Iron deficient Medicago scutellata grown in nutrient solution at high pH accumulates and secretes large amounts of flavins.

Flavin synthesis and secretion is an integral part of the toolbox of root-borne Fe facilitators used by Strategy I species upon Fe deficiency. The Fe-deficiency responses of the wild legume Medicago scutellata grown in nutrient solution have been studied at two different pH values (5.5 and 7.5). Parameters studied include leaf chlorophyll, nutrient solution pH, concentrations and contents of micronutrients, flavin accumulation in roots, flavin export to the medium, and root ferric chelate reductase and acidification activities. Results show that M. scutellata behaves upon Fe deficiency as a Strategy I species, with a marked capacity for synthesizing flavins (riboflavin and three hydroxylated riboflavin derivatives), which becomes more intense at high pH. Results also show that this species is capable of exporting a large amount of flavins to the external medium, both at pH 5.5 and 7.5. This is the first report of a species having a major flavin secretion at pH 7.5, in contrast with the very low flavin secretion found in other flavin-producing species such as Beta vulgaris and M. truncatula. These results provide further support to the hypothesis that flavin secretion is relevant for Fe acquisition at high pH, and open the possibility to improve the Fe-efficiency responses in legumes of agronomic interest.

Multi-metal Restriction by Calprotectin Impacts De Novo Flavin Biosynthesis in Acinetobacter baumannii.

Calprotectin (CP) inhibits bacterial viability through extracellular chelation of transition metals. However, how CP influences general metabolism remains largely unexplored. We show here that CP restricts bioavailable Zn and Fe to the pathogen Acinetobacter baumannii, inducing an extensive multi-metal perturbation of cellular physiology. Proteomics reveals severe metal starvation, and a strain lacking the candidate ZnII metallochaperone ZigA possesses altered cellular abundance of multiple essential Zn-dependent enzymes and enzymes in de novo flavin biosynthesis. The ΔzigA strain exhibits decreased cellular flavin levels during metal starvation. Flavin mononucleotide provides regulation of this biosynthesis pathway, via a 3,4-dihydroxy-2-butanone 4-phosphate synthase (RibB) fusion protein, RibBX, and authentic RibB. We propose that RibBX ensures flavin sufficiency under CP-induced Fe limitation, allowing flavodoxins to substitute for Fe-ferredoxins as cell reductants. These studies elucidate adaptation to nutritional immunity and define an intersection between metallostasis and cellular metabolism in A. baumannii.

Flavin Storage and Sequestration by Mycobacterium tuberculosis Dodecin.

Dodecins are small flavin binding proteins occurring in archaea and bacteria. They are remarkable for binding dimers of flavins with their functional relevant aromatic isoalloxazine rings deeply covered. Bacterial dodecins are widely spread and found in a large variety of pathogens, among them Pseudomonas aeruginosa, Streptococcus pneumonia, Ralstonia solanacearum, and Mycobacterium tuberculosis ( M. tuberculosis). In this work, we seek to understand the function of dodecins from M. tuberculosis dodecin. We describe flavin binding in thermodynamic and kinetic properties and achieve mechanistic insight in dodecin function by applying spectroscopic and electrochemical methods. Intriguingly, we reveal a significant pH dependence in the affinity and specificity of flavin binding. Our data give insight in M. tuberculosis dodecin function and advance the current understanding of dodecins as flavin storage and sequestering proteins. We suggest that the dodecin in M. tuberculosis may specifically be important for flavin homeostasis during the elaborate lifestyle of this organism, which calls for the evaluation of this protein as drug target.

The UbiX-UbiD system: The biosynthesis and use of prenylated flavin (prFMN).

The UbiX-UbiD system consists of the flavin prenyltransferase UbiX that produces prenylated FMN that serves as the cofactor for the (de)carboxylase UbiD. Recent developments have provided structural insights into the mechanism of both enzymes, detailing unusual chemistry in each case. The proposed reversible 1,3-dipolar cycloaddition between the cofactor and substrate serves as a model to explain many of the key UbiD family features. However, considerable variation exists in the many branches of the UbiD family tree.

