Significance of C-terminal sequence elements for Petunia flavanone 3beta-hydroxylase activity

The time-specific requirement of Rho proteins for the S phase progression of vascular endothelial cells was determined by reversibly introducing inhibitor proteins with a cell-penetrating peptide. We found evidence of the reversibility of protein transduction. The removal of extracellular protein caused the transduced protein to decay in a manner sensitive to low temperatures. The time required for a 50% decay correlated with the protein size. The time-specific transduction of the inhibitor proteins uncovered a critical period requiring Rho proteins in the G₁–S transition phase. Reversible protein transduction may thus be a powerful tool to investigate the time-specific role of signaling proteins.     

Purification of flavanone 3 beta-hydroxylase from Petunia hybrida: antibody preparation and characterization of a chemogenetically defined mutant

Flavanone 3 beta-hydroxylase from Petunia hybrida has been purified to apparent homogeneity utilizing an improved purification protocol including chromatography of the partially purified enzyme on hydroxyapatite, chromatofocusing, and hydrophobic interaction chromatography on phenyl-Superose. The specificity of mouse and rabbit polyclonal antisera directed to flavanone 3 beta-hydroxylase was demonstrated by Western blotting and immunotitration with crude extracts from wild-type flowers of P. hybrida and with the purified enzyme. Cross-reactivity was observed with flavanone 3 beta-hydroxylase from extracts of illuminated parsley cells. A Petunia mutant with white flowers, previously shown to lack 3 beta-hydroxylase activity and to accumulate flavanone glycosides, showed complete absence of the enzyme protein.     

The monomeric polypeptide comprises the functional flavanone 3beta-hydroxylase from Petunia hybrida

Flavanone 3beta-hydroxylase catalyzes the Fe(II)/oxoglutarate-dependent hydroxylation of (2S)-flavanones to (2R,3R)-dihydroflavonols in the biosynthesis of flavonoids, catechins and anthocyanidins. The enzyme had been partially purified from Petunia hybrida and proposed to be active as a dimer of roughly 75 kDa in size. More recently, the Petunia 3beta-hydroxylase was cloned and shown to be encoded in a 41655 Da polypeptide. In order to characterize the molecular composition, the enzyme was expressed in a highly active state in Escherichia coli and purified to apparent homogeneity. Size exclusion chromatographies of the pure, recombinant enzyme revealed that this flavanone 3beta-hydroxylase exists in functional monomeric and oligomeric forms. Protein cross-linking experiments employing a specific homobifunctional sulfhydryl group reagent or the photochemical activation of tryptophan residues confirmed the tendency of the enzyme to aggregate to oligomeric complexes in solution. Thorough equilibrium sedimentation analyses, however, revealed a molecular mass of 39. 2+/-12 kDa for the recombinant flavanone 3beta-hydroxylase. The result implies that the monomeric polypeptide comprises the catalytically active flavanone 3beta-hydroxylase of P. hybrida, which may readily associate in vivo with other proteins.     

Evolution of flavone synthase I from parsley flavanone 3beta-hydroxylase by site-directed mutagenesis

Flavanone 3beta-hydroxylase (FHT) and flavone synthase I (FNS I) are 2-oxoglutarate-dependent dioxygenases with 80% sequence identity, which catalyze distinct reactions in flavonoid biosynthesis. However, FNS I has been reported exclusively from a few Apiaceae species, whereas FHTs are more abundant. Domain-swapping experiments joining the N terminus of parsley (Petroselinum crispum) FHT with the C terminus of parsley FNS I and vice versa revealed that the C-terminal portion is not essential for FNS I activity. Sequence alignments identified 26 amino acid substitutions conserved in FHT versus FNS I genes. Homology modeling, based on the related anthocyanidin synthase structure, assigned seven of these amino acids (FHT/FNS I, M106T, I115T, V116I, I131F, D195E, V200I, L215V, and K216R) to the active site. Accordingly, FHT was modified by site-directed mutagenesis, creating mutants encoding from one to seven substitutions, which were expressed in yeast (Saccharomyces cerevisiae) for FNS I and FHT assays. The exchange I131F in combination with either M106T and D195E or L215V and K216R replacements was sufficient to confer some FNS I side activity. Introduction of all seven FNS I substitutions into the FHT sequence, however, caused a nearly complete change in enzyme activity from FHT to FNS I. Both FHT and FNS I were proposed to initially withdraw the beta-face-configured hydrogen from carbon-3 of the naringenin substrate. Our results suggest that the 7-fold substitution affects the orientation of the substrate in the active-site pocket such that this is followed by syn-elimination of hydrogen from carbon-2 (FNS I reaction) rather than the rebound hydroxylation of carbon-3 (FHT reaction).     

