Site-directed Mutagenesis of Two Zinc-binding Centers of the NADH-dependent Phenylacetaldehyde Reductase from Styrene-assimilating Corynebacterium sp. Strain ST-10

Phenylacetaldehyde reductase (PAR) with a unique and wide substrate range from styrene-assimilating Corynebacterium sp. strain ST-10, which is a useful biocatalyst producing chiral alcohols, has been found to belong to a family of zinc-containing, long-chain alcohol dehydrogenases (ADHs) on the basis of the primary structure similarity. The enzyme contains 2 moles of zinc per mole of subunit. The amino acid residues assumed to be three catalytic and four structural zinc-binding ligands were characterized by site-directed mutagenesis, compared with other zinc-containing, long-chain ADHs. Sixteen PAR mutants gave measurable but rather low activities toward phenylacetaldehyde, n-hexyl aldehyde, and 2-heptanone, although they maintained the activities of 8 to 16% of that of wild-type PAR for an acetophenone substrate except that the D153N mutant showed quite low activity. The results suggested that the seven residues present in PAR were probably zinc-binding ligands, and mutation in these residues caused a change in activities for some substrates.     

Artificial redox coenzymes: biomimetic analogues of NAD+

A range of biomimetic analogues of the nicotinamide nucleotide coenzymes NAD(P)(H) have been developed based on the structure of a triazine dye template. These biomimetic redox coenzymes are relatively straightforward and inexpensive to synthesise and display NAD⁺-like activity with different dehydrogenases, despite their apparently minimal structural similarity to the native coenzyme NAD⁺. Horse liver alcohol dehydrogenase oxidises butan-1-ol, using the most active biomimetic coenzyme (Nap 1), with a k _cat value an order of magnitude lower and a K _m for the coenzyme two orders of magnitude higher than those using native NAD⁺. The enzymatically reduced biomimetic coenzymes may be reoxidised by phenazine methosulfate. We believe that these coenzymes may find applications in biotransformations and biosensors, and in the development of biomimetic catalysts where the redox enzyme itself is replaced by a synthetic binding site. Received: 26 October 1998 / Received revision: 25 January 1999 / Accepted: 31 January 1999     

Site-directed mutagenesis of two zinc-binding centers of the NADH-dependent phenylacetaldehyde reductase from styrene-assimilating Corynebacterium sp. strain ST-10

Phenylacetaldehyde reductase (PAR) with a unique and wide substrate range from styrene-assimilating Corynebacterium sp. strain ST-10, which is a useful biocatalyst producing chiral alcohols, has been found to belong to a family of zinc-containing, long-chain alcohol dehydrogenases (ADHs) on the basis of the primary structure similarity. The enzyme contains 2 moles of zinc per mole of subunit. The amino acid residues assumed to be three catalytic and four structural zinc-binding ligands were characterized by site-directed mutagenesis, compared with other zinc-containing, long-chain ADHs. Sixteen PAR mutants gave measurable but rather low activities toward phenylacetaldehyde, n-hexyl aldehyde, and 2-heptanone, although they maintained the activities of 8 to 16% of that of wild-type PAR for an acetophenone substrate except that the D153N mutant showed quite low activity. The results suggested that the seven residues present in PAR were probably zinc-binding ligands, and mutation in these residues caused a change in activities for some substrates.     

Enzymic and Structural Studies on Drosophila Alcohol Dehydrogenase and Other Short-Chain Dehydrogenases/Reductases

