Preparation and characterization of the NAD vinylogue, 3-pyridylacryloamide adenine dinucleotide

The vinylogue of NAD, 3-pyridylacryloamide adenine dinucleotide, was prepared from NAD and 3-pyridylacryloamide through the snake venom NADase-catalyzed transglycosidation reaction. The analog, purified by ion-exchange chromatography, was obtained in a 55% yield. The cyanide adduct and reduced form of the analog exhibited absorbance maxima at 358 nm and 378 nm, respectively, with extinction coefficients in each case being 2.3-times higher than those reported for the corresponding NAD derivatives. 3-Pyridylacryloamide adenine dinucleotide served as a coenzyme with bovine liver glutamic dehydrogenase and to a lesser extent with malate and lactate dehydrogenases. The analog was not reduced in reactions catalyzed by yeast and horse liver alcohol dehydrogenases, sheep liver sorbitol dehydrogenase, and rabbit muscle glycerophosphate dehydrogenase. Substitution of the pyridylacryloamide analogs for NAD and NADH in the assay of substrates for glutamic dehydrogenase was demonstrated.     

A novel diazonium-sulfhydryl reaction in the inactivation of yeast alcohol dehydrogenase by diazotized 3-aminopyridine adenine dinucleotide.

Diazotized 3-aminopyridine adenine dinucleotide has been found to modify four sulfhydryl groups per molecule of enzyme during the complete inactivation of yeast alcohol dehydrogenase. The reaction of sulfhydryl groups was indicated by titration studies with 5,5-dithiobis(2-nitrobenzoic acid) as well as isolation and quantitation of the cysteinyl derivative released by acid hydrolysis of the modified enzyme. The cysteinyl derivative was identified as S-(3-pyridyl)cysteine. Authentic S-(3-pyridyl)cystein was synthesized and structurally characterized for these studies. Diazonium-sulfhydryl reactions were demonstrated for a number of diazonium derivatives with cysteine, homocysteine, glutathione, and mercaptoethanol at 0-4 degrees and neutral pH. Second order rate constants were determined in reactions of these sulfhydryl compounds with diazotized 1-methyl-3-aminopyridinium chloride, diazotized 3-aminopyridine adenine dinucleotide, and diazotized 3-aminopyridine adenine dinucleotide phosphate.     

Studies of 3-Aminopyridine adenine dinucleotide phosphate

3-Aminopyridine adenine dinucleotide phosphate (AADP) was prepared from NADP and 3-amino-pyridine through the pig brain NADase-catalyzed pyridine base exchange reaction. The purified dinucleotide was chemically characterized and spectral properties of the compound were determined. The importance of the application of AADP in studies of NADP-requiring biochemical processes was indicated by the demonstration of AADP as an effective inhibitor of five NADP-requiring enzymes, by the demonstration of the fluorescence enhancement on the binding of AADP to yeast glucose-6-phosphate dehydrogenase when glucose-6-phosphate is present, and by the functioning of AADP as a fluorimetric substrate for snake venom nucleotide pyrophosphatase.     

Reactions of the Neurospora crassa nitrate reductase with NAD(P) analogs.

The assimilatory NADPH-nitrate reductase (NADPH:nitrate oxidoreductase, EC 1.6.6.3) from Neurospora crassa is competitively inhibited by 3-aminopyridine adenine dinucleotide (AAD) and 3-aminopyridine adenine dinucleotide phosphate (AADP) which are structural analogs of NAD and NADP, respectively. The amino group of the pyridine ring of AAD(P) can react with nitrous acid to yield the diazonium derivative which may covalently bind at the NAD(P) site. As a result of covalent attachment, diazotized AAD(P) causes time-dependent irreversible inactivation of nitrate reductase. However, only the NADPH-dependent activities of the nitrate reductase, i.e. the overall NADPH-nitrate reductase and the NADPH-cytochrome c reductase activities, are inactivated. The reduced methyl viologen- and reduced FAD-nitrate reductase activities which do not utilize NADPH are not inhibited. This inactivation by diazotized AADP is prevented by 1 mM NADP. The inclusion of 1 muM FAD can also prevent inactivation, but the FAD effect differs from the NADP protection in that even after removal of the exogenous FAD by extensive dialysis or Sephadex G-25 filtration chromatography, the enzyme is still protected against inactivation. The FAD-generated protected form of nitrate reductase could again be inactivated if the enzyme was treated with NADPH, dialyzed to remove the NADPH, and then exposed to diazotized AADP. When NADP was substituted for NADPH in this experiment, the enzyme remained in the FAD-protected state. Difference spectra of the inactivated nitrate reductase demonstrated the presence of bound AADP, and titration of the sulfhydryl groups of the inactivated enzyme revealed that a loss of accessible sulfhydryls had occurred. The hypothesis generated by these experiments is that diazotized AADP binds at the NADPH site on nitrate reductase and reacts with a functional sulfhydryl at the site. FAD protects the enzyme against inactivation by modifying the sulfhydryl. Since NADPH reverses this protection, it appears the modifications occurring are oxidation-reduction reactions. On the basis of these results, the physiological electron flow in the nitrate reductase is postulated to be from NADPH via sulfhydryls to FAD and then the remainder of the electron carriers as follows: NADPH leads to -SH leads to FAD leads to cytochrome b-557 leads to Mo leads to NO-3.     