Purification of recombinant flavanone 3beta-hydroxylase from petunia hybrida and assignment of the primary site of proteolytic degradation

Flavanone 3beta-hydroxylase catalyzes the Fe(II)/oxoglutarate-dependent hydroxylation of (2S)-flavanones to (2R,3R)-dihydroflavonols in the course of flavonol/anthocyanin or catechin biosynthesis. The enzyme from Petunia hybrida consists of a 41,655-Da polypeptide that is prone to rapid proteolysis in crude plant extracts as well as on expression in Escherichia coli, and commercial protease inhibitors were inefficient in stopping the degradation. To pinpoint the primary site of proteolysis and to improve the activity yields, two revised schemes of purification were developed for the recombinant polypeptides. Applying a four-step protocol based on extraction and ion-exchange chromatography at pH 7.5, the primary, catalytically inactive proteolytic enzyme fragment (1.1 mg) was isolated and shown to cross-react on Western blotting as one homogeneous band of about 38 kDa. Mass spectrometric analysis assigned a mass of 37,820 +/- 100 Da to this fragment, and partial sequencing revealed an unblocked amino terminus identical to that of the native 3beta-hydroxylase. Thus, the native enzyme had been degraded by proteolysis of a small carboxy-terminal portion, and the primary site of cleavage must be assigned most likely to the Glu 337-Leu 338 bond, accounting for a loss of about 3800 Da. Alternatively, the enzyme degradation was greatly reduced when the extraction of recombinant bacteria was carried out with phosphate buffer at pH 5.5 followed by size exlusion and anion-exchange chromatography. This rapid, two-step purification resulted in a homogeneous 3beta-hydroxylase of high specific acitivity (about 32 mkat/kg) at roughly 5% yield, and the procedure is a major breakthrough in mechanistic investigations of this class of labile dioxygenases.     

Biochemical characterization of ClpB3, a chloroplastic disaggregase from Arabidopsis thaliana

Plant Molecular Biology - The first biochemical characterization of a chloroplastic disaggregase is reported (Arabidopsis thaliana ClpB3). ClpB3 oligomerizes into active hexamers that resolubilize...     

Site-directed mutagenesis of the active site serine290 in flavanone 3beta-hydroxylase from Petunia hybrida.

Flavanone 3beta-hydroxylase (FHT) catalyzes a pivotal reaction in the formation of flavonoids, catechins, proanthocyanidins and anthocyanidins. In the presence of oxygen and ferrous ions the enzyme couples the oxidative decarboxylation of 2-oxoglutarate, releasing carbon dioxide and succinate, with the oxidation of flavanones to produce dihydroflavonols. The hydroxylase had been cloned from Petunia hybrida and expressed in Escherichia coli, and a rapid isolation method for the highly active, recombinant enzyme had been developed. Sequence alignments of the Petunia hydroxylase with various hydroxylating 2-oxoglutarate-dependent dioxygenases revealed few conserved amino acids, including a strictly conserved serine residue (Ser290). This serine was mutated to threonine, alanine or valine, which represent amino acids found at the corresponding sequence position in other 2-oxoglutarate-dependent enzymes. The mutant enzymes were expressed in E. coli and purified to homogeneity. The catalytic activities of [Thr290]FHT and [Ala290]FHT were still significant, albeit greatly reduced to 20 and 8%, respectively, in comparison to the wild-type enzyme, whereas the activity of [Val290]FHT was negligible (about 1%). Kinetic analyses of purified wild-type and mutant enzymes revealed the functional significance of Ser290 for 2-oxoglutarate-binding. The spatial configurations of the related Fe(II)-dependent isopenicillin N and deacetoxycephalosporin C synthases have been reported recently and provide the lead structures for the conformation of other dioxygenases. Circular dichroism spectroscopy was employed to compare the conformation of pure flavanone 3beta-hydroxylase with that of isopenicillin N synthase. A double minimum in the far ultraviolet region at 222 nm and 208-210 nm and a maximum at 191-193 nm which are characteristic for alpha-helical regions were observed, and the spectra of the two dioxygenases fully matched revealing their close structural relationship. Furthermore, the spectrum remained unchanged after addition of either ferrous ions, 2-oxoglutarate or both of these cofactors, ruling out a significant conformational change of the enzyme on cofactor-binding.     

