Characterization of the peptide-bound flavin of a bacterial sarcosine dehydrogenase.

As in the case of the succinate and sarcosine dehydrogenases of liver mitochondria, the flavin prosthetic group of the bacterial sarcosine dehydrogenase can be released from the enzyme by proteolytic digestion with trypsin and chymotrypsin. The flavin, isolated in the dinucleotide form and covalently bound to a peptide fragment, is converted to the mononucleotide and purified by sequential chromatography on Sephadex G-25, DEAE-Sephadex A-25, followed by preparative paper chromatography and high voltage electrophoresis.The absorption maxima of the purified flavin at pH 7 are found at 268, 350, and 447 nm, with 268:447 nm and 350:447 nm ratios of 3.08 and 0.79, respectively. The fluorescence excitation and emission maxima, 450 and 530 nm, respectively, are similar to those of flavin mononucleotide. The fluorescence of the flavin-peptide is maximal at pH 3.0–3.1.Amino acid analysis of the flavin-peptide (riboflavin form) gave the following molar ratios of amino acids to flavin: Lys(1), Asp(2), Thr(1), Ser(1), Glu(1), Gly(1), and Ala(1). Aspartic acid was the N-terminal amino acid. Upon more extensive hydrolysis, histidine was obtained in 71–84% yields. Employing aminopeptidase M, the partial sequence of amino acids in the flavin-peptide was determined to be as follows: Flavin

-Asp-Lys-Ser-Glu-Gly-His-(Asp,Ala,Thr)-Evidence is presented that the isoalloxazine ring is linked covalently via its 8 α-methyl group to N-3 of histidine.     

Enzymatic properties of dimethylglycine dehydrogenase and sarcosine dehydrogenase from rat liver

Dimethylglycine dehydrogenase (EC 1.5.99.2) and sarcosine dehydrogenase (EC 1.5.99.1) are flavoproteins which catalyze the oxidative demethylation of dimethylglycine to sarcosine and sarcosine to glycine, respectively. During these reactions tightly bound tetrahydropteroylpentaglutamate (H4PteGlu5) is converted to 5,10-methylene tetrahydropteroylpentaglutamate (5,10-CH2-H4PteGlu5), although in the absence of H4PteGlu5, formaldehyde is produced. Single turnover studies using substrate levels of the enzyme (2.3 microM) showed pseudo-first-order kinetics, with apparent first-order rate constants of 0.084 and 0.14 s-1 at 23 and 48.3 microM dimethylglycine, respectively, for dimethylglycine dehydrogenase and 0.065 s-1 at 47.3 microM sarcosine for sarcosine dehydrogenase. The rates were identical in the absence or presence of bound tetrahydropteroylglutamate (H4PteGlu). Titration of the enzymes with substrate under anaerobic conditions did not disclose the presence of an intermediate semiquinone. The effect of dimethylglycine concentration upon the rate of the dimethylglycine dehydrogenase reaction under aerobic conditions showed nonsaturable kinetics suggesting a second low-affinity site for the substrate which increases the enzymatic rate. The Km for the high-affinity active site was 0.05 mM while direct binding for the low-affinity site could not be measured. Sarcosine and dimethylthetin are poor substrates for dimethylglycine dehydrogenase and methoxyacetic acid is a competitive inhibitor at low substrate concentrations. At high dimethylglycine concentrations, increasing the concentration of methoxyacetic acid produces an initial activation and then inhibition of dimethylglycine dehydrogenase activity. When these compounds were added in varying concentrations to the enzyme in the presence of dimethylglycine, their effects upon the rate of the reaction were consistent with the presence of a second low-affinity binding site on the enzyme which enhances the reaction rate. When sarcosine is used as the substrate for sarcosine dehydrogenase the kinetics are Michaelis-Menten with a Km of 0.5 mM for sarcosine. Also, methoxyacetic acid is a competitive inhibitor of sarcosine dehydrogenase with a Ki of 0.26 mM. In the absence of folate, substrate and product determinations indicated that 1 mol of formaldehyde and of sarcosine or glycine were produced for each mole of dimethylglycine or sarcosine consumed with the concomitant reduction of 1 mol of bound FAD.     