Enzymic and structural studies on Drosophila alcohol dehydrogenases and other short-chain dehydrogenases/reductases (SDRs) are presented. Like alcohol dehydrogenases from other Drosophila species, the enzyme from D. simulans is more active on secondary than on primary alcohols, although ethanol is its only known physiological substrate. Several secondary alcohols were used to determine the kinetic parameters k_cat and K_m. The results of these experiments indicate that the substrate-binding region of the enzyme allows optimal binding of a short ethyl side-chain in a small binding pocket, and of a propyl or butyl side-chain in large binding pocket, with stereospecificity for R(−) alcohols. At a high concentration of R(−) alcohols substrate activation occurs. The k_cat and K_m values determined under these conditions are about two-fold, and two orders of magnitude, respectively, higher than those at low substrate concentrations. Sequence alignment of several SDRs of known, and unknown three-dimensional structures, indicate the presence of several conserved residues in addition to those involved in the catalyzed reactions. Structural roles of these conserved residues could be derived from observations made on superpositioned structures of several SDRs with known structures. Several residues are conserved in tetrameric SDRs, but not in dimeric ones. Two halohydrin-halide-lyases show significant homology with SDRs in the catalytic domains of these enzymes, but they do not have the structural features required for binding NAD⁺. Probably these lyases descend from an SDR, which has lost the capability to bind NAD⁺, but the enzyme reaction mechanisms may still be similar. Received: 23 May 2000 / Accepted: 4 January 2001     

Maize glutathione-dependent formaldehyde dehydrogenase: protein sequence and catalytic properties

Glutathione-dependent formaldehyde dehydrogenase (FDH; EC 1.2.1.1) has been purified 3900-fold from maize cell-suspension cultures to a specific activity of 4.68 μmol (mg protein)⁻¹ min⁻¹. The homogeneous enzyme consisted of two identical subunits with a molecular mass of 42 kDa, and an isoelectric point of 5.8. Eight tryptic peptides were sequenced and gave a perfect fit to the protein sequence derived from maize Fdh cDNA (J. Fliegmann and H. Sandermann, 1997, Plant Mol Biol 34: 843–854). There was 62% identity with the eucaryotic FDH consensus sequence. Michaelis constants of approx. 20 μm (formaldehyde), approx. 50 μm (glutathione) and approx. 31 μm (NAD⁺) were determined for the maize enzyme as well as for FDH partially purified from dog lung. Besides S-hydroxymethylglutathione, pentanol-1, octanol-1, and ω-hydroxyfatty acids served as substrates for both FDH preparations. The unusual substrate specificity indicates that FDH may be involved in the detoxification of long-chain lipid peroxidation products. Received: 1 April 1998 / Accepted: 18 November 1998     

Cloning and functional expression of the d-β-hydroxybutyrate dehydrogenase gene of Rhodobacter sp. DSMZ 12077

Nucleotide sequence and biochemical analysis of d-β-hydroxybutyrate dehydrogenase (EC 1.1.1.30), isolated from Rhodobacter sp., indicate functional oligomers composed of subunits of 257 amino acids with a calculated M _r of 26,800 and a pI of 5.90. Compared to mammalian short-chain alcohol dehydrogenases, the bacterial enzyme lacks a C-terminal lipid anchor domain and was found to be highly active upon expression in Escherichia coli even without lipid supplement. The recombinant enzyme could be highly enriched using a single chromatography step and was shown to be stable over a broad range of pH and temperature. Received: 1 April 1999 / Received last revision: 11 June 1999 / Accepted: 11 June 1999     

Two malate dehydrogenases in Methanobacterium thermoautotrophicum

Methanobacterium thermoautotrophicum (strain Marburg) was found to contain two malate dehydrogenases, which were partially purified and characterized. One was specific for NAD⁺ and catalyzed the dehydrogenation of malate at approximately one-third of the rate of oxalacetate reduction, and the other could equally well use NAD⁺ and NADP⁺ as coenzyme and catalyzed essentially only the reduction of oxalacetate. Via the N-terminal amino acid sequences, the encoding genes were identified in the genome of M. thermoautotrophicum (strain ΔH). Comparison of the deduced amino acid sequences revealed that the two malate dehydrogenases are phylogenetically only distantly related. The NAD⁺-specific malate dehydrogenase showed high sequence similarity to l-malate dehydrogenase from Methanothermus fervidus, and the NAD(P)⁺-using malate dehyrogenase showed high sequence similarity to l-lactate dehydrogenase from Thermotoga maritima and l-malate dehydrogenase from Bacillus subtilis. A function of the two malate dehydrogenases in NADPH:NAD⁺ transhydrogenation is discussed. Received: 29 December 1997 / Accepted: 4 March 1998     