UbiX is a flavin prenyltransferase required for bacterial ubiquinone biosynthesis.

Ubiquinone (also known as coenzyme Q) is a ubiquitous lipid-soluble redox cofactor that is an essential component of electron transfer chains. Eleven genes have been implicated in bacterial ubiquinone biosynthesis, including ubiX and ubiD, which are responsible for decarboxylation of the 3-octaprenyl-4-hydroxybenzoate precursor. Despite structural and biochemical characterization of UbiX as a flavin mononucleotide (FMN)-binding protein, no decarboxylase activity has been detected. Here we report that UbiX produces a novel flavin-derived cofactor required for the decarboxylase activity of UbiD. UbiX acts as a flavin prenyltransferase, linking a dimethylallyl moiety to the flavin N5 and C6 atoms. This adds a fourth non-aromatic ring to the flavin isoalloxazine group. In contrast to other prenyltransferases, UbiX is metal-independent and requires dimethylallyl-monophosphate as substrate. Kinetic crystallography reveals that the prenyltransferase mechanism of UbiX resembles that of the terpene synthases. The active site environment is dominated by π systems, which assist phosphate-C1' bond breakage following FMN reduction, leading to formation of the N5-C1' bond. UbiX then acts as a chaperone for adduct reorientation, via transient carbocation species, leading ultimately to formation of the dimethylallyl C3'-C6 bond. Our findings establish the mechanism for formation of a new flavin-derived cofactor, extending both flavin and terpenoid biochemical repertoires.

New cofactor supports α,ß-unsaturated acid decarboxylation via 1,3-dipolar cycloaddition.

The bacterial ubiD and ubiX or the homologous fungal fdc1 and pad1 genes have been implicated in the non-oxidative reversible decarboxylation of aromatic substrates, and play a pivotal role in bacterial ubiquinone (also known as coenzyme Q) biosynthesis or microbial biodegradation of aromatic compounds, respectively. Despite biochemical studies on individual gene products, the composition and cofactor requirement of the enzyme responsible for in vivo decarboxylase activity remained unclear. Here we show that Fdc1 is solely responsible for the reversible decarboxylase activity, and that it requires a new type of cofactor: a prenylated flavin synthesized by the associated UbiX/Pad1. Atomic resolution crystal structures reveal that two distinct isomers of the oxidized cofactor can be observed, an isoalloxazine N5-iminium adduct and a N5 secondary ketimine species with markedly altered ring structure, both having azomethine ylide character. Substrate binding positions the dipolarophile enoic acid group directly above the azomethine ylide group. The structure of a covalent inhibitor-cofactor adduct suggests that 1,3-dipolar cycloaddition chemistry supports reversible decarboxylation in these enzymes. Although 1,3-dipolar cycloaddition is commonly used in organic chemistry, we propose that this presents the first example, to our knowledge, of an enzymatic 1,3-dipolar cycloaddition reaction. Our model for Fdc1/UbiD catalysis offers new routes in alkene hydrocarbon production or aryl (de)carboxylation.

The flavoproteome of the yeast Saccharomyces cerevisiae.

Genome analysis of the yeast Saccharomyces cerevisiae identified 68 genes encoding flavin-dependent proteins (1.1% of protein encoding genes) to which 47 distinct biochemical functions were assigned. The majority of flavoproteins operate in mitochondria where they participate in redox processes revolving around the transfer of electrons to the electron transport chain. In addition, we found that flavoenzymes play a central role in various aspects of iron metabolism, such as iron uptake, the biogenesis of iron-sulfur clusters and insertion of the heme cofactor into apocytochromes. Another important group of flavoenzymes is directly (Dus1-4p and Mto1p) or indirectly (Tyw1p) involved in reactions leading to tRNA-modifications. Despite the wealth of genetic information available for S. cerevisiae, we were surprised that many flavoproteins are poorly characterized biochemically. For example, the role of the yeast flavodoxins Pst2p, Rfs1p and Ycp4p with regard to their electron donor and acceptor is presently unknown. Similarly, the function of the heterodimeric Aim45p/Cir1p, which is homologous to the electron-transferring flavoproteins of higher eukaryotes, in electron transfer processes occurring in the mitochondrial matrix remains to be elucidated. This lack of information extends to the five membrane proteins involved in riboflavin or FAD transport as well as FMN and FAD homeostasis within the yeast cell. Nevertheless, several yeast flavoproteins, were identified as convenient model systems both in terms of their mechanism of action as well as structurally to improve our understanding of diseases caused by dysfunctional human flavoprotein orthologs.