Covalent binding of folic acid to dimethylglycine dehydrogenase

Dimethylglycine dehydrogenase (EC 1.5.99.2) carries out the oxidative demethylation of dimethylglycine to sarcosine in liver mitochondria. In vivo, the enzyme uses tightly bound tetrahydropteroyl pentaglutamate (H₄PteGlu₅) as an acceptor of the one-carbon group generated during the reaction. The purified enzyme can use, but does not require, H₄PteGluB and under these conditions formaldehyde is the one-carbon unit produced. It is reported that folic acid may be covalently linked to dimethylglycine dehydrogenase in a specific and saturable manner so that only 1 mole of folic acid is bound per mole of enzyme. Covalently bound folic acid blocks the subsequent binding of H₄PteGlu, and does not inhibit the rate of dimethylglycine dehydrogenase activity in vitro.     

Cloning and Mapping of the cDNA for Human Sarcosine Dehydrogenase, a Flavoenzyme Defective in Patients with Sarcosinemia

Sarcosine dehydrogenase is a liver mitochondrial matrix flavoenzyme that is defective in patients with sarcosinemia, a rare autosomal metabolic defect characterized by elevated levels of sarcosine in blood and urine. Some patients also exhibit mental retardation and growth failure. A full-length cDNA for human sarcosine dehydrogenase was isolated from an adult liver cDNA library. The first 22 residues in the deduced amino acid sequence exhibit features expected for a mitochondrial targeting sequence. The predicted mass of the mature human liver sarcosine dehydrogenase (99,505 Da) is in good agreement with that observed for rat liver sarcosine dehydrogenase ( approximately 100,000 Da). Human sarcosine dehydrogenase exhibits 89% identity with rat liver sarcosine dehydrogenase and strong homology ( approximately 35% identity) with rat liver dimethylglycine dehydrogenase, a sarcosine dehydrogenase-related protein from Rhodobacter capsulatus, and the regulatory subunit from bovine pyruvate dehydrogenase phosphatase. The human sarcosine dehydrogenase gene is at least 75.3 kb long and located on chromosome 9q34. The adult human liver clone is assembled from 21 exons (1-6, 7a, 8a, 9-21). Two smaller cDNA clones, isolated from adult liver and infant brain libraries, were assembled from the same sarcosine dehydrogenase gene by the use of alternate polyadenylation and splice sites. This is the first report of the genomic structure of the sarcosine dehydrogenase gene in any species. The observed chromosomal location is consistent with genetic studies with a mouse model for sarcosinemia that map the mouse gene to a region of mouse chromosome 2 syntenic with human 9q33-q34. The availability of the SDH gene sequence will enable characterization of the genotypes of sarcosinemia patients with different phenotypes.     

Monomeric sarcosine oxidase: structure of a covalently flavinylated amine oxidizing enzyme.

BACKGROUND: Monomeric sarcosine oxidases (MSOXs) are among the simplest members of a recently recognized family of eukaryotic and prokaryotic enzymes that catalyze similar oxidative reactions with various secondary or tertiary amino acids and contain covalently bound flavins. Other members of this family include heterotetrameric sarcosine oxidase, N-methyltryptophan oxidase and pipecolate oxidase. Mammalian sarcosine dehydrogenase and dimethylglycine dehydrogenase may be more distantly related family members. RESULTS: The X-ray crystal structure of MSOX from Bacillus sp. B-0618, expressed in Escherichia coli, has been solved at 2.0 A resolution by multiwavelength anomalous dispersion (MAD) from crystals of the selenomethionine-substituted enzyme. Fourteen selenium sites, belonging to two MSOX molecules in the asymmetric unit, were used for MAD phasing and to define the local twofold symmetry axis for electron-density averaging. The structures of the native enzyme and of two enzyme-inhibitor complexes were also determined. CONCLUSIONS: MSOX is a two-domain protein with an overall topology most similar to that of D-amino acid oxidase, with which it shares 14% sequence identity. The flavin ring is located in a very basic environment, making contact with sidechains of arginine, lysine, histidine and the N-terminal end of a helix dipole. The flavin is covalently attached through an 8alpha-S-cysteinyl linkage to Cys315 of the catalytic domain. Covalent attachment is probably self-catalyzed through interactions with the positive sidechains and the helix dipole. Substrate binding is probably stabilized by hydrogen bonds between the substrate carboxylate and two basic sidechains, Arg52 and Lys348, located above the re face of the flavin ring.     

