Tetrazolium-dye-linked alcohol dehydrogenase of the methylotrophic actinomycete Amycolatopsis methanolica is a three-component complex.

Tetrazolium-dye-linked alcohol dehydrogenase (TD-ADH) of Amycolatopsis methanolica could be resolved into three protein components, which have been purified. Each of the components has the ability to reconstitute TD-ADH activity when combined with the other two. Component 1 is identical to the previously characterized methanol:N,N'-dimethyl-4-nitrosoaniline oxidoreductase (MNO), a decameric protein with 50-kDa subunits, each carrying a tightly bound NADPH. Component 2 is a high molecular mass (> 640 kDa) protein with subunits of 44 kDa and 72 kDa, and which possesses a low tetrazolium-dye-linked NADH dehydrogenase activity. The protein contains a yellow chromophore of unknown identity. Component 3 is a low molecular mass (15 kDa) protein containing a 5'-deazaflavin and at least one other low-molecular-mass compound with properties similar, but not identical, to those of nicotinamide coenzymes. The results suggest that alcohol oxidation by the TD-ADH complex is carried out by component 1 (MNO), after which transfer of the reducing equivalents (mediated by component 3) occurs to component 2, which (in vitro) is linked to the tetrazolium dye. Fractionation of A. methanolica extracts showed that most of the 5'-deazaflavin was present in component 3. Other gram-positive bacteria having a TD-ADH complex also produced 5'-deazaflavin. It is concluded that oxidation of primary aliphatic alcohols by A. methanolica, and probably also by other gram-positive bacteria containing MNO or TD-ADH, proceeds via TD-ADH. The likeliness of 5'-deazaflavin participation in this process is discussed.

Properties of an NAD(H)-containing methanol dehydrogenase and its activator protein from Bacillus methanolicus.

Oxidation of C1-C4 primary alcohols in thermotolerant Bacillus methanolicus strains is catalyzed by an NAD-dependent methanol dehydrogenase (MDH), composed of ten identical 43,000-Mr subunits. Each MDH subunit contains a tightly, but non-covalently, bound NAD(H) molecule, in addition to 1 Zn2+ and 1-2 Mg2+ ions. The NAD(H) cofactor is oxidized and reduced by formaldehyde and methanol, respectively, while it remains bound to the enzyme. Incubation of MDH with methanol and exogenous NAD (coenzyme) results in reduction of this NAD coenzyme. Both NAD species are not exchanged during catalysis. NAD thus plays two different and important roles in the MDH-catalyzed reaction, with the bound NAD cofactor acting as primary electron acceptor and the NAD coenzyme being responsible for reoxidation of the reduced cofactor. MDH obeys a ping-pong type reaction mechanism, which is consistent with such a temporary parking of reducing equivalents at the MDH-bound cofactor. Spectral studies show that, in the presence of exogenous NAD and Mg2+ ions, MDH interacts with a previously identified 50,000-Mr activator protein. The activator protein appears to facilitate the oxidation of the reduced NADH cofactor of MDH, which results in a strongly increased turnover rate of MDH.

Production of actinorhodin-related "blue pigments" by Streptomyces coelicolor A3(2).

The genetically well-known strain Streptomyces coelicolor A3(2) produces the pH indicator (red/blue) antibiotic actinorhodin, but not all the "blue pigment" produced by this strain is actinorhodin. When the organism was subjected to various nutrient limitations (ammonium, nitrate, phosphate, or trace elements), and also during growth cessation caused by a relatively low medium pH, blue pigment production was initiated but the pigment and its location varied. At pH 4.5 to 5.5, significant formation of actinorhodin occurred and was located exclusively intracellularly. At pH 6.0 to 7.5 a different blue pigment was produced intracellularly as well as extracellularly. It was purified and identified as gamma-actinorhodin (the lactone form of actinorhodin). Analysis of act mutants of S. coelicolor A3(2) confirmed that both pigments are derived from the act biosynthetic pathway. Mutants with lesions in actII-ORF2, actII-ORF3, or actVA-ORF1, previously implicated or suggested to be involved in actinorhodin export, were impaired in production of gamma-actinorhodin, suggesting that synthesis of gamma-actinorhodin from actinorhodin is coupled to its export from the cell. However, effects on the level of actinorhodin production were also found in some mutants.