Activation of genes involved in xenobiotic metabolism is a shared signature of mouse models with extended lifespan.

Xenobiotic metabolism has been proposed to play a role in modulating the rate of aging. Xenobiotic metabolizing enzymes (XME) are expressed at higher levels in calorically restricted mice (CR) and in GH/IGF-I-deficient, long-lived mutant mice. In this study, we show that many phase I XME genes are similarly upregulated in additional long-lived mouse models, including "crowded litter" (CL) mice, whose lifespan has been increased by food restriction limited to the first 3 wk of life, and in mice treated with rapamycin. Induction in the CL mice lasts at least through 22 mo of age, but induction by rapamycin is transient for many of the mRNAs. Cytochrome P-450s, flavin monooxygenases, hydroxyacid oxidase, and metallothioneins were found to be significantly elevated in similar proportions in each of the models of delayed aging tested, whether these were based on mutation, diet, drug treatment, or transient early intervention. The same pattern of mRNA elevation could be induced by 2 wk of treatment with tert-butylhydroquinone, an oxidative toxin known to activate Nrf2-dependent target genes. These results suggest that elevation of phase I XMEs is a hallmark of long-lived mice and may facilitate screens for agents worth testing in intervention-based lifespan studies.

Genetic control of biosynthesis and transport of riboflavin and flavin nucleotides and construction of robust biotechnological producers.

Riboflavin [7,8-dimethyl-10-(1'-d-ribityl)isoalloxazine, vitamin B2] is an obligatory component of human and animal diets, as it serves as the precursor of flavin coenzymes, flavin mononucleotide, and flavin adenine dinucleotide, which are involved in oxidative metabolism and other processes. Commercially produced riboflavin is used in agriculture, medicine, and the food industry. Riboflavin synthesis starts from GTP and ribulose-5-phosphate and proceeds through pyrimidine and pteridine intermediates. Flavin nucleotides are synthesized in two consecutive reactions from riboflavin. Some microorganisms and all animal cells are capable of riboflavin uptake, whereas many microorganisms have distinct systems for riboflavin excretion to the medium. Regulation of riboflavin synthesis in bacteria occurs by repression at the transcriptional level by flavin mononucleotide, which binds to nascent noncoding mRNA and blocks further transcription (named the riboswitch). In flavinogenic molds, riboflavin overproduction starts at the stationary phase and is accompanied by derepression of enzymes involved in riboflavin synthesis, sporulation, and mycelial lysis. In flavinogenic yeasts, transcriptional repression of riboflavin synthesis is exerted by iron ions and not by flavins. The putative transcription factor encoded by SEF1 is somehow involved in this regulation. Most commercial riboflavin is currently produced or was produced earlier by microbial synthesis using special selected strains of Bacillus subtilis, Ashbya gossypii, and Candida famata. Whereas earlier RF overproducers were isolated by classical selection, current producers of riboflavin and flavin nucleotides have been developed using modern approaches of metabolic engineering that involve overexpression of structural and regulatory genes of the RF biosynthetic pathway as well as genes involved in the overproduction of the purine precursor of riboflavin, GTP.

Candida albicans Hap43 is a repressor induced under low-iron conditions and is essential for iron-responsive transcriptional regulation and virulence.