Cloning of dimethylglycine dehydrogenase and a new human inborn error of metabolism, dimethylglycine dehydrogenase deficiency

Dimethylglycine dehydrogenase (DMGDH) (E.C. number 1.5.99.2) is a mitochondrial matrix enzyme involved in the metabolism of choline, converting dimethylglycine to sarcosine. Sarcosine is then transformed to glycine by sarcosine dehydrogenase (E.C. number 1.5.99.1). Both enzymes use flavin adenine dinucleotide and folate in their reaction mechanisms. We have identified a 38-year-old man who has a lifelong condition of fishlike body odor and chronic muscle fatigue, accompanied by elevated levels of the muscle form of creatine kinase in serum. Biochemical analysis of the patient's serum and urine, using (1)H-nuclear magnetic resonance NMR spectroscopy, revealed that his levels of dimethylglycine were much higher than control values. The cDNA and the genomic DNA for human DMGDH (hDMGDH) were then cloned, and a homozygous A-->G substitution (326 A-->G) was identified in both the cDNA and genomic DNA of the patient. This mutation changes a His to an Arg (H109R). Expression analysis of the mutant cDNA indicates that this mutation inactivates the enzyme. We therefore confirm that the patient described here represents the first reported case of a new inborn error of metabolism, DMGDH deficiency.     

Cloning and overexpression of Lactobacillus helveticus D-lactate dehydrogenase gene in Escherichia coli.

NAD(+)-dependent D-lactate dehydrogenase from Lactobacillus helveticus was purified to apparent homogeneity, and the sequence of the first 36 amino acid residues determined. Using forward and reverse oligonucleotide primers, based on the N-terminal sequence and amino acid residues 220-215 of the Lactobacillus bulgaricus enzyme [Kochhar, S., Hunziker, P. E., Leong-Morgenthaler, P. & Hottinger, H. (1992) J. Biol. Chem. 267, 8499-8513], a 0.6-kbp DNA fragment was amplified from L. helveticus genomic DNA by the polymerase chain reaction. This amplified DNA fragment was used as a probe to identify two recombinant clones containing the D-lactate dehydrogenase gene. Both plasmids overexpressed D-lactate dehydrogenase (greater than 60% total soluble cell protein) and were stable in Escherichia coli, compared to plasmids carrying the L. bulgaricus and Lactobacillus plantarum genes. The entire nucleotide sequence of the L. helveticus D-lactate dehydrogenase gene was determined. The deduced amino acid sequence indicated a polypeptide consisting of 336 amino acid residues, which showed significant amino acid sequence similarity to the recently identified family of D-2-hydroxy-acid dehydrogenases [Kochhar, S., Hunziker, P. E., Leong-Morgenthaler, P. & Hottinger, H. (1992) Biochem. Biophys. Res. Commun. 184, 60-66]. The physicochemical and catalytic properties of recombinant D-lactate dehydrogenase were identical to those of the wild-type enzyme, e.g. alpha 2 dimeric subunit structure, isoelectric pH, Km and Kcat for pyruvate and other 2-oxo-acid substrates. The kinetic profiles of 2-oxo-acid substrates showed some marked differences from that of L-lactate dehydrogenase, suggesting different mechanisms for substrate binding and specificity.     

Isolation and characterization of cDNA clones for rat liver 10-formyltetrahydrofolate dehydrogenase.