Electron microscopic analysis and structural characterization of novel NADP(H)-containing methanol: N,N'-dimethyl-4-nitrosoaniline oxidoreductases from the gram-positive methylotrophic bacteria Amycolatopsis methanolica and Mycobacterium gastri MB19.

The quaternary protein structure of two methanol:N,N'-dimethyl-4-nitrosoaniline (NDMA) oxidoreductases purified from Amycolatopsis methanolica and Mycobacterium gastri MB19 was analyzed by electron microscopy and image processing. The enzymes are decameric proteins (displaying fivefold symmetry) with estimated molecular masses of 490 to 500 kDa based on their subunit molecular masses of 49 to 50 kDa. Both methanol:NDMA oxidoreductases possess a tightly but noncovalently bound NADP(H) cofactor at an NADPH-to-subunit molar ratio of 0.7. These cofactors are redox active toward alcohol and aldehyde substrates. Both enzymes contain significant amounts of Zn2+ and Mg2+ ions. The primary amino acid sequences of the A. methanolica and M. gastri MB19 methanol:NDMA oxidoreductases share a high degree of identity, as indicated by N-terminal sequence analysis (63% identity among the first 27 N-terminal amino acids), internal peptide sequence analysis, and overall amino acid composition. The amino acid sequence analysis also revealed significant similarity to a decameric methanol dehydrogenase of Bacillus methanolicus C1.

Dye-linked dehydrogenase activities for formate and formate esters in Amycolatopsis methanolica. Characterization of a molybdoprotein enzyme active with formate esters and aldehydes.

Cell-free extracts of methanol-grown Amycolatopsis methanolica contain dye-linked dehydrogenase activities for formate and methyl formate. Fractionation of the extracts revealed that the (unstable) activity for formate resides in membrane particles, while that for methyl formate belongs to a soluble enzyme that was purified and characterized. The enzyme, indicated as formate-ester dehydrogenase, appeared to be a molybdoprotein (4 Fe, 3 or 4 S, 1 Mo and 1 FAD were found for each enzyme molecule), with a molecular mass of 186 kDa and consisting of two subunits of equal size. Product identification suggests that the formate moiety in the ester becomes hydroxylated to a carbonate group after which the unstable alkyl carbonate decomposes into CO2 and the alcohol moiety. Based on structural and catalytic characteristics, the enzyme appears to be very similar to an enzyme isolated from Comamonas testosteroni [Poels, P. A., Groen, B. W. & Duine, J. A. (1987) Eur. J. Biochem. 166, 575-579] which was at that time considered to be an aldehyde dehydrogenase. Formate-ester dehydrogenase activity appeared to be present in several other bacteria. Possible roles for the A. methanolica enzyme in C1 dissimilation (oxidation of methyl formate to methanol and CO2 or a factor-formate adduct to factor plus CO2) or in general aldehyde oxidation, are discussed.

Modification of flavin adenine dinucleotide in alcohol oxidase of the yeast Hansenula polymorpha.

Alcohol oxidase, a major peroxisomal protein of methanol-utilizing yeasts, may possess two different forms of flavin adenine dinucleotide, classical FAD and so-called modified FAD (mFAD). Conversion of FAD into mFAD was observed both in purified preparations of the enzyme and in cells grown in batch and continuous culture. The relative amount of mFAD in the enzyme varied from 5 to 95%, depending on the growth or storage conditions. The presence of mFAD led to a slight decrease in Vmax and a significant (about one order) decrease in the Km of alcohol oxidase with respect to methanol. The kinetics of modification measured in purified preparations of the enzyme obeyed first-order kinetics (k = 0.78 h-1). The modification process was strongly inhibited by methanol, formaldehyde or hydroxylamine. Modification observed in continuous culture under steady state conditions depended on the dilution rate and could also be described as a spontaneous first-order reaction (kapp = 0.27 h-1). FAD modification could only be detected in alcohol oxidase and not in other yeast peroxisomal flavoenzymes, such as D-amino acid oxidase from Candida boidinii.

Production of metabolites by methylotrophic yeasts.

Recent advances in biotechnology of methanol-utilizing yeasts are briefly summarized. The emphasis is given to production of some fine and commercial chemicals such as formaldehyde, formate, hydrogen peroxide, dihydroxyacetone, ATP, FAD as well as proteins, specifically alcohol oxidase. The advantages of mutants and recombinants derived from methylotrophic yeasts for efficient production of various useful materials are demonstrated.