Biochemical characterization of the aba2 and aba3 mutants in Arabidopsis thaliana.

Abscisic acid (ABA)-deficient mutants in a variety of species have been identified by screening for precocious germination and a wilty phenotype. Mutants at two new loci, aba2 and aba3, have recently been isolated in Arabidopsis thaliana (L.) Hynh. (K.M. Léon-Kloosterziel, M. Alvarez-Gil, G.J. Ruijs, S.E. Jacobsen, N.E. Olszewski, S.H. Schwartz, J.A.D. Zeevaart, M. Koornneef [1996] Plant J 10: 655-661), and the biochemical characterization of these mutants is presented here. Protein extracts from aba2 and aba3 plants displayed a greatly reduced ability to convert xanthoxin to ABA relative to the wild type. The next putative intermediate in ABA synthesis, ABA-aldehyde, was efficiently converted to ABA by extracts from aba2 but not by extracts from aba3 plants. This indicates that the aba2 mutant is blocked in the conversion of xanthoxin to ABA-aldehyde and that aba3 is impaired in the conversion of ABA-aldehyde to ABA. Extracts from the aba3 mutant also lacked additional activities that require a molybdenum cofactor (Moco). Nitrate reductase utilizes a Moco but its activity was unaffected in extracts from aba3 plants. Moco hydroxylases in animals require a desulfo moiety of the cofactor. A sulfido ligand can be added to the Moco by treatment with Na2S and dithionite. Treatment of aba3 extracts with Na2S restored ABA-aldehyde oxidase activity. Therefore, the genetic lesion in aba3 appears to be in the introduction of S into the Moco.     

Molecular cloning, sequence analysis, and in vitro expression of flavanone 3 beta-hydroxylase from Petunia hybrida.

A cDNA encoding flavanone 3 beta-hydroxylase was isolated from petals of Petunia hybrida. The open reading frame of the nearly full length cDNA coded for a 369-amino acid polypeptide with a calculated Mr of 41,466. The function of this nucleotide sequence was verified by comparison with amino acid sequence of the amino terminus and tryptic peptides from purified plant enzyme, by Northern blotting with RNA from wild type and mutant plants, and by prokaryotic expression yielding an enzymatically active hydroxylase. Computer-aided sequence analysis revealed high similarity (73.5%) to flavanone 3 beta-hydroxylase from barley. Genomic Southern blot analysis showed the presence of only one gene for flavanone 3 beta-hydroxylase in P. hybrida.     

Human succinate dehydrogenase: Biochemical and genetic characterization

A simple procedure for the preparation of soluble human succinate dehydrogenase is described. These preparations have proved suitable for analysis by zone electrophoresis, using a specific stain to detect activity after separation. In a survey of succinate dehydrogenase from various tissues and different individuals, no evidence for genetic heterogeneity due to the expression of either multiple loci or alternative alleles at the succinate dehydrogenase locus was found. However, epigenetic heterogeneity in both molecular size and charge was seen and various explanations for the occurrence of the isoenzymes are explored. Estimates of molecular size (93,300 ± 9100) suggest that the smallest active unit of succinate dehydrogenase accounts for the major part of the solubilized activity. Kinetic studies have shown that the apparent K _m values for succinate (0.9mm) and PMS (0.4mm) are comparable to those previously described for the beef heart enzyme, and these parameters were not significantly altered when the enzyme was removed from the membrane milieu. However a marked non-succinate-dependent activation of the membrane-associated enzyme at 38C is apparently lost on solubilization, and this observation may have some bearing on earlier reports of an apparent decrease in V _max on solubilization of succinate dehydrogenase.     

Arabidopsis mutants deficient in polyunsaturated fatty acid synthesis. Biochemical and genetic characterization of a plant oleoyl-phosphatidylcholine desaturase.