Two δ1-pyrroline-5-carboxylate dehydrogenase isoforms are expressed in cultured Nicotiana plumbaginifolia cells and are differentially modulated during the culture growth cycle

δ¹-Pyrroline-5-carboxylate (P5C) dehydrogenase (EC 1.5.1.12) activity was measured in extracts from cultured tobacco (Nicotiana plumbaginifolia Viviani) cells. Two putative isozymes were resolved by anion-exchange fast protein liquid chromatography. These enzyme forms showed different patterns of expression during the culture growth cycle: activity-I increased in exponentially growing cells and declined rapidly in late logarithmic phase, while activity-II was found at substantial level only in cells which were entering the stationary phase. Both P5C dehydrogenases were partially purified and characterized with respect to kinetic and biochemical properties. They showed similar molecular masses as judged from retention patterns upon gel-filtration chromatography. The in vitro activity of both enzymes had a broad maximum around pH 7.4, and was progressively inhibited by Cl⁻ at concentrations ranging from 0.1 to 1 M. A pronounced difference was found between their apparent K _m values for the two substrates, P5C and NAD⁺, the higher affinities being shown by activity-I. Regulation of P5C dehydrogenase during salt-stress-induced proline accumulation was investigated. Following the addition of 175 mM NaCl to the culture medium the level of activity-I was substantially unaffected, while the specific activity of the other isozyme failed to increase even after the onset of the stationary phase of growth. Possible roles for P5C dehydrogenase isozymes in proline and arginine metabolism are discussed. Received: 23 May 1996 / Accepted: 18 December 1996     

Characterization of alcohol dehydrogenase 1 and 3 from <Emphasis Type="Italic">Neurospora crassa</Emphasis> FGSC2489

Alcohol dehydrogenase (ADH) is a key enzyme in the production and utilization of alcohols. Some also catalyze the formation of carboxylate esters from alcohols and aldehydes. The ADH1 and ADH3 genes of Neurospora crassa FGSC2489 were cloned and expressed in recombinant Escherichia coli to investigate their alcohol dehydrogenation and carboxylate ester formation abilities. Homology analysis and sequence alignment of amino acid sequence indicated that ADH1 and ADH3 of N. crassa contained a zinc-binding consensus sequence and a NAD⁺-binding motif and showed 54–75% identity with fungi ADHs. N. crassa ADH1 was expressed in E. coli to give a specific activity of 289 ± 9 mU/mg using ethanol and NAD⁺ as substrate and cofactor, respectively. Corresponding experiments on the expression and activity of ADH3 gave 4 mU/mg of specific activity. N. crassa ADH1 preferred primary alcohols containing C3–C8 carbons to secondary alcohols such as 2-propanol and 2-butanol. N. crassa ADH1 possessed 5.3 mU/mg of specific carboxylate ester-forming activity accumulating 0.4 mM of ethyl acetate in 18 h. Substrate specificity of various linear alcohols and aldehydes indicated that short chain-length alcohols and aldehydes were good substrates for carboxylate ester production. N. crassa ADH1 was a primary alcohol dehydrogenase using cofactor NAD⁺ preferably and possessed carboxylate ester-forming activity with short chain alcohols and aldehydes.     

Carbohydrate metabolism in Thermoproteus tenax: in vivo utilization of the non-phosphorylative Entner-Doudoroff pathway and characterization of its first enzyme, glucose dehydrogenase