We have isolated and characterized cDNA clones encoding rat liver cytosol 10-formyltetrahydrofolate dehydrogenase (EC 1.5.1.6). An open reading frame of 2706 base pairs encodes for 902 amino acids of Mr 99,015. The deduced amino acid sequence contains exact matches to the NH2-terminal sequence (28 residues) and the sequences of five peptides derived from cyanogen bromide cleavage of the purified protein. The amino acid sequence of 10-formyltetrahydrofolate dehydrogenase has three putative domains. The NH2-terminal sequence (residues 1-203) is 24-30% identical to phosphoribosylglycinamide formyltransferase (EC 2.1.2.2) from Bacillus subtilis (30%), Escherichia coli (24%), Drosophila melanogaster (24%), and human hepatoma HepG2 (27%). Residues 204-416 show no extensive homology to any known protein sequence. Sequence 417-900 is 46% (mean) identical to the sequences of a series of aldehyde dehydrogenase (NADP+) (EC 1.2.1.3). Intact 10-formyltetrahydrofolate dehydrogenase exhibits NADP-dependent aldehyde dehydrogenase activity. The sequence identity to phosphoribosylglycinamide formyltransferase is discussed, and a binding region for 10-formyltetrahydrofolate is proposed.     

Molecular cloning and characterization of (R)-3-hydroxybutyrate dehydrogenase from human heart.

The complete amino acid sequence of human heart (R)-3-hydroxybutyrate dehydrogenase (EC 1.1.1.30) has been deduced from the nucleotide sequence of cDNA clones. This mitochondrial enzyme has an absolute and specific requirement of phosphatidylcholine for enzymic activity (allosteric activator) and is an important prototype of lipid-requiring enzymes. Despite extensive studies, the primary sequence has not been available and is now reported. The mature form of the enzyme consists of 297 amino acids (predicted M(r) of 33,117), does not appear to contain any transmembrane helices, and is homologous with the family of short-chain alcohol dehydrogenases (SC-ADH) (Persson, B., Krook, M., and J?rnvall, H. (1991) Eur. J. Biochem. 200, 537-543) (30% residue identity with human 17 beta-hydroxysteroid dehydrogenase). The first two-thirds of the enzyme includes both putative coenzyme binding and active site conserved residues and exhibits a predicted secondary structure motif (alternating alpha-helices and beta-sheet) characteristic of SC-ADH. Bovine heart peptide sequences (174 residues in nine sequences determined by microsequencing) have extensive homology (89% identical residues) with the deduced human heart sequence. The C-terminal third (Asn-194 to Arg-297) shows little sequence homology with the SC-ADH and likely contains elements that determine the substrate specificity for the enzyme including the phospholipid (phosphatidylcholine) binding site(s). Northern blot analysis identifies a 1.3-kilobase mRNA encoding the enzyme in heart tissue.     

Mammalian electron transferring flavoprotein.flavoprotein dehydrogenase complexes observed by microelectrospray ionization-mass spectrometry and surface plasmon resonance

Microelectrospray ionization-mass spectrometry was used to directly observe electron transferring flavoprotein.flavoprotein dehydrogenase interactions. When electron transferring flavoprotein and porcine dimethylglycine dehydrogenase or sarcosine dehydrogenase were incubated together in the absence of substrate, a relative molecular mass corresponding to the flavoprotein.electron transferring flavoprotein complex was observed, providing the first direct observation of these mammalian complexes. When an acyl-CoA dehydrogenase family member, human short chain acyl-CoA dehydrogenase, was incubated with dimethylglycine dehydrogenase and electron transferring flavoprotein, the microelectrospray ionization-mass spectrometry signal for the dimethylglycine dehydrogenase.electron transferring flavoprotein complex decreased, indicating that the acyl-CoA dehydrogenases have the ability to compete with the dimethylglycine dehydrogenase/sarcosine dehydrogenase family for access to electron transferring flavoprotein. Surface plasmon resonance solution competition experiments revealed affinity constants of 2.0 and 5.0 microm for the dimethylglycine dehydrogenase-electron transferring flavoprotein and short chain acyl-CoA dehydrogenase-electron transferring flavoprotein interactions, respectively, suggesting the same or closely overlapping binding motif(s) on electron transferring flavoprotein for dehydrogenase interaction.     