The overall fatty acid composition of leaf lipids in a mutant of Arabidopsis thaliana was characterized by reduced levels of polyunsaturated 18-carbon fatty acids and an increased proportion of oleate as a consequence of a single recessive nuclear mutation. Quantitative analysis of the fatty acid composition of individual lipids demonstrated that all the major phospholipids of the extrachloroplast membranes are affected by the mutation, whereas the chlorplast lipids show fatty acid compositions only slightly different from those of wild type plants. These results are consistent with the parallel operation of two pathways of lipid synthesis in plant leaf cells (the prokaryotic pathway in the chloroplast and the eukaryotic pathway in the endoplasmic reticulum) and with genetic evidence (Browse, J., Kunst, L., Anderson, S., Hugly, S., and Somerville, C.R. (1989) Plant Physiol 90, 522-529) that an independent 18:1/16:1 desaturase operates on chloroplast membrane lipids. Direct enzyme assays confirmed that the mutant plants are deficient in the activity of a microsomal oleoyl-phosphatidycholine desaturase and demonstrated that this desaturase is the major enzyme responsible for the synthesis of polyunsaturated phospholipids. Despite this deficiency in 18:1-desaturase activity, mutant plants contained relatively high levels of 18:3 in their leaf phospholipids. This finding is interpreted as additional evidence that considerable two-way exchange of lipid occurs between the chloroplast and endoplasmic reticulum and that this exchange allows the chloroplast desaturases to provide lipids containing 18:3 to the extrachloroplast compartment, thus partially alleviating the deficiency in 18:1 desaturase activity.     

Biochemical characterization of Arabidopsis wild-type and mutant phytochrome B holoproteins.

Although phytochrome B (phyB) plays a particularly important role throughout the life cycle of a plant, it has not been studied in detail at the molecular level due to its low abundance. Here, we report on the expression, assembly with chromophore, and purification of epitope-tagged Arabidopsis phyB. In addition, we have reconstructed two missense mutations, phyB-4 and phyB-101, isolated in long hypocotyl screens. We show that mutant proteins phyB-4 and phyB-101 exhibit altered spectrophotometric and biochemical properties relative to the wild-type protein. In particular, we demonstrate that phyB-101 Pfr exhibits rapid nonphotochemical (dark) reversion to Pr that results in a lower photoequilibrium level of the active Pfr form. We conclude that this occurs in vivo as well because phyB-101 mutants are shown to lack an end-of-day-far-red hypocotyl elongation response that requires a stable Pfr species. We propose that this Pfr instability may be the primary molecular mechanism underlying the phyB-101 mutant phenotype.     

Biochemical and molecular characterization of a hydroxyjasmonate sulfotransferase from Arabidopsis thaliana

12-Hydroxyjasmonate, also known as tuberonic acid, was first isolated from Solanum tuberosum and was shown to have tuber-inducing properties. It is derived from the ubiquitously occurring jasmonic acid, an important signaling molecule mediating diverse developmental processes and plant defense responses. We report here that the gene AtST2a from Arabidopsis thaliana encodes a hydroxyjasmonate sulfotransferase. The recombinant AtST2a protein was found to exhibit strict specificity for 11- and 12-hydroxyjasmonate with K(m) values of 50 and 10 microm, respectively. Furthermore, 12-hydroxyjasmonate and its sulfonated derivative are shown to be naturally occurring in A. thaliana. The exogenous application of methyljasmonate to A. thaliana plants led to increased levels of both metabolites, whereas treatment with 12-hydroxyjasmonate led to increased level of 12-hydroxyjasmonate sulfate without affecting the endogenous level of jasmonic acid. AtST2a expression was found to be induced following treatment with methyljasmonate and 12-hydroxyjasmonate. In contrast, the expression of the methyljasmonate-responsive gene Thi2.1, a marker gene in plant defense responses, is not induced upon treatment with 12-hydroxyjasmonate indicating the existence of independent signaling pathways responding to jasmonic acid and 12-hydroxyjasmonic acid. Taken together, the results suggest that the hydroxylation and sulfonation reactions might be components of a pathway that inactivates excess jasmonic acid in plants. Alternatively, the function of AtST2a might be to control the biological activity of 12-hydroxyjasmonic acid.     

Phytochrome regulation and differential expression of gibberellin 3beta-hydroxylase genes in germinating Arabidopsis seeds.