Alcohol oxidase of methylotrophic thermo- and acidotolerant yeast Hansenula sp.

Electrophoretic analysis of alcohol oxidase purified from the methylotrophic thermo- and acidotolerant yeast Hansenula sp. revealed the presence of two active forms of the enzyme with molar mass 440 kg/mol (major component) and 724 kg/mol (minor component). A subunit M of the enzyme was found to be 72 kg/mol. Two active forms of the enzyme found by electrophoresis seem to be caused by dissociation of the octameric form to the tetramer under alkaline conditions. Studies of alcohol oxidase showed a kinetic variability of the enzyme with respect to its Km. It is proposed that the variability of Km is caused by enzyme binding to formaldehyde.

Transformation of methylotrophic yeast Hansenula polymorpha: cloning and expression of genes.

We developed a host-vector system for transformation and gene cloning experiments using the methylotrophic yeast Hansenula polymorpha. Regeneration of protoplasts in a medium containing polyethylene glycol before plating made transformation more efficient and reproducible (2 to 3 x 10(4) micrograms DNA). The frequency of transformation was significantly lower when dominant resistance marker Cup1r was used for transformant selection. The transformation system developed was used to clone the DNA fragment which complements functionally the defect in the dihydroxyacetone kinase (DHAK*) activity of a H. polymorpha mutant strain. The DNA insert isolated was shown to increase by up to ten times the activity of DHAK in transformants carrying recombinant plasmids. When recombinant plasmids were introduced into S. cerevisiae, the transformants obtained acquired the ability to grow on the medium with dihydroxyacetone as a sole carbon source and the activity of DHAK was observed.

[Purification and several properties of dihydroxyacetone kinase from the methylotrophic yeast Candida boidinii]. / Ochistka i nekotorye svoistva dioksiatsetonkinazy metilotrofnykh drozhzhei Candida boidinii.

A procedure for isolation of a homogeneous dihydroxyacetone kinase including fractionation by polyethylene glycol and ion-exchange chromatography on polyethylenimine-Biogel has been developed. The enzyme is a dimer with Mr = 139 000 (2.71 000 according to SDS disc electrophoresis) and has a pI of 4.64 and pH optimum of 7.8-8.2. The enzyme phosphorylates dihydroxyacetone and, in a lesser degree, glyceraldehyde. ATP is the most efficient phosphate group donor for the enzyme. When ITP, GTP, CTP and UTP are used, the dihydroxyacetone kinase activity is about 30%.

[Glutathione participation in the regulation of methanol metabolism in yeasts]. / Uchastie glutationa v reguliatsii metabolizma metanola u drozhzhei.

The activity of enzymes involved in methanol oxidation and assimilation as well as the levels of formaldehyde and glutathione were determined during batch cultivation of Candida boidinii KD1 in a medium with methanol. The distribution of [14C]methanol between oxidative and biosynthetic processes in the yeast was analysed. Changes in the concentrations of formaldehyde and glutathione were found to correlate with the activity of formaldehyde dehydrogenase. The results indicate that an increase in the concentration of reduced glutathione (GSH) at the early logarithmic phase of the yeast growth stimulates formaldehyde oxidation via formate to carbon dioxide whereas a subsequent decrease in the concentration of GSH favours formaldehyde assimilation.

[Dihydroxyacetone synthase from the methanol-utilizing yeast Candida boidinii]. / Dioksiatsetonsintaza metilotrofnykh drozhzhei Candida boidinii.

A procedure for purification of dihydroxyacetone synthase catalyzing the formation of dihydroxyacetone and glyceraldehyde 3-phosphate from formaldehyde and xylulose 5-phosphate has been developed. Using ion-exchangers with increasing affinity for dihydroxyacetone synthase, a homogenous preparation of the enzyme with specific activity of 2 u./mg has been obtained. The enzyme is made up of 2 subunits with m. w. of 76,000, contains thiamine pyrophosphate, requires Mg2+ for its activity and differs from yeast transketolase by substrate specificity and some other properties. The role of dihydroxyacetone synthase in metabolism of methanol-utilizing yeasts is discussed.