 1984). The upper sequence from each pair is the experimentally determined sequence of a tryptic peptide from the human enzyme, and is designated according to chromatographic isolation. The lower sequences are segments from the yeast enzyme, designated according to their position number in the complete structure. Identical residues are marked with an asterisk. Peptide
Sequence
% Identity
E-23 176 191
Y A V E L E G Q Q P I Y A S D L YAQQ P v
S v L X(Y) s RADG
F-19 204-209
F C Y A D K F V Y A V S
50
G-17 342-350
S A F E Y GGQK G T F E F QGQK
55
G-19 131-139
A A D M L SGPR A A D L I STKY
45
G-23 102-110
A I E A A LAAR V M N A V KAAK
33
G-26 521-537
S T G S I V G Q Q P r G G A(G) C T G A V V S Q Q PWGGA P
60
G-27 125-130
A Q I F L K S A I F L K
66
G-48 252-280
E A G L P P N I E A G L P KGV
J-32 357-368
L Y V P H S L W P Q I K L Y L P E S K S E E F L
I Q F V PADG I N F I LGD
buffer. GSDH was eluted with a linear salt gradient (0-1 M NaC1). Peptide analysis
The purified ALDH4 protein was S-(~4C) carboxymethylated and cleaved with trypsin as described for the human liver ALDHI (Hempel et al., 1984). The tryptic digest was prefractionated by ion exchange HPLC on a 4 x 250 mm poly (2-sulfoethyl aspartamide)-silica column (Nest Group, Southboro, MA) in 5mM potassium phosphate/25% CH3CN, pH 3.0 at 1 ml/min. A gradient of the same solvent containing 0.4 M KCI was applied over 1 hr (Alpert and Andrews, 1988). The eluate was pooled according to u.v. absorbing peaks and selected pools were separately applied to a C-18 reversed-phase HPLC column (Ultrapack, LKB, 4.6 x 250 mm) in 0.1% trifluoroacetic acid and eluted with a linear gradient of acetontrile, with u.v.-monitoring at 214 and 280 nm. Aliquots from u.v.-absorbing peaks were taken for hydrolysis (6N HC1, 0.5% phenol, 110°C in vacuo, 24 hr) and compositional analysis (Beckman 6300 amino acid analyser) to judge peptide purity. Material from selected peaks was submitted to automated Edman degradation in either a Beckman 890M or Porton 2090E sequenator with identification of the derivatives by HPLC.
RESULTS
The preparation was found to be homogeneous by SDS-electrophoresis, and by a lack of cross-reaction with antisera raised against other proteins. Material from 10 different reversed-phase H P L C peaks was submitted to Edman degradation. Two samples were impure, with two peptides each, although it was possible to unambiguously separate two sequences
44
P L FG EXV P VQV TDQV
39 33
from C-terminal part of one of the impure samples after re-digestion with endoprotease Glu-C and separation of the products by rechromatography on the C-18 column. These two sequences and the remaining nine peptides provided 12 sequences of six or more residues, unequivocally covering over 120 residues. O f these, nine sequences are placed relatively unambiguously versus the sequence of the yeast enzyme (Krzywicki and Brandriss, 1984), with % matching residues ranging from 33 to 66% (Fig. 1). The three other sequences (not shown) as yet have no discernable counterpart in the yeast enzyme.
DISCUSSION
A structure has been known for an enzyme from yeast (Krzywicki and Brandriss, 1984), which catalyzes the same reaction as the present G S D H or A L D H 4 from human liver. Thus it was of immediate interest to compare the sequences obtained to the yeast sequence. The ability to readily place several peptides vs the yeast P5C dehydrogenase deduced sequence suggests a c o m m o n ancestor for the enzymes from these two sources. The range in per cent matching residues, and the presence of three sequences not yet alignable with the yeast enzyme suggests that some regions are substantially more conserved than others. Some of these sequences are being used as the basis for the design of oligonucleotide probes for P C R amplification of the G S D H ( A L D H 4 ) gene (Cooper and Isola, 1990). When the entire sequences become known, it is anticipated that
Human A1DH4 peptides the three sequences for which no yeast enzyme counterpart was found will be incorporated into the entire sequence. REFERENCES
Agarwal D. P., Eckey R., Rudnay A.-C., Volkens T. and Goedde H. W. (1989) High Km aldehyde dehydrogenase isozymes in human tissues. In Enzymology and Molecular Biology of Carbonyl Metabolism 2 (Edited by Weiner H. and Flynn T. G.), pp. 119-131. Alan R. Liss, New York. Alpert A. I. and Andrews P. C, (1988) Cation exchange chromatography of peptides on poly(2-sulfoethyl aspartamide)-silica. J. Chromat. 443, 85-96. Cooper D. L. and Isola N. (1990) Full-length cDNA cloning utilizing the polymerase chain reaction. Biotech. 9, 60-65. Eckey R., Agarwal D. P., Volkens T. and Goedde H. W. (1988) In Alcohol Toxicity and Free Radicals Mechanisms (Edited by Nordmann R., Ribiere C. and Rouach H.) pp. 171-176. Pergamon Press, Oxford. Eckey R., Timmann R., Hempel J., Agarwal D. P. and Goedde H. W. (1991) Biochemical, immunological and molecular characterization of a "high Kin" aldehyde dehydrogenase. In Enzymology and Molecular Biology of Carbonyl Metabolism 3 (Edited by Weiner H., Wermuth B. and Crabb D. W.), pp. 43-52. Plenum Press, New York.
793
Forte-McRobbie C. M. and Pietruszko R. (1986) Characterization of human liver "high Kin" aldehyde dehydrogenase as glutamic v-semialdehyde dehydrogenase. J. biol. Chem. 261, 2154-2163. Harada S., Agarwal D. P. and Goedde H. W. (1980) Electrophoretic and biochemical studies of human aldehyde dehydrogenase. Life Sci. 26, 1771-1780. Hempel J., von Bahr-Lindstr6m H. and J6rnvall H. (1984) Aldehyde dehydrogenase from human liver: primary structure of the cytosolic enzyme. Eur. J. Biochem. 141, 21-35. J6rnvall H., Persson B., Krook M. and Hempel J. (1991) Alcohol and aldehyde dehydrogenases. In The Molecular Pathology of Alcoholism (Edited by Palmer N.), pp. 130-156. Oxford University Press, Oxford. Krzywicki K. A. and Brandriss M. C. (1984) Primary structures of the nuclear PUT2 gene involved in the mitochondrial pathway for proline utilization in S. cerevisiae. Mol. Cell. Biol. 4, 2837-2842. Lindahl R. and Hempel J. (1991) Aldehyde dehydrogenases: what can be learned from a baker's dozen sequences. In Enzymology and Molecular Biology or Carbonyl Metabolism 3 (Edited by Weiner H., Wermuth B. and Crabb D. W.), pp. 1-8. Plenum Press, New York. Pietruszko R., Ryzlak M. T. and Forte-McRobbie C. M. (1987) Multiplicity and identity of human aldehyde dehydrogenases. Alcohol Alcoholism. Suppl. 1, 175-179.