Inactivation of chicken liver d-3-phosphoglycerate dehydrogenase by N-alkylmaleimides

Chicken liver d-3-phosphoglycerate dehydrogenase was effectively inhibited at 25 °C by micromolar concentrations of N-ethyl-, N-butyl-, N-pentyl-, N-heptyl-, and N-phenylmaleimide. The rates of inactivation of the enzyme did not vary with chain length of the N-alkylmaleimide derivative. Saturation kinetics in the same concentration range was observed with each maleimide derivative studied. A maximum pseudo-first-order rate constant of 0.1 min^?1 was determined for all of the maleimide inactivation reactions. Compounds shown to bind at the coenzyme binding site such as NAD, 3-aminopyridine adenine dinucleotide, adenosine diphosphoribose, and adenosine diphosphate did not protect the enzyme against N-ethylmaleimide inactivation. AMP was demonstrated to be a substrate-competitive inhibitor of the enzyme. AMP and 3-phosphoglycerate both effectively protected the enzyme against N-ethylmaleimide inactivation. Diazotized 3-aminopyridine adenine dinucleotide, a sulfhydryl modifying, site-labeling reagent for several pyridine nucleotide-dependent enzymes, did not inactivate the phosphoglycerate dehydrogenase but functioned rather as a reversible coenzyme-competitive inhibitor.     

Studies of NAD kinase and NMN:ATP adenylyltransferase in Haemophilus influenzae

Previous studies of Haemophilus influenzae documented the importance of several pyridine nucleotide-dependent enzymes in processing extracellular NAD and NMN to satisfy the V-factor growth requirement of the organism. The substrate specificities of two of these enzymes. NMN:ATP adenylyltransferase and NAD kinase, were investigated following partial purification. The ability of the transferase to utilize 3-acetylpyridine mononucleotide and 3-aminopyridine mononucleotide as substrates for the synthesis of the corresponding dinucleotides was demonstrated. The NAD kinase was observed to accept 3-acetylpyridine adenine dinucleotide as a substrate but failed to utilize 3-aminopyridine adenine dinucleotide. The mononucleotides of 3-acetylpyridine and 3-aminopyridine were shown to be as effective as the corresponding dinucleotides in the support of growth and inhibition of growth of H. influenzae, respectively. Inhibition of growth of H. influenzae by submicromolar 3-aminopyridine adenine dinucleotide was shown to occur because 3-aminopyridine mononucleotide was produced from it in reactions catalysed by the H. influenzae periplasmic nucleotide pyrophosphatase. The presence of an additional important pyridine nucleotide-dependent enzyme, NMN glycohydrolase, is also reported.     

Periodate-oxidized 3-aminopyridine adenine dinucleotide phosphate as a fluorescent affinity label for pigeon liver malic enzyme