Candida albicans is an opportunistic fungal pathogen that exists as normal flora in healthy human bodies but causes life-threatening infections in immunocompromised patients. In addition to innate and adaptive immunities, hosts also resist microbial infections by developing a mechanism of "natural resistance" that maintains a low level of free iron to restrict the growth of invading pathogens. C. albicans must overcome this iron-deprived environment to cause infections. There are three types of iron-responsive transcriptional regulators in fungi; Aft1/Aft2 activators in yeast, GATA-type repressors in many fungi, and HapX/Php4 in Schizosaccharomyces pombe and Aspergillus species. In this study, we characterized the iron-responsive regulator Hap43, which is the C. albicans homolog of HapX/Php4 and is repressed by the GATA-type repressor Sfu1 under iron-sufficient conditions. We provide evidence that Hap43 is essential for the growth of C. albicans under low-iron conditions and for C. albicans virulence in a mouse model of infection. Hap43 was not required for iron acquisition under low-iron conditions. Instead, it was responsible for repression of genes that encode iron-dependent proteins involved in mitochondrial respiration and iron-sulfur cluster assembly. We also demonstrated that Hap43 executes its function by becoming a transcriptional repressor and accumulating in the nucleus in response to iron deprivation. Finally, we found a connection between Hap43 and the global corepressor Tup1 in low-iron-induced flavinogenesis. Taken together, our data suggest a complex interplay among Hap43, Sfu1, and Tup1 to coordinately regulate iron acquisition, iron utilization, and other iron-responsive metabolic activities.

Secretion of flavins by three species of methanotrophic bacteria.

We detected flavins in the growth medium of the methanotrophic bacterium Methylocystis species strain M. Flavin secretion correlates with growth stage and increases under iron starvation conditions. Two other methanotrophs, Methylosinus trichosporium OB3b and Methylococcus capsulatus (Bath), secrete flavins, suggesting that flavin secretion may be common to many methanotrophic bacteria.

A key enzyme for flavin synthesis is required for nitric oxide and reactive oxygen species production in disease resistance.

Nitric oxide (NO) and reactive oxygen species (ROS) play key roles in plant immunity. However, the regulatory mechanisms of the production of these radicals are not fully understood. Hypersensitive response (HR) cell death requires the simultaneous and balanced production of NO and ROS. In this study we indentified NbRibAencoding a bifunctional enzyme, guanosine triphosphate cyclohydrolase II/3,4-dihydroxy-2-butanone-4-phosphate synthase, which participates in the biosynthesis of flavin, by screening genes related to mitogen-activated protein kinase-mediated cell death, using virus-induced gene silencing. Levels of endogenous riboflavin and its derivatives, flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD), which are important prosthetic groups for several enzymes participating in redox reactions, decreased in NbRibA-silenced Nicotiana benthamiana. Silencing NbRibA compromised not only HR cell death, but also the NO and ROS production induced by INF1 elicitin and a constitutively active form of NbMEK2 (NbMEK2DD), and also induced high susceptibility to oomycete Phytophthora infestans and ascomycete Colletotrichum orbiculare. Compromised radical production and HR cell death induced by INF1 in NbRibA-silenced leaves were rescued by adding riboflavin, FMN or FAD. These results indicate that flavin biosynthesis participates in regulating NO and ROS production, and HR cell death.

RibR, a possible regulator of the Bacillus subtilis riboflavin biosynthetic operon, in vivo interacts with the 5'-untranslated leader of rib mRNA.

RibR is a minor cryptic flavokinase (EC 2.7.1.26) of the Gram-positive bacterium Bacillus subtilis with an unknown cellular function. The flavokinase activity appears to be localized to the N-terminal domain of the protein. Using the yeast three-hybrid system, it was shown that RibR specifically interacts in vivo with the nontranslated wild-type leader of the mRNA of the riboflavin biosynthetic operon. This interaction is lost partially when a leader containing known cis-acting deregulatory mutations in the so-called RFN element is tested. The RFN element is a sequence within the rib-leader mRNA reported to serve as a receptor for an FMN-dependent 'riboswitch'. In RibR itself, interaction was localized to the carboxy-terminate part of the protein, a segment of unknown function that does not show similarity to other proteins in the public databases. Analysis of a ribR-defective strain revealed a mild deregulation with respect to flavin (riboflavin, FMN and FAD) biosynthesis. The results indicate that the RNA-binding protein RibR may be involved in the regulation of the rib genes.