Despite extensive studies on the roles of phytochrome in photostimulated seed germination, the mechanisms downstream of the photoreceptor that promote germination are largely unknown. Previous studies have indicated that light-induced germination of Arabidopsis seeds is mediated by the hormone gibberellin (GA). Using RNA gel blot analyses, we studied the regulation of two Arabidopsis genes, GA4 and GA4H (for GA4 homolog), both of which encode GA 3beta-hydroxylases that catalyze the final biosynthetic step to produce bioactive GAs. The newly isolated GA4H gene was expressed predominantly during seed germination. We show that expression of both GA4 and GA4H genes in imbibed seeds was induced within 1 hr after a brief red (R) light treatment. In the phytochrome B-deficient phyB-1 mutant, GA4H expression was not induced by R light, but GA4 expression still was, indicating that R light-induced GA4 and GA4H expression is mediated by different phytochromes. In contrast to the GA4 gene, the GA4H gene was not regulated by the feedback inhibition mechanism in germinating seeds. Our data demonstrate that expression of GA 3beta-hydroxylase genes is elevated by R light, which may result in an increase in biosynthesis of active GAs to promote seed germination. Furthermore, our results suggest that each GA 3beta-hydroxylase gene plays a unique physiological role during light-induced seed germination.     

Mechanistic studies on three 2-oxoglutarate-dependent oxygenases of flavonoid biosynthesis: anthocyanidin synthase, flavonol synthase, and flavanone 3beta-hydroxylase

Anthocyanidin synthase (ANS), flavonol synthase (FLS), and flavanone 3beta-hydroxylase (FHT) are involved in the biosynthesis of flavonoids in plants and are all members of the family of 2-oxoglutarate- and ferrous iron-dependent oxygenases. ANS, FLS, and FHT are closely related by sequence and catalyze oxidation of the flavonoid "C ring"; they have been shown to have overlapping substrate and product selectivities. In the initial steps of catalysis, 2-oxoglutarate and dioxygen are thought to react at the ferrous iron center producing succinate, carbon dioxide, and a reactive ferryl intermediate, the latter of which can then affect oxidation of the flavonoid substrate. Here we describe work on ANS, FLS, and FHT utilizing several different substrates carried out in 18O2/16OH2, 16O2/18OH2, and 18O2/18OH2 atmospheres. In the 18O2/16OH2 atmosphere close to complete incorporation of a single 18O label was observed in the dihydroflavonol products (e.g. (2R,3R)-trans-dihydrokaempferol) from incubations of flavanones (e.g. (2S)naringenin) with FHT, ANS, and FLS. This and other evidence supports the intermediacy of a reactive oxidizing species, the oxygen of which does not exchange with that of water. In the case of products formed by oxidation of flavonoid substrates with a C-3 hydroxyl group (e.g. (2R,3R)-trans-dihydroquercetin), the results imply that oxygen exchange can occur at a stage subsequent to initial oxidation of the C-ring, probably via an enzyme-bound C-3 ketone/3,3-gem-diol intermediate.     

Biochemical and genetic characterization of kynurenine formamidase from Drosophila melanogaster

The molecular weight forms of kynurenine formamidase were studied both genetically and biochemically. Formamidase I (native molecular weight 60,000) was purified using (NH₄)₂SO₄ and pH fractionation, DEAE-cellulose chromatography at two different pH's, hydroxylapatite chromatography, and Sephadex G-100 gel filtration. Its subunit molecular weight, as determined by SDS gel electrophoresis, is 34,000, indicating that formamidase I is a dimer. Its K _m is 1.87×10^–3 m. Its isoelectric point is pH 5.3. Its amino acid composition is reported. Formamidase II (native molecular weight 31,000) was partially purified using techniques similar to those above. Its K _m is 2.31×10^–3 m. The response of formamidase activity to change in gene dosage was measured in segmental aneuploids generated in the second, third, and X chromosomes. Two separate chromosomal regions were identified which when present in extra dosage result in an elevation of the level of formamidase activity close to that predicted for the addition of a structural gene in a two-gene system. These tentative map positions were substantiated by demonstration that addition of one of the regions, 25A–27E, causes a 50% elevation in the relative amount of formamidase II. Addition of the other region, 91B–93F, causes a similar elevation in the relative amount of formamidase I. A model of the evolutionary origin of the two forms is presented, and the significance of these results to this model is discussed.This work was supported by USPHS Grant GM-21286.     