Treatment of 3-aminopyridine adenine dinucleotide phosphate with sodium periodate resulted in oxidation of the ribose linked to 3-aminopyridine ring and cleavage of the dinucleotide into 3-aminopyridine and adenosine moieties. These two moieties were separated by thin layer chromatography and were synergistically bound to pigeon liver malic enzyme (EC 1.1.1.40), causing inactivation of the enzyme. The inactivation showed saturation kinetics. The apparent binding constant for the reversible enzyme-reagent binary complex (KI) and the maximum inactivation rate constant at saturating reagent concentration (kmax) were found to be 1.1 +/- 0.02 mM and 0.068 +/- 0.001 min-1, respectively. L-Malate at low concentration enhanced the inactivation rate by lowering the KI value whereas high malate concentration increased the kmax. Mn2+ or NADP+ partially protected the enzyme from the inactivation and gave additive protection when used together. L-Malate eliminated the protective effect of NADP+ or Mn2+. Maximum and synergistic protection was afforded by NADP+, Mn2+ plus L-malate (or tartronate). Oxidized and cleaved 3-aminopyridine adenine dinucleotide phosphate was also found to be a competitive inhibitor versus NADP+ in the oxidative decarboxylation reaction catalyzed by malic enzyme with a Ki value of 4.1 +/- 0.1 microM. 3-Aminopyridine adenine dinucleotide phosphate or its periodate-oxidized cleaved products bound to the enzyme anticooperatively. Oxidized 3-aminopyridine adenine dinucleotide phosphate labeled the nucleotide binding site of the enzyme with a fluorescent probe which may be readily traced or quantified. The completely inactivated enzyme incorporated 2 mol of reagent/mol of enzyme tetramer. The inactivation was partially reversible by dilution and could be made irreversible by treating the modified enzyme with sodium borohydride. This fluorescent compound and its counterpart-oxidized 3-aminopyridine adenine dinucleotide may be a potential affinity label for all other NAD(P)+-dependent dehydrogenases.     

Nonpolar interactions in the modification of an essential sulfhydryl of sorbitol dehydrogenase by N-alkylmaleimides

A series of N-alkylmaleimides varying in chainlength from N-methyl- to N-octylmaleimide inclusive was shown to effectively inactivate sheep liver sorbitol dehydrogenase at pH 7.5 and 25 degrees C. The apparent second-order rate constants for inactivation increased with increasing chainlength of the N-alkylmaleimide used. Positive chainlength effects were also indicated by the Kd values for the N-ethyl and N-heptyl derivatives obtained from studies of the saturation kinetics observed for inactivation of the enzyme at high concentrations of these maleimides. The complete inactivation of sorbitol dehydrogenase was demonstrated to occur through the selective covalent modification of one cysteine residue per subunit of enzyme. The stoichiometry of enzyme inactivation was supported on the one hand by fluorescence titration with fluorescein mercuric acetate of the native and the inactivated enzyme, and, on the other hand, by the simultaneous inactivation of the enzyme with selective modification of one sulfhydryl per subunit by N-[p-(2-benzoxazolyl)phenyl]maleimide. Protection of the enzyme from N-alkylmaleimide inactivation was observed with the binding of NADH, whereas both NAD and sorbitol were ineffective as protecting ligands. Diazotized 3-aminopyridine adenine dinucleotide, in contrast to previous studies of this reagent with yeast alcohol dehydrogenase and rabbit muscle glycerophosphate dehydrogenase, did not function as a site-labeling reagent for sorbitol dehydrogenase.     

Adenosine diphosphoribose transfer reactions catalyzed by Bungarus fasciatus venom NAD glycohydrolase

Reactions catalyzed by purified Bungarus fasciatus venom NAD glycohydrolase were demonstrated to include ADP-ribose transfer from NAD to alcohols and to imidazole derivatives to produce a variety of ADP-ribosides. The formation of products was monitored by high performance liquid chromatography. In the enzyme-catalyzed alcoholysis of NAD, the ratio of n-alkyl-ADP-riboside formed to the hydrolytic product, ADP-ribose, increased linearly with alcohol concentration. The effectiveness of alcohols as acceptors of the ADP-ribose moiety in these reactions increased with increasing chainlength of the alcohol used. Linear positive chainlength effects extended from methanol to pentanol suggesting facilitation of these reactions by nonpolar interactions. In the methanolysis reaction, NADP, thionicotinamide adenine dinucleotide, nicotinamide-1, N6-ethenoadenine dinucleotide, and 3-acetylpyridine adenine dinucleotide were shown to be as effective as NAD as donor substrates. The NAD glycohydrolase-catalyzed ADP-ribose transfer to pyridine bases to form NAD analogs was studied at pyridine base concentrations above those determined to be saturating for the base exchange reaction. Under these conditions, the ratio of base exchange to hydrolysis of NAD was directly related to the pKa of the ring nitrogen of the pyridine base employed. In addition to alcoholysis and pyridine-base exchange reactions, the snake venom enzyme was demonstrated to catalyze an ADP-ribose transfer reaction to imidazole derivatives. Arginine methyl ester was ineffective as an ADP-ribose acceptor molecule in these reactions.     