Purification and molecular cloning of succinyltransferase of the rat alpha-ketoglutarate dehydrogenase complex. Absence of a sequence motif of the putative E3 and/or E1 binding site

Full-length cDNA clones for succinyltransferase of the rat alpha-ketoglutarate dehydrogenase complex were isolated from rat heart cDNA libraries in lambda gt11. The cDNA clones were identified as those for rat succinyltransferase by the identity of their predicted amino acid sequence with the NH2-terminal amino acid sequence of rat succinyltransferase determined by protein chemical analysis and the known amino acid sequence of bovine succinyltransferase. The clone with the longest cDNA consisted of 2747 base pairs and coded for a leader peptide of 56 amino acid residues and a mature protein of 386 amino acid residues. The primary structure of rat succinyltransferase showed close similarity to Escherichia coli and Azotobacter vinelandii succinyltransferases, in the COOH-terminal part forming the lipoyl-binding domain and the NH2-terminal part forming the inner core-catalytic domain. However, the rat succinyltransferase did not contain a sequence motif that has been found as an E3- and/or E1-binding site in the dihydrolipoamide acyltransferases of three alpha-ketoacid dehydrogenase complexes (Hummel, K. B., Litwer, S., Bradford, A. P., Aitken, A., Danner, D. J., and Yeaman, S. J. (1988) J. Biol. Chem. 263, 6165-6168, Reed, L. J., and Hackert, M. L. (1990) J. Biol. Chem. 265, 8971-8974). The absence of this sequence was confirmed by direct sequencing of the polymerase chain reaction product of rat heart mRNA and by computer analysis. These results show that the rat succinyltransferase does not have the sequence motif of the putative E3- and/or E1-binding site.     

Organization of the genes involved in dimethylglycine and sarcosine degradation in Arthrobacter spp.: implications for glycine betaine catabolism.

The nucleotide sequences of two cloned DNA fragments containing the structural genes of heterotetrameric sarcosine oxidase (soxBDAG) and dimethylglycine dehydrogenase (dmg) from Arthrobater spp. 1-IN and Arthrobacter globiformis, respectively, have been determined. Open reading frames were identified in the soxBDAG operon corresponding to the four subunits of heterotetrameric sarcosine oxidase by comparison with the N-terminal amino-acid sequences and the subunit relative molecular masses of the purified enzyme. Alignment of the deduced sarcosine oxidase amino-acid sequence with amino-acid sequences of functionally related proteins indicated that the arthrobacterial enzyme is highly homologous to sarcosine oxidase from Corynebacterium P-1. Deletion and expression analysis, and alignment of the deduced amino-acid sequence of the dmg gene, showed that dmg encodes a novel dimethylglycine oxidase, which is related to eukaryotic dimethylglycine dehydrogenase, and contains nucleotide-binding, flavinylation and folate-binding motifs. The recombinant dimethylglycine oxidase was purified to homogeneity and characterized. The DNA located upstream and downstream of both the soxBDAG and dmg genes is predicted to encode enzymes involved in the tetrahydrofolate-dependent assimilation of methyl groups. Based on the sequence analysis reported herein, pathways are proposed for glycine betaine catabolism in Arthrobacter species, which involve the identified folate-dependent enzymes.     

Nicotinamide adenine dinucleotide-specific glutamate dehydrogenase of Neurospora crassa. Isolation and sequences of several cyanogen bromide peptides from the NH2-terminal portion of the peptide chain

Previous studies of the amino acid sequence of the NAD-specific glutamate dehydrogenase of Neurospora crassa (EC 1.4.1.2) resulted in the assignments of peptides to four fragments, the longest being the COOH-terminal 669 residues of the protein. A further study of peptides derived by cyanogen bromide cleavage by different separation methods has yielded additional peptides that have provided new information concerning the sequence and has given overlaps of previously known sequences. This has permitted establishment of 313 residues in one sequence (fragment II). This is in addition to a sequence of 43 residues (fragment I) at the NH2-terminal end and a sequence of 669 residues (fragment III) previously established at the COOH-terminal end of the molecule. The present status of our knowledge of the overall sequence is given in the accompanying papers, together with some views regarding the conformation of the protein (Haberland, M.E., Chen, C.-W., and Smith, E.L. (1980) J. Biol. Chem. 255, 7993-8000, and Austen, B.M., Haberland, M.E., and Smith, E.L. (1980) J. Biol. Chem. 255, 8001-8004).     