Biochemical and genetic characterization of -glucosidase mutants in Saccharomyces lactis

Mutants with reduced activity for beta-glucosidase (beta-d-glucoside glucohydrolase EC 3.2.1.21) were isolated from the haploid yeast Saccharomyces lactis. Tetrad analysis indicated that in each mutant a single genetic factor, closely linked or allelic to the structural gene for beta-glucosidase (B locus), is responsible for the decreased activity. beta-Glucosidases produced by wild-type and mutant strains are similar in molecular size and charge but differ in catalytic properties, thermal stability, and serological specificity, indicating that mutants are in the structural gene. All mutants retained their capacity to be induced by either methyl-beta-d-glucoside or glucose. In all cases, the mutant phenotype was dominant in heterozygous diploids.     

Biochemical and genetic characterization of the three metabolic routes in Thermococcus kodakarensis linking glyceraldehyde 3-phosphate and 3-phosphoglycerate

In the classical Embden-Meyerhof (EM) pathway for glycolysis, the conversion between glyceraldehyde 3-phosphate (GAP) and 3-phosphoglycerate (3-PGA) is reversibly catalysed by phosphorylating GAP dehydrogenase (GAPDH) and phosphoglycerate kinase (PGK). In the Euryarchaeota Thermococcus kodakarensis and Pyrococcus furiosus, an additional gene encoding GAP:ferredoxin oxidoreductase (GAPOR) and a gene similar to non-phosphorylating GAP dehydrogenase (GAPN) are present. In order to determine the physiological roles of the three routes that link GAP and 3-PGA, we individually disrupted the GAPOR, GAPN, GAPDH and PGK genes (gor, gapN, gapDH and pgk respectively) of T. kodakarensis. The Δgor strain displayed no growth under glycolytic conditions, confirming its proposed function to generate reduced ferredoxin for energy generation in glycolysis. Surprisingly, ΔgapN cells also did not grow under glycolytic conditions, suggesting that GAPN plays a key role in providing NADPH under these conditions. Disruption of gor and gapN had no effect on gluconeogenic growth. Growth experiments with the ΔgapDH and Δpgk strains indicated that, unlike their counterparts in the classical EM pathway, GAPDH/PGK play a major role only in gluconeogenesis. Biochemical analyses indicated that T. kodakarensis GAPN did not recognize aldehyde substrates other than d-GAP, preferred NADP(+) as cofactor and was dramatically activated with glucose 1-phosphate.     

Biochemical and genetic characterization of arazyme, an extracellular metalloprotease produced from Serratia proteamaculans HY-3

Serratia proteamaculans HY-3 isolated from the digestive tract of a spider produces an extracellular protease named arazyme, with an estimated molecular mass of 51.5 kDa. The purified enzyme was characterized as having high activities at wide pH and temperature ranges. We further characterized biochemical features of the enzymatic reactions under various reaction conditions. The protease efficiently hydrolyzed a broad range of protein substrates including albumin, keratin, and collagen. The dependence of enzymatic activities on the presence of metal ions such as calcium and zinc indicated that the enzyme is a metalloprotease, together with the previous observation that the proteolytic activity of the enzyme was not inhibited by aspartate, cysteine, or serine protease inhibitors, but strongly inhibited by 1,10-phenanthroline and EDTA. The araA gene encoding the exoprotease was isolated as a 5.6 kb BamHl fragment after PCR amplification using degenerate primers and subsequent Southern hybridization. The nucleotide sequence revealed that the deduced amino acid sequences shared extensive similarity with those of the serralysin family of metalloproteases from other enteric bacteria. A gene (inh) encoding a putative protease inhibitor was also identified immediately adjacent to the araA structural gene.     

Biochemical and genetic characterization of the structure of yeast ornithine decarboxylase

The ornithine decarboxylase gene of S. cerevisiae encodes a predicted protein of approximately 53 kD highly homologous with the ornithine decarboxylase of other species. However, the native enzyme has been reported as an 86 kD protein. Our molecular sieve analysis indicated a Mr = 110,000 for the native enzyme. SDS-PAGE analysis of [H3]-alpha-difluoromethylornithine labelled enzyme demonstrated a subunit Mr of approximately 50 kD and suggested the native enzyme is a dimer. Genetic analyses support this conclusion. The complementary, ornithine decarboxylase deficient mutations spe 1A and spe 1B were mapped to the enzyme structural gene by linkage analysis and gene conversion mapping. This demonstrated that the mutations exhibit intragenic complementation which suggests protein-protein interactions and an oligomeric structure for the yeast enzyme. We conclude that yeast ornithine decarboxylase is a dimeric enzyme of 53 kD subunits.