Thermoproteus tenax is a hyperthermophilic, facultative heterotrophic archaeum. In this organism the utilization of the two catabolic pathways, a variant of the Embden-Meyerhof-Parnas (EMP) pathway and the modified (nonphosphorylative) Entner-Doudoroff (ED) pathway, was investigated and the first enzyme of the ED pathway, glucose dehydrogenase, was characterized. The distribution of the ¹³C label in alanine synthesized by cells grown with [1-¹³C]glucose indicated that in vivo the EMP pathway and the modified ED pathway operate parallel, with glucose metabolization via the EMP pathway being prominent. To initiate studies on the regulatory mechanisms governing carbon flux via these pathways, the first enzyme of the ED pathway, glucose dehydrogenase, was purified to homogeneity and its phenotypic properties were characterized. The pyridine-nucleotide-dependent enzyme used both NAD⁺ and NADP⁺ as cosubstrates, showing a 100-fold higher affinity for NADP⁺. Besides glucose, xylose was used as substrate, but with significantly lower affinity. These data suggest that the physiological function of the enzyme is the oxidation of glucose by NADP⁺. A striking feature was the influence of NADP⁺ and NAD⁺ on the quaternary structure and activity state of the enzyme. Without cosubstrate, the enzyme was highly aggregated (mol. mass > 600 kDa) but inactive, whereas in the presence of the cosubstrate the aggregates dissociated into enzymatically active, homomeric dimers with a mol. mass of 84 kDa (mol. mass of subunits: 41 kDa). The N-terminal amino acid sequence showed striking similarity to the respective partial sequences of alcohol dehydrogenases and sorbitol dehydrogenases, but no resemblance to the known pyridine-nucleotide-dependent archaeal and bacterial glucose dehydrogenases. Received: 25 October 1996 / Accepted: 15 April 1997     

A cold-active and thermostable alcohol dehydrogenase of a psychrotorelant from Antarctic seawater, <Emphasis Type="Italic">Flavobacterium frigidimaris</Emphasis> KUC-1

An NAD⁺-dependent alcohol dehydrogenase of a psychrotorelant from Antarctic seawater, Flavobacterium frigidimaris KUC-1 was purified to homogeneity with an overall yield of about 20% and characterized enzymologically. The enzyme has an apparent molecular weight of 160k and consists of four identical subunits with a molecular weight of 40k. The pI value of the enzyme and its optimum pH for the oxidation reaction were determined to be 6.7 and 7.0, respectively. The enzyme contains 2 gram-atoms Zn per subunit. The enzyme exclusively requires NAD⁺ as a coenzyme and shows the pro-R stereospecificity for hydrogen transfer at the C4 position of the nicotinamide moiety of NAD⁺. F. frigidimaris KUC-1 alcohol dehydrogenase shows as high thermal stability as the enzymes from thermophilic microorganisms. The enzyme is active at 0 to over 85°C and the most active at 70°C. The half-life time and k _cat value at 60°C were calculated to be 50 min and 27,400 min⁻¹, respectively. The enzyme also shows high catalytic efficiency at low temperatures (0–20°C) (k _cat/K _m at 10°C; 12,600 mM⁻¹ min⁻¹) similar to other cold-active enzymes from psychrophiles. The alcohol dehydrogenase gene is composed of 1,035 bp and codes 344 amino acid residues with an estimated molecular weight of 36,823. The sequence identities were found with the amino acid sequences of alcohol dehydrogenases from Moraxella sp. TAE123 (67%), Pseudomonas aeruginosa (65%) and Geobacillus stearothermophilus LLD-R (56%). This is the first example of a cold-active and thermostable alcohol dehydrogenase.     

Microbial production, purification, and characterization of (S)-specific N-acetyl-2-amino-1-phenyl-4-pentene amidohydrolase from Rhodococcus globerulus K1/1