Carbanicotinamide adenine dinucleotide: synthesis and enzymological properties of a carbocyclic analogue of oxidized nicotinamide adenine dinucleotide

The dinucleotide carbanicotinamide adenine dinucleotide (carba-NAD), in which a 2,3-dihydroxycyclopentane ring replaces the beta-D-ribonucleotide ring of the nicotinamide ribonucleoside moiety of NAD, has been synthesized and characterized enzymologically. The synthesis begins with the known 1-aminoribose analogue (+/-)-4 beta-amino-2 alpha,3 alpha-dihydroxy-1 beta-cyclopentanemethanol. The pyridinium ring is first introduced and the resultant nucleoside analogue specifically 5'-phosphorylated. Coupling the racemic carbanicotinamide 5'-mononucleotide with adenosine 5'-monophosphate produces two diastereomeric carba-NAD analogues which are chromatographically separable. Only one diastereomer is a substrate for alcohol dehydrogenase and on this basis is assigned a configuration analogous to D-ribose. The reduced dinucleotide carba-NADH was characterized by fluorescence spectroscopy and found to adopt a "stacked" conformation similar to that of NADH. The analogue is reduced by both yeast and horse liver alcohol dehydrogenase with Km and Vmax values for the analogue close to those observed for NAD. Carba-NAD is resistant to cleavage by NAD glycohydrolase, and the analogue has been demonstrated to noncovalently inhibit the soluble NAD glycohydrolase from Bungarus fasciatus venom at low concentrations (less than or equal to 100 microM).     

Properties and applications of immobilized snake venom NAD glycohydrolase

The NAD glycohydrolase (NADase) from Bungarus fasciatus snake venom was adsorbed on concanavalin A-Sepharose, and demonstrated to retain both hydrolase and transglycosidase activities in the bound form. The matrix-bound enzyme was stable to repeated washing with buffer and storage at 4°C. The bound enzyme exhibited the same K_m value for hydrolysis of nicotinamide-1,N⁶-ethenoadenine dinucleotide as previously measured with the soluble, purified form of the enzyme. The bound NADase was used repeatedly for a preparative-scale synthesis of 3-acetylpyridine adenine dinucleotide. It was further demonstrated that the immobilized enzyme could be prepared directly from crude snake venom, thus avoiding the time required for purification. The application of the immobilized snake venom NADase for the preparation of pyridine nucleotide coenzyme analogs has many advantages over procedures used previously for analog synthesis.     

Inactivation of yeast alcohol dehydrogenase by a reactive coenzyme analogue: 3-chloroacetyl pyridine adenine dinucleotide

Yeast alcohol dehydrogenase is very rapidly and irreversibly inactivated by 3-chloroacetyl pyridine adenine dinucleotide, a reactive NAD+-analogue (Biellmann et al., 1974, FEBS Lett. 40, 29-32). Kinetic investigations with this compound, and structurally related compounds, show that this inactivation, against which NAD+ provides a complete protection, corresponds to an affinity label. The incorporation of the coenzyme analogue correlates linearly with the enzyme inactivation, the total inactivation corresponding to one mole of inactivator per coenzyme binding site. The pH-dependence of the inactivation rates of the enzyme by this coenzyme analogue and by its reduced form reflects exactly the pH variation of their respective dissociation constants. In spite of a good stability of the label in the non denatured inactivated enzyme, no modified amino-acid residue could be identified. Considering the affinity of this analogue for yeast alcohol dehydrogenase and the strict steric requirements of this enzyme towards its ligands, the nature of the inactivation reaction as well as different possibilities of the loss of the label in the inactivated enzyme are discussed.     

Oxidation and cleavage of 3-aminopyridine adenine dinucleotide phosphate with periodate

Treatment of 3-aminopyridine adenine dinucleotide phosphate with sodium periodate in the neutral pH resulted in oxidation of the ribose linked to 3-aminopyridine and cleavage of the dinucleotide into adenosine- and 3-aminopyridine-containing moieties. Separation of these moieties was afforded by thin-layer chromatography, high-performance liquid chromatography, and fast protein liquid chromatography. From fast atom bombardment mass spectra and nuclear magnetic resonance spectra, the adenosine-containing moiety was identified as 2'-phosphoadenosine 5'-phosphate while the aminopyridine moiety was present in a mixture of the hydrated 3-aminopyridine mononucleotide/nucleoside dialdehyde. Separation of the completely oxidized product by Pharmacia fast protein liquid chromatography gave three major peaks corresponding to 2'-phosphoadenosine 5'-phosphate, 2'-phosphoadenosine 5'-diphosphate and oxidized 3-aminopyridine nucleoside, with minor amount of oxidized 3-aminopyridine mononucleotide. Thus the oxidized 3-aminopyridine adenine dinucleotide phosphate was shown to cleave by two pathways: it may either undergo beta-elimination to give 2'-phosphoadenosine 5'-diphosphate and oxidized 3-aminopyridine nucleoside; or the phosphodiester linkage may be hydrolyzed to give 2'-phosphoadenosine 5'-phosphate and oxidized 3-aminopyridine mononucleotide. The latter compound may further undergo beta-elimination and eventually give oxidized 3-aminopyridine nucleoside. Hydrolysis could be prevented by storing the sample as lyophilized powder, while beta-elimination was diminished by lowering the storage temperature. We found that the lyophilized powder of oxidized 3-aminopyridine adenine dinucleotide phosphate can be stored at -50 degrees C for several months with minimum decomposition.     