The cDNA and protein sequences of human lactate dehydrogenase B.

Human lactate dehydrogenase B (LDH-B) cDNA was isolated and sequenced. The LDH-B cDNA insert consists of the protein-coding sequence (999 bp), the 5' (54 bp) and 3' (203 bp) non-coding regions, and the poly(A) tail (50 bp). The predicted sequence of 333 amino acid residues was confirmed by amino acid composition and/or sequence analyses of a total of 185 (56%) residues from tryptic peptides of human LDH-B protein. The nucleotide and amino acid sequences of the human LDH-B coding region show 68% and 75% homologies respectively with those of the human LDH-A. The peptide map and amino acid composition data have been deposited as Supplementary Publication SUP 50139 (7 pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K., from whom copies are available on prepayment [see Biochem. J. (1987) 241, 5].     

The amino acid sequence of tauropine dehydrogenase from the polychaete Arabella iricolor

The amino acid sequence of tauropine dehydrogenase (EC 1.5.1.23) from the polychaete Arabella iricolor was determined by automated sequencing of fragments obtained by cleavage with lysyl endopeptidase, endoproteinase Glu-C, and cyanogen bromide. The purified enzyme contained two isoforms that differ only in the 41st amino acid residue (Thr or Ile). Although the sequence contained eight Cys residues, intrachain disulfide bonds were not found. Two possible N-linked glycosylation sites occur in the sequences, but the enzyme does not appear to contain bound carbohydrates. Based on these data, the two isoforms of Arabella tauropine dehydrogenase are simple proteins consisted of 396 amino acid residues with calculated molecular masses of 43,085.7 Da (Thr41 isoform) and 43,097.8 Da (Ile41 isoform).     

L-galactono-gamma-lactone dehydrogenase from sweet potato: purification and cDNA sequence analysis

L-Galactono-gamma-lactone dehydrogenase (EC 1.3.2.3, GLDHase) was partially purified from mitochondria of sweet potato tuberous roots over 600-fold on a specific activity basis, followed by purification of the enzyme protein of 56 kDa by a preparative SDS-PAGE. The absorption spectrum of the hydroxylapatite column-purified GLDH-ase showed peaks at 448 and 373 nm, suggesting the presence of flavin as a prosthetic group. The activity of GLDH-ase was inhibited by lycorine, an alkaloid which inhibits ascorbic acid biosynthesis in vivo. N-terminal partial sequences of four internal polypeptides generated by partial digestion of GLDHase with V8 protease were determined. The deduced nucleotide sequences were used to amplify a cDNA fragment of the GLDHase gene. The clone encoded a polypeptide of 581 amino acid residues with a molecular mass of 66 kDa. The deduced amino acid sequence showed 77% identity with that of cauliflower GLDHase, and significant homology to those of L-gulono-gamma-lactone oxidase (22% identity) from rat and L-galactono-gamma-lactone oxidase from yeast (17% identity), which are enzymes involved in L-ascorbic acid biosynthesis in these organisms. The absorption spectrum and cDNA sequence suggested that the flavin group bound noncovalently. We conclude that GLDHase, L-gulono-gamma-lactone oxidase and L-galactono-gamma-lactone oxidase are homologous in spite of the difference in substrates and electron acceptors. Genomic Southern analysis suggested that GLDHase gene exists as a single copy in the genome of sweet potato.     