Kinetic and spectroscopic characterization of type II isopentenyl diphosphate isomerase from Thermus thermophilus: evidence for formation of substrate-induced flavin species.

Type II isopentenyl diphosphate (IPP) isomerase catalyzes the interconversion of IPP and dimethylallyl diphosphate (DMAPP). Although the reactions catalyzed by the type II enzyme and the well-studied type I IPP isomerase are identical, the type II protein requires reduced flavin for activity. The chemical mechanism, including the role of flavin, has not been established for type II IPP isomerase. Recombinant type II IPP isomerase from Thermus thermophilus HB27 was purified by Ni2+ affinity chromatography. The aerobically purified enzyme was inactive until the flavin cofactor was reduced by NADPH or dithionite or photochemically. The inactive oxidized flavin-enzyme complex bound IPP in a Mg2+-dependent manner for which KD approximately KmIPP, suggesting that the substrate binds to the inactive oxidized and active reduced forms of the protein with similar affinities. N,N-Dimethyl-2-amino-1-ethyl diphosphate (NIPP), a transition state analogue for the type I isomerase, competitively inhibits the type II enzyme, but with a much lower affinity. pH-dependent spectral changes indicate that the binding of IPP, DMAPP, and a saturated analogue isopentyl diphosphate promotes protonation of anionic reduced flavin. Electron paramagnetic resonance (EPR) and UV-visible spectroscopy show a substrate-dependent accumulation of the neutral flavin semiquinone during both the flavoenzyme reduction and reoxidation processes in the presence of IPP and related analogues. Redox potentials of IPP-bound enzyme indicate that the neutral semiquinone state of the flavin is stabilized thermodynamically relative to free FMN in solution.

Blue light perception in plants. Detection and characterization of a light-induced neutral flavin radical in a C450A mutant of phototropin.

The LOV2 domain of Avena sativa phototropin and its C450A mutant were expressed as recombinant fusion proteins and were examined by optical spectroscopy, electron paramagnetic resonance, and electron-nuclear double resonance. Upon irradiation (420-480 nm), the LOV2 C450A mutant protein gave an optical absorption spectrum characteristic of a flavin radical even in the absence of exogenous electron donors, thus demonstrating that the flavin mononucleotide (FMN) cofactor in its photogenerated triplet state is a potent oxidant for redox-active amino acid residues within the LOV2 domain. The FMN radical in the LOV2 C450A mutant is N(5)-protonated, suggesting that the local pH close to the FMN is acidic enough so that the cysteine residue in the wild-type protein is likely to be also protonated. An electron paramagnetic resonance analysis of the photogenerated FMN radical gave information on the geometrical and electronic structure and the environment of the FMN cofactor. The experimentally determined hyperfine couplings of the FMN radical point to a highly restricted delocalization of the unpaired electron spin in the isoalloxazine moiety. In the light of these results a possible radical-pair mechanism for the formation of the FMN-C(4a)-cysteinyl adduct in LOV domains is discussed.

Stimulated biosynthesis of flavins in Photobacterium phosphoreum IFO 13896 and the presence of complete rib operons in two species of luminous bacteria.