New coenzymically-active soluble and insoluble macromolecular NAD+ derivatives.

Reaction in dimethyl sulfoxide of nicotinamide 8-bromoadenine dinucleotide with the disodium salt of 3-mercaptopropionic acid afforded nicotinamide-8-(2-carboxyethylthio)adenine dinucleotide, a new NAD+ analogue functionalized at the adenine C-8 position by an omega-carboxylic side chain. Carbodimide coupling of the latter derivative to high-molecular-weight water-soluble (polyethyleneimine, polylysine) and insoluble (aminohexy)-Sepharose) polymers gave the corresponding macromolecular NAD+ analogues. These derivatives have been shown to be enzymically reducible. The polyethyleneimine analogue showed a substantial degree of efficiency relative to free NAD+ with yeast alcohol dehydrogenase (47%) but a considerably lower one with rabbit muscle lactate dehydrogenase (3%); the polylysine analogue showed a low degree of efficiency with both enzymes (5-6%).     

The stereochemical course of nucleotidyl transfer catalysed by NAD pyrophosphorylase

NAD pyrophosphorylase catalyses nucleotidyl transfer from adenosine (R)-5'-[alpha-17O]triphosphate to nicotinamide mononucleotide with inversion of configuration at the alpha-P giving (S)-[17O]NAD+. The simplest interpretation of this observation is that the adenylyl group is transferred directly from ATP to the co-substrate by an 'in line' mechanism. It is also shown that snake venom phosphodiesterase hydrolyses NAD+ regio-specifically at the adenylyl terminus of the pyrophosphate bond.     

Synthesis of coenzymically active soluble and insoluble macromolecularized NAD+ derivatives.

Alkylation at N-1 of the NAD+ adenine ring with 3,4-epoxybutanoic acid, followed by chemical reduction to the alkali-stable NADH form and alkaline Dimroth rearrangement, gave the NADH derivative alkylated at the exocyclic adenine amino group. Enzymic reoxidation of the latter derivative gave nicotinamide-6-(2-hydroxy-3-carboxypropylamino)purine dinucleotide, a functionalized NAD+ analogue carrying an omega-carboxyalkyl side-chain at the exocyclic adenine amino group. Carbodiimide coupling of the latter derivative to high-molecular-weight water-soluble (polyethyleneimine, polylysine) and insoluble (aminohexyl-Sepharose) polymers gave the corresponding macromolecularized NAD+ analogues. These derivatives have been shown to be enzymically reducible. The polyethyleneimine and polylysine analogues showed a substantial degree of efficiency relative to free NAD+ with rabbit muscle lactate dehydrogenase (60 and 25% respectively) but a lower one with yeast alcohol dehydrogenase and Bacillus subtilis alanine dehydrogenase (2-7%). The polyethyleneimine derivative entrapped in cellulose triacetate fibres together with the lactate dehydrogenase was operationally stable during repetitive use.     

The conformation of adenosine diphosphoribose and 8-bromoadenosine diphosphoribose when bound to liver alcohol dehydrogenase.

8-Bromo-adenosine diphosphoribose (br8 ADP-Rib) and nicotinamide 8-bromoadenine dinucleotide (Nbr8AD+) which are analogues of the coenzyme NAD+, were prepared and their liver alcohol dehydrogenase complexes studied by crystallographic methods. Nbr8AD+ is active in alcohol dehydrogenase complexes studied by crystallographic methods. Nbr8AD+ is active in hydrogen transport and br8ADP-Rib is a coenzyme competitive inhibitor for the enzymes liver alcohol dehydrogenase and yeast alcohol dehydrogenase. X-ray data were obtained for the complex between liver alcohol dehydrogenase and br8ADP-Rib to 0.45 nm resolution and for the liver alcohol dehydrogenase-adenosine diphosphoribose complex to 0.29-nm resolution. The conformations of these analogues were determined from the X-ray data. It was found that ADP-Rib had a conformation very similar to the corresponding part of NAD+, when NAD+ is bound to lactate and malate dehydrogenase. br8ADP-Rib had the same anti conformation of the adenine ring with respect to the ribose as ADP-Rib and NAD+, in contrast to the syn conformation found in 8-bromo-adenosine. The overcrowding at the 8-position is relieved in br8ADP-Rib by having the ribose in the 2' endo condormation instead of the usual 3' endo as in ADP-Rib and NAD+.     