Rhodococcus globerulus K1/1 was found to express an inducible (S)-specific N-acetyl-2-amino-1-phenyl-4-pentene amidohydrolase. Optimal bacterial growth and amidohydrolase expression were both observed at about pH 6.5. Purification of the enzyme to a single band in a Coomassie blue-stained SDS-PAGE gel was achieved by nucleic acid and ammonium sulfate precipitation of Rhodococcus globerulus K1/1 crude extract and column chromatography on TSK Butyl-650(S) Fractogel and Superose 12HR. The amidohydrolase was purified to a homogeneity leading to a tenfold increase of the specific activity with a recovery rate of 65%. At pH 7.0 and 23 °C the enzyme showed no loss of activity after 30 days incubation. The amidohydrolase was stable up to 55 °C. The enzyme was inhibited strongly only by 10 mM Zn²⁺ among the tested metal cations and was inhibited 100% by 0.01 mM phenylmethanesulfonyl fluoride. The molecular weight of the native enzyme was estimated to be 92 kDa by gel filtration and 55 kDa by SDS-PAGE, suggesting a homodimeric structure. Received: 8 February 1999 / Received revision: 3 May 1999 / Accepted: 7 May 1999     

Glucoamylase production in batch, chemostat and fed-batch cultivations by an industrial strain of Aspergillus niger

The Aspergillus niger strain BO-1 was grown in batch, continuous (chemostat) and fed-batch cultivations in order to study the production of the extracellular enzyme glucoamylase under different growth conditions. In the pH range 2.5–6.0, the specific glucoamylase productivity and the specific growth rate of the fungus were independent of pH when grown in batch cultivations. The specific glucoamylase producivity increased linearly with the specific growth rate in the range 0–0.1 h⁻¹ and was constant in the range 0.1–0.2 h⁻¹. Maltose and maltodextrin were non-inducing carbon sources compared to glucose, and the maximum specific growth rate was 0.19 ± 0.02 h⁻¹ irrespective of whether glucose or maltose was the carbon source. In fed-batch cultivations, glucoamylase titres of up to 6.5 g l⁻¹ were obtained even though the strain contained only one copy of the glaA gene. Received: 5 May 1999 / Received revision: 7 September 1999 / Accepted: 17 September 1999     

A neutral carrier-based liquid membrane microelectrode for divalent putrescine cations

A new ion-selective liquid membrane microelectrode, based on the neutral carrier 1,1′-bis(2,3-naphtho-18-crown-6), is described that shows the dependence of EMF on the activity of divalent putrescine cations a _Put, with the linear slope s _Put = 26 ± 3 mV/decade (mean ± SD, N = 18), in the range 10⁻⁴–10⁻¹ M at 25 ± 1 °C. Values of potentiometric putrescine cation selectivity coefficients of logK ^Pot _{Put j} (mean ± SD, N) are obtained by the separate solution method for the ions K⁺ (1.0 ± 0.4, 10), Na⁺ (−1.2 ± 0.4, 8), Ca²⁺ (−2.3 ± 0.5, 10) and Mg²⁺ (−2.5 ± 0.5, 7). The microelectrode can be applied for the direct analysis of the activities of free divalent putrescine cations in the range 5 × 10⁻⁴ to 10⁻¹ M in an extracellular ionic environment. Established analytical methods, e.g. high performance liquid chromatography, determine the total concentration of the derivatives of free and bound putrescine. Received: 20 December 1998 / Revised version: 7 May 1999 / Accepted: 27 May 1999     

Δ5-3β-Hydroxysteroid dehydrogenase from Digitalis lanata Ehrh. – a multifunctional enzyme in steroid metabolism?

Δ⁵-3β-Ηydroxysteroid dehydrogenase (Δ⁵-3β-HSD; EC 1.1.1.145), an enzyme converting pregn-5-ene-3β-ol-20-one (pregnenolone) to pregn-5-ene-3,20-dione (isoprogesterone), was isolated from the soluble fraction of suspension-cultured cells of Digitalis lanata L. strain VIII. Starting with acetone dry powder the enzyme was purified in three steps using column chromatography on Fractogel-TSK DEAE, hydroxyapatite and Sephacryl G-200. Fractions with highest Δ⁵-3β-HSD activity were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. After in-situ digestion the resulting bands were sequenced N-terminally. The 29-kDa band yielded three fragments with high sequence homology to members of the superfamily of short-chain dehydrogenases/reductases. High similarity was found to microbial hydroxysteroid dehydrogenases. The band may therefore represent the Δ⁵-3β-HSD. The purified enzyme was characterized with respect to kinetic parameters, substrate specificity and localization. The function of the enzyme in steroid metabolism is discussed. Received: 20 January 1999 / Accepted: 5 May 1999     