Photobacterium phosphoreum IFO 13896 emits light strongly when cultured in medium containing 3% NaCl, but only weakly in medium containing 1% NaCl. It is known that dim or dark mutants appear frequently and spontaneously from this parent strain. To confirm that riboflavin biosynthesis is stimulated when the lux operon is active, the amount of light emitted and flavins synthesized under strongly or weakly light emitting conditions was determined. In comparison with the parent strain cultured in 3% NaCl, the same strain cultured in 1% NaCl emitted 1/36 the light and produced 1/4 the flavins, while three dim or dark mutants, M1, M2 and M3 cultured in 3% NaCl, emitted almost no light, 1/58 the light and 1/10 the light and produced 1/8, 1/5 and 1/3 the amount of flavins, respectively. From these results, we deduced that the genes for riboflavin synthesis, rib genes, are organized in an operon in this strain. In P. phosphoreum NCMB 844, it has been reported that a rib gene cluster is present just downstream of the lux operon. However, among rib genes, the gene for pyrimidine deaminase/pyrimidine reductase, ribD, was not found in this cluster. Because a complete rib operon seems to be necessary for efficient regulation at the transcriptional level, we expected ribD to be present downstream of this cluster and sequenced this region, using SUGDAT, Sequencing Using Genomic DNA As a Template. We could not find this gene but found a gene for hybrid-cluster protein (prismane protein). To find ribD in a different region, a partial ribD sequence was amplified and sequenced using a PCR-based method, and subsequently the genomic DNA was sequenced in both directions from this partial sequence using SUGDAT. Because ribC was found just downstream of ribD, we sequenced further downstream of ribC and confirmed that another complete set of rib genes, ribD, ribC, ribBA, and ribE, is present in P. phosphoreum. The presence of a complete rib operon in P. phosphoreum explains why this species can synthesize flavins at enhanced levels to sustain a strong light emission. Furthermore, we sequenced the rib operon in Vibrio fischeri, another representative luminous bacterium, in a manner similar to that described above, and confirmed that a complete operon is present also in this species. The organization of rib genes in an operon in the Proteobacteria gamma-subdivision is discussed.

Influence of type and concentration of flavinogenic factors on production of riboflavin by Eremothecium ashbyii NRRL 1363.

A study was conducted to determine the effect of various low cost organic wastes as flavinogenic factors and the various concentrations at which they induced flavinogenecity resulting in higher yields of riboflavin. A high-yielding riboflavin strain; Eremothecium ashbyii NRRL 1363 was chosen to determine the flavinogenicity. Carbon source at 50 g l(-1) (dextrose equivalents) of molasses and nitrogen source at 50 g l(-1) (weight/volume) of peanut seed cake were found to be optimal levels to yield higher riboflavin. Among the organic wastes, (beef extract, hog casings, blood meal, fish meal) hog casings in association with fish meal supported the highest yield of riboflavin. Among the different recovery processes studied, a vacuum drying process was the most efficient allowing maximum yield, followed by drying at 90 degrees C and freeze-drying. It is apparent from this study that inexpensive or waste organic materials could induce E. ashbyii to synthesize and secrete riboflavin at higher levels in the medium and this could be purified using a vacuum drying process. This bioconversion process allows us to recycle the biomaterials and produce a value-added product of economic importance.

Cloning, sequencing, and tissue-dependent expression of flavin-containing monooxygenase (FMO) 1 and FMO3 in the dog.

The expression of flavin-containing monooxygenases (FMOs) in dog liver microsomes was suggested by a high methimazole S-oxidase activity. When the reaction was catalyzed by dog liver microsomes, apparent V(max) and K(m) values were 6.3 nmol/min/mg and 14 microM, respectively. This reaction was highly inhibited (73%) in the presence of imipramine, but it was also weakly affected by trimethylamine, suggesting the involvement of different isoforms. The sequences of dog FMO1 and FMO3 were obtained by reverse transcription-polymerase chain reaction and 5'/3' terminal extension. The cDNAs of dog FMO1 and dog FMO3 encode proteins of 532 amino acids, which contain the NADPH- and FAD-binding sites. The dog FMO1 amino acid sequence is 88, 86, and 89% identical to sequences of human, rabbit, and pig FMO1, respectively. The dog FMO3 amino acid sequence is 83, 84, and 82% identical to sequences of human, rabbit, and rat FMO3, respectively. Dog FMO1 and dog FMO3 exhibited only 56% identities. The FMO1 and FMO3 recombinant proteins and the FMO1 and FMO3 microsomal proteins migrated with the same mobility (56 kDa), as determined in SDS-polyacrylamide gel electrophoresis and immunoblotting. By Western blotting, dog FMO1 and dog FMO3 were detected in microsomes from liver and lung but not in kidney microsomes. By Northern blotting, the probe for FMO1 specifically hybridized a 2.6-kilobase (kb) transcript in liver and lung samples only. The probe for FMO3 hybridized two transcripts of approximately 3 and 4.2 kb in the liver and lung samples.