Involvement of the protocatechuate pathway in the metabolism of mandelic acid by Aspergillus niger

Cell-free extracts of Aspergillus niger UBC 814 grown in the presence of dl-mandelate oxidized both d(-)- and l(+)-mandelate via benzoylformate and benzaldehyde to benzoate. dl-p-Hydroxymandelate was oxidized, presumably through a parallel pathway, to p-hydroxybenzoate. A particulate d(-)-mandelate dehydrogenase and a supernatant fraction l(+)-mandelate dehydrogenase converted their respective substrates to benzoylformate. Both flavine adenine dinucleotide and flavine mononucleotide showed a stimulatory effect on the activity of the l(+)-mandelate dehydrogenase. Benzoylformate was decarboxylated to benzaldehyde by an enzyme requiring thiamine pyrophosphate for maximal activity. Two benzaldehyde dehydrogenases dependent on nicotinamide adenine dinucleotide (NAD) and nicotinamide adenine dinucleotide phosphate (NADP), respectively, for their activity dehydrogenated benzaldehyde to benzoate. In the presence of reduced NADP (NADPH), benzoate was oxidized via p-hydroxybenzoate and protocatechuate. Reduced NAD could not replace NADPH. Sensitive methods of assay for d(-)-mandelate dehydrogenase and benzoylformate decarboxylase are described. The fungal pathway is compared with these systems in bacteria.     

Mono ADP-ribosylation of transducin catalyzed by rod outer segment extract.

Transducin is the retinal rod outer segment (ROS)-specific G protein coupling the photoexcited rhodopsin to cyclic GMP-phosphodiesterase. The alpha subunit of transducin is known to be ADP-ribosylated by bacterial toxins. We investigated the possibility that transducin is modified in vitro by an endogenous ADP-ribosyltransferase activity. By using either ROS, cytosolic extract of ROS or purified transducin in the presence of [alpha-32P]nicotinamide adenine dinucleotide (NAD+), the alpha and beta subunits of transducin were found to be radiolabeled. The labeling was decreased by snake venom phosphodiesterase I (PDE I). The modification was shown to be mono ADP-ribosylation by analyses on thin layer chromatography of the PDE I-hydrolyzed products which revealed only 5'AMP residues. In addition we report that sodium nitroprusside activates the ADP-ribosylation of transducin.     

Regulation of flavin biosynthesis in the methylotrophic yeast Hansenula polymorpha

Under various conditions of growth of the methylotrophic yeast Hansenula polymorpha, a tight correlation was observed between the levels of flavin adenine dinucleotide (FAD)-containing alcohol oxidase, and the levels of intracellularly bound FAD and flavin biosynthetic enzymes. Adaptation of the organism to changes in the physiological requirement for FAD was by adjustment of the levels of the enzymes catalyzing the last three steps in flavin biosynthesis, riboflavin synthetase, riboflavin kinase and flavin mononucleotide adenylyltransferase. The regulation of the synthesis of the latter enzymes in relation to that of alcohol oxidase synthesis was studied in experiments involving addition of glucose to cells of H. polymorpha growing on methanol in batch cultures or in carbon-limited continuous cultures. This resulted not only in selective inactivation of alcohol oxidase and release of FAD, as previously reported, but invariably also in repression/inactivation of the flavin biosynthetic enzymes. In further experiments involving addition of FAD to the same type of cultures it became clear that inactivation of the latter enzymes was not caused directly by glucose, but rather by free FAD that accumulated intracellularly. In these experiments no repression or inactivation of alcohol oxidase occurred and it is therefore concluded that the synthesis of this enzyme and the flavin biosynthetic enzymes is under separate control, the former by glucose (and possibly methanol) and the latter by intracellular levels of free FAD.Abbreviations FAD Flavin adenine dinucleotide - FMN riboflavin-5-phosphate; flavin mononucleotide - Rf riboflavin