Biotransformation of eugenol to vanillin by a mutant of Pseudomonas sp. strain HR199 constructed by disruption of the vanillin dehydrogenase (vdh) gene

The catabolism of eugenol in Pseudomonas sp. strain HR199 (DSM7063) proceeds via coniferyl alcohol, coniferyl aldehyde, ferulic acid, vanillin, vanillate and protocatechuate, which is further degraded by the ortho-cleavage pathway. The vanillin dehydrogenase of Pseudomonas sp. strain HR199, which catalyses the NAD⁺-dependent oxidation of vanillin to vanillate, was inactivated by the insertion of omega elements into the vdh gene, which was characterized recently. Omega elements conferring resistance against kanamycin (ΩKm) or gentamycin (ΩGm) were constructed by polymerase chain reaction amplification of the aminoglycoside 3′-O-phosphotransferase gene and the gentamycin- 3-acetyltransferase gene, using the plasmids pSUP5011 and pBBR1MCS-5 respectively as template DNA. A 211-bp BssHII fragment of the vdh gene was substituted by ΩKm or ΩGm, and the functional vdh gene was replaced by vdhΩKm or vdhΩGm in Pseudomonas sp. strain HR199 by homologous recombination. Cells of the mutant Pseudomonas sp. strain HRvdhΩKm, pregrown on gluconate, accumulated up to 2.9 mM vanillin during incubation in mineral medium with 6.5 mM eugenol. As a result of another vanillin dehydrogenase activity (VDH-II), the accumulated vanillin was further degraded, when coniferyl aldehyde was exhausted from the medium. Characterization of the purified VDH-II revealed the identity of this enzyme with the recently characterized coniferyl-aldehyde dehydrogenase. Received: 19 March 1999 / Received revision: 31 June 1999 / Accepted: 5 July 1999     

Kinetic properties of N-(2-carboxyethyl)-NAD(H) and poly(ethylene glycol)-bound NAD(H) for alcohol, lactate, malate and glyceraldehyde-3-phosphate dehydrogenase from different organisms

The steady-state kinetics of alcohol dehydrogenases (alcohol:NAD⁺ oxidoreductase, EC 1.1.1.1 and alcohol:NADP⁺ oxidoreductase, EC 1.1.1.2), lactate dehydrogenases (l-lactate:NAD⁺ oxidoreductase, EC 1.1.1.27 and d-lactate:NAD⁺ oxidoreductase, EC 1.1.1.28), malate dehydrogenase (l-malate:NAD⁺ oxidoreductase, EC 1.1.1.37), and glyceraldehyde-3-phosphate dehydrogenases [d-glyceraldehyde-3-phosphate:NAD⁺ oxidoreductase (phosphorylating), EC 1.2.1.12] from different sources (prokaryote and eukaryote, mesophilic and thermophilic organisms) have been studied using NAD(H), N⁶-(2-carboxyethyl)-NAD(H), and poly(ethylene glycol)-bound NAD(H) as coenzymes. The kinetic constants for NAD(H) were changed by carboxyethylation of the 6-amino group of the adenine ring and by conversion to macromolecular form. Enzymes from thermophilic bacteria showed especially high activities for the derivatives. The relative values of the maximum velocity (NAD = 1) of Thermus thermophilus malate dehydrogenase for N⁶-(2-carboxyethyl)-NAD and poly(ethylene glycol)-bound NAD were 5.7 and 1.9, respectively, and that of Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase for poly(ethylene glycol)-bound NAD was 1.9.     