L-Mandelate dehydrogenase from Rhodotorula graminis: cloning, sequencing and kinetic characterization of the recombinant enzyme and its independently expressed flavin domain.

The l-mandelate dehydrogenase (L-MDH) from the yeast Rhodotorula graminis is a mitochondrial flavocytochrome b2 which catalyses the oxidation of mandelate to phenylglyoxylate coupled with the reduction of cytochrome c. We have used the N-terminal sequence of the enzyme to isolate the gene encoding this enzyme using the PCR. Comparison of the genomic sequence with the sequence of cDNA prepared by reverse transcription PCR revealed the presence of 11 introns in the coding region. The predicted amino acid sequence indicates a close relationship with the flavocytochromes b2 from Saccharomyces cerevisiae and Hansenula anomala, with about 40% identity to each. The sequence shows that a key residue for substrate specificity in S. cerevisiae flavocytochrome b2, Leu-230, is replaced by Gly in L-MDH. This substitution is likely to play an important part in determining the different substrate specificities of the two enzymes. We have developed an expression system and purification protocol for recombinant L-MDH. In addition, we have expressed and purified the flavin-containing domain of L-MDH independently of its cytochrome domain. Detailed steady-state and pre-steady-state kinetic investigations of both L-MDH and its independently expressed flavin domain have been carried out. These indicate that L-MDH is efficient with both physiological (cytochrome c, kcat=225 s-1 at 25 degrees C) and artificial (ferricyanide, kcat=550 s-1 at 25 degrees C) electron acceptors. Kinetic isotope effects with [2-2H]mandelate indicate that H-C-2 bond cleavage contributes somewhat to rate-limitation. However, the value of the isotope effect erodes significantly as the catalytic cycle proceeds. Reduction potentials at 25 degrees C were measured as -120 mV for the 2-electron reduction of the flavin and -10 mV for the 1-electron reduction of the haem. The general trends seen in the kinetic studies show marked similarities to those observed previously with the flavocytochrome b2 (L-lactate dehydrogenase) from S. cerevisiae.

Differential expression and activity of flavin-containing monooxygenases in euryhaline and stenohaline flatfishes indicates potential osmoregulatory role.

N,N-Dimethylaniline (DMA) N-oxidase activity indicative of flavin-containing monooxygenase (FMO) was examined in four tissues (liver, gill, muscle, and kidney) of the flounder (Platichthys flesus). Gill microsomes had the highest levels of activity (456 +/- 343 pmol/min/mg), while kidney (121 +/- 109) and liver (67 +/- 26) had levels just above detection. A single faint band of a 56 kD protein was observed in liver and gill microsomes following Western blot analyses with polyclonal antibodies to FMO 1. DMA N-oxidase activity in gill and liver directly correlated with the expression of the 56 kD protein recognized by polyclonal antibodies against FMO form 1. Likewise a mRNA band of approximately 2.5 kilobases was higher in gill than a 3.0 kb band in liver following hydridization with an FMO 1 cDNA probe. Gill and liver microsomal DMA N-oxidase from the euryhaline P. flesus was compared with that of the stenohaline turbot (Scophthalmus maximus). DMA oxidase activity, FMO protein and mRNA were significantly greater in the gill of P. flesus, while S. maximus had higher levels of enzyme activity in the liver, but also significant levels in gill. Comparison of the enzymatic properties of the P. flesus gill and S. maximus liver enzymes indicated dramatic differences in Km between gill and liver, but were both inhibited by equimolar concentrations of trimethylamine (TMA). Gill microsomal activity in each species was unaffected by the mammalian FMO 2 substrate (competitive inhibitor) n-octylamine. Differential expression of FMO in tissues from stenohaline and euryhaline fish suggests a functional relationship between FMO and osmoregulation.