The complete primary structure of the allosteric L-lactate dehydrogenase from Lactobacillus casei

The polypeptide chain of the allosteric L-lactate dehydrogenase (EC 1.1.1.27) of Lactobacillus casei consists of 325 amino acid residues. Despite the strikingly different enzymatic characteristics of the allosteric L-lactate dehydrogenase of L. casei and of the non-allosteric vertebrate enzymes, the sequence of the allosteric enzyme shows a distinct homology with that of the non-allosteric vertebrate enzymes (average identity: 37%). An especially high sequence homology can be identified within the active center (average identity: 70%). A clear deviation of the L. casei enzyme from the vertebrate enzyme is the lack of the first 12 amino acid residues at the N terminus and an additional 7 amino acid residues at the C terminus. The localization of the binding site of the allosteric effector D-fructose 1,6-bisphosphate and pH and effector-induced changes of the spectroscopic properties are discussed on the basis of the primary structure.     

Chloroplast glyceraldehyde-3-phosphate dehydrogenase (NADP): amino acid sequence of the subunits from isoenzyme I and structural relationship with isoenzyme II

The structural relationship between isoenzymes I and II of chloroplast glyceraldehyde-3-phosphate dehydrogenase (D-glyceraldehyde-3-phosphate: NADP+ oxidoreductase (phosphorylating) EC 1.2.1.13) has been established at the protein level. The complete primary structure of subunits A and B of glyceraldehyde-3-phosphate dehydrogenase I from Spinacia oleracea has been determined by sequence analysis of the corresponding tryptic peptides, aligned by fragments derived from cyanogen bromide and Staphylococcus proteinase V8 digestions and by partially sequencing each intact subunit. Subunit A has an Mr of 36,225 and consists of 337 amino acid residues, whilst subunit B (Mr 39,355) consists of 368 residues. The amino acid sequence of subunit B, as determined through direct analysis of the protein, is identical to that recently deduced at cDNA level (Brinkmann et al. (1989) Plant Mol. Biol. 13, 81-94). The two subunits share a common portion of amino acid sequence which differs by 66 amino acid residues. Subunit B has an extra C-terminal sequence of 31 amino acid residues. Chloroplast glyceraldehyde-3-phosphate dehydrogenase II was partially characterized by sequencing the N-terminal portion of the intact protein and some of its tryptic peptides. The sequences of all the examined fragments fit precisely that of the corresponding regions of subunit A from glyceraldehyde-3-phosphate dehydrogenase I.     

Structural studies on glyceraldehyde-phosphate dehydrogenase from rat skeletal muscle

The NH2-terminal amino acid sequence of rat skeletal muscle glyceraldehydephosphate dehydrogenase (D-glyceraldehyde-3-phosphate : NAD+ oxidoreductase(physphorylating), EC 1.2.1.12) was determined to be Val-Lys-Val-Gly-Val-Asn-Gly-Phe-Gly-Arg-Ile-Gly-Arg-Leu-Val-Thr-Arg-Ala-Ala-Phe-Ser-Ser-(-)-(-)--Val-Asx-Ile-Val-Ala-Ile. The presence of Asn instead of Asp in position 6 differentiates this enzyme from other glyceraldehyde-3-phosphate dehydrogenases so far sequenced with the exception of the enzymes isolated from liver. The location of Asn in position 6 has been considered as a specific property of liver glyceraldehyde-3-phosphate dehydrogenase (Kulbe, K.D., Jackson, K.W. and Tang, J. (1975) Biochem. Biophys. Res. Commun. 67, 35--42); this suggestion is not sustained by the results of the present investigation. The amino acid composition of the rat skeletal muscle dehydrogenase demonstrates the unusually low histidine content of this enzyme as compared to other mammalian muscle glyceraldehyde-phosphate dehydrogenases.     

Dihydrolipoamide dehydrogenase from the halophilic archaebacterium Haloferax volcanii: characterization and N-terminal sequence.

Dihydrolipoamide dehydrogenase, a flavin disulfide reductase, has been purified and characterized from Haloferax volcanii. The enzyme is a dimer of relative mass 128,000, with an optimal activity at pH 9.0 in 1 M NaCl. Following reduction with its substrate, dihydrolipoamide, the enzyme is inactivated through covalent bond formation with the trivalent arsenical p-aminophenyl arsenoxide. The amino acid composition and the amino acid sequence of the first 49 residues of the N-terminus have been determined.