Propionic acid fermentation of glycerol and glucose by Propionibacterium acidipropionici and Propionibacterium freudenreichii ssp.shermanii

A comparative study was carried out in anaerobic batch cultures on 20 g/l of either glycerol or glucose using two propionibacteria strains, Propionibacterium acidipropionici and Propionibacterium freudenreichii ssp. shermanii. In all cases, fermentation end-products were the same and consisted of propionic acid as the major product, acetic acid as the main by-product and two minor metabolites, n-propanol and succinic acid. Evidence was provided that greater production of propionic acid by propionibacteria was obtained with glycerol as carbon and energy sources. P. acidipropionici showed higher efficiency in glycerol conversion to propionic acid with a faster substrate consumption (0.64 g l⁻¹ h⁻¹) and a higher propionic acid production (0.42 g l⁻¹ h⁻¹ and 0.79 mol/mol). The almost exclusive production of propionic acid from glycerol by this bacterium suggested an homopropionic tendency of this fermentation. Acetic acid final concentration was two times lower on glycerol (2 g/l) than on glucose (4 g/l) for both micro-organisms. P. freudenreichii ssp. shermanii exhibited a glycerol fermentation pattern typical of non-associated glycerol-consumption-product formation. This could indicate a particular metabolism for P. freudenreichii ssp. shermanii oriented towards the production of other specific components. These results tend to show that glycerol could be an excellent alternative to conventional carbon sources such as carbohydrates for propionic acid production. Received: 21 May 1999 / Accepted: 1 November 1999     

Biochemical characterization of ethanol-dependent reduction of furfural by alcohol dehydrogenases

Lignocellulosic biomass is usually converted to hydrolysates, which consist of sugars and sugar derivatives, such as furfural. Before yeast ferments sugars to ethanol, it reduces toxic furfural to non-inhibitory furfuryl alcohol in a prolonged lag phase. Bioreduction of furfural may shorten the lag phase. Cupriavidus necator JMP134 rapidly reduces furfural with a Zn-dependent alcohol dehydrogenase (FurX) at the expense of ethanol (Li et al. 2011). The mechanism of the ethanol-dependent reduction of furfural by FurX and three homologous alcohol dehydrogenases was investigated. The reduction consisted of two individual reactions: ethanol-dependent reduction of NAD⁺ to NADH and then NADH-dependent reduction of furfural to furfuryl alcohol. The kinetic parameters of the coupled reaction and the individual reactions were determined for the four enzymes. The data indicated that limited NADH was released in the coupled reaction. The enzymes had high affinities for NADH (e.g., K _d of 0.043 μM for the FurX-NADH complex) and relatively low affinities for NAD⁺ (e.g., K _d of 87 μM for FurX-NAD⁺). The kinetic data suggest that the four enzymes are efficient “furfural reductases” with either ethanol or NADH as the reducing power. The standard free energy change (ΔG°′) for ethanol-dependent reduction of furfural was determined to be −1.1 kJ mol⁻¹. The physiological benefit for ethanol-dependent reduction of furfural is likely to replace toxic and recalcitrant furfural with less toxic and more biodegradable acetaldehyde.     

Cytosolic NADPH balancing in <Emphasis Type="Italic">Penicillium chrysogenum</Emphasis> cultivated on mixtures of glucose and ethanol

The in vivo flux through the oxidative branch of the pentose phosphate pathway (oxPPP) in Penicillium chrysogenum was determined during growth in glucose/ethanol carbon-limited chemostat cultures, at the same growth rate. Non-stationary ¹³C flux analysis was used to measure the oxPPP flux. A nearly constant oxPPP flux was found for all glucose/ethanol ratios studied. This indicates that the cytosolic NADPH supply is independent of the amount of assimilated ethanol. The cofactor assignment in the model of van Gulik et al. (Biotechnol Bioeng 68(6):602–618, 2000) was supported using the published genome annotation of P. chrysogenum. Metabolic flux analysis showed that NADPH requirements in the cytosol remain nearly the same in these experiments due to constant biomass growth. Based on the cytosolic NADPH balance, it is known that the cytosolic aldehyde dehydrogenase in P. chrysogenum is NAD⁺ dependent. Metabolic modeling shows that changing the NAD⁺-aldehyde dehydrogenase to NADP⁺-aldehyde dehydrogenase can increase the penicillin yield on substrate.