Isolation of the flavodehydrogenase domain of Hansenula anomala flavocytochrome b2 after mild proteolysis by an H. anomala proteinase

The protomeric chain of Hansenula anomala flavocytochrome b2 was previously shown to be built as the covalent association of two functional domains: an L-lactate dehydrogenase domain and a cytochrome c reductase domain, joined together by a proteolytically sensitive zone. This paper concerns the specific cleavage of this latter zone with a H. anomala proteinase(s) preparation and the purification of the resulting L-lactate dehydrogenase moiety of the molecule with at least 25% recovery, (i.e. one order of magnitude more than for the previously published method). A preliminary characterization of this dehydrogenase domain indicates that it is a tetramer (Mr = 4 x 39000) containing FMN as expected and not heme. It has high L-lactate:ferricyanide oxidoreductase activity (about 70% that of the whole flavocytochrome b2) and the same Km for L(+)-lactate as flavocytochrome b2, but it has no L-lactate:cytochrome c oxidoreductase activity. Its flavin semiquinone is stabilized in the presence of pyruvate as in flavocytochrome b2. The subcellular origin of the H. anomala proteinase in the preparation has not yet been elucidated.     

Degradation of biphenyl by Mycobacterium sp. strain PYR-1

The metabolism of biphenyl by Mycobacterium sp. PYR-1 was investigated. The Mycobacterium sp. degraded >98% of the biphenyl added within 72 h. Analysis of ethyl acetate extracts of the culture medium by HPLC indicated that benzoic acid was the major metabolite. Other products were 4-hydroxybiphenyl, 4-hydroxybenzoic acid, and 5-oxo-5-phenylpentanoic acid. The metabolites were characterized by mass and 1H NMR spectrometry. Identification of benzoic acid and 5-oxo-5-phenylpentanoic acid indicates that biphenyl degradation by Mycobacterium sp. PYR-1 is generally similar to known pathways. A novel alternative metabolic pathway consisted of monooxygenation at C-4 of biphenyl to give 4-hydroxybiphenyl, with subsequent degradation via ring cleavage to 4-hydroxybenzoic acid.     

Aggregation and separability of the shikimate pathway enzymes in yeasts

Gel filtration was employed to estimate the molecular weights and to determine possible physical aggregation of enzymes [5-dehydroquinate synthase (DHQ synthase), 5-dehydroquinase (DHQase, EC 4.2.1.10), shikimate: NADP oxidoreductase (EC 1.1.1.25), shikimate kinase (EC 2.7.1.71), 3-enolpyruvylshikimate 5-phosphate synthase (EPSP synthase)] in the shikimate pathway in eleven species of yeasts. The five enzymes were not aggregated in extracts of Hansenula henricii, H. fabianii, H. anomala, Candida utilis, Pichia guilliermondii, and Lodderomyces elongisporus. Two enzymes (DHQase and shikimate:NADP oxidoreductase) were not separable by this method and by ion exchange chromatography, and we conclude that they exist as an aggregate in these yeasts. Evidence is presented for an enzyme aggregate containing five activities, with a molecular weight of approximately 280,000 in Rhodosporidium spaerocarpum, Rh. toruloides, Rhodotorula rubra, Saccharomycopsis lipolytica, and Saccharomyces cerevisiae. Similarities between the enzymes in the shikimate pathway of plants, bacteria, and other fungi and those of investigated yeasts are discussed.     

Stereochemistry of the hydrogen transfer to NAD catalyzed by (S)alanine dehydrogenase from Bacillus subtilis.

The stereochemistry of the hydrogen transfer to NAD catalyzed by (S)alanine dehydrogenase [ (S)alanine: NAD oxidoreductase (EC 1.4.1.1) ] from B. subtilis was investigated. The label at C-2 of (S) [2,3--3H] alanine was enzymatically transferred to NAD, and the [4--3H]NADH produced isolated and the stereochemistry at C-4 investigated. It was found that the label was exclusively located at the (R) position which indicates that (S)alanine dehydrogenase is an A-type enzyme. This result was confirmed in an alternate way by reducing enzymatically [4--3H]NAD with non labeled (S)alanine and (S)alanine dehydrogenase and investigating the stereochemistry of the ]4--3H]NADH produced. As expected, the label was now exclusively located at the (S) position. This proves that (S)alanine dehydrogenase isolated from B. subtilis should be classified as an A-enzyme with regard to the stereochemistry of the hydrogen transfer to NAD.     

Breeding of wastewater treatment yeasts that accumulate high concentrations of phosphorus

Inorganic phosphate is an essential nutrient. In general, microorganisms take up phosphorus when the extracellular phosphorus concentration is low, but not when it is high. In Saccharomyces cerevisiae, the major phosphate transporters, such as Pho84p, and acid phosphatases (APases), such as Pho5p, are regulated in parallel by the phosphate signal transduction pathway (PHO pathway). We found that PHO mutants expressing PHO84 and PHO5, even under high-P conditions, could take up phosphorus at twice the rate of the wild-type strain. The regulatory pathway for phosphorus accumulation in two wastewater treatment yeasts, Hansenula fabianii J640 and Hansenula anomala J224-1, was found to be similar to that in S. cerevisiae. We screened for mutants of these yeasts that constitutively expressed APase. Such mutants formed blue colonies on high phosphorus concentration agar plates containing 5-bromo-4-chloro-3-indolylphosphate (X-phosphate). We found four mutants of H. fabianii J640 and one mutant of H. anomala J224-1 that accumulated from 2.2 to 3.5 times more phosphorus than the parent strains. The growth rates and abilities to remove dissolved total nitrogen and dissolved organic carbon of the mutants were similar to those of the parent strains. In addition, the mutants removed 95% of dissolved total phosphorus from shochu wastewater, while the parent strain removed only 50%.     

Synthesis of enantiopure (S)-2-hydroxyphenylbutanoic acid using novel hydroxy acid dehydrogenase from Enterobacter sp. BK2K

Enterobacter sp. BK2K, screened from soil samples, can enantioselectively reduce 2-oxo-4-phenylbutanoic acid into (S)-2-hydroxy-4-phenylbutanoic acid. alpha-Hydroxy acid dehydrogenase (HADH) (specific activity 62.6 U/mg) was purified from the crude extract of Enterobacter sp. BK2K, and its gene was cloned and functionally expressed in E. coli BL21. The optimal pH and temperature for the HADH activity were 6.5 and 30 degrees C, respectively. The purified enzyme catalyzes the reduction of various aromatic and aliphatic 2-oxo carboxylic acids to the corresponding (S)-2-hydoxy carboxylic acids using NADH as cofactor. For example, the Km and kcat/Km for 2-oxo-4-phenylbutaonoic acid in the presence of 2 mM NADH were 6.8 mM and 350 M-1 min-1, respectively. For practical applications, a NADH recycle system employing the recombinant formate dehydrogenase from E. coli K12 was coupled with HADH in E. coli BL21. Using the recombinant HADH (110 U of 11 U/mg crude cell extract) and formate dehydrogenase (670 U of 67 U/mg crude cell extract) in 10 mL of 500 mM phosphate buffer (pH 6.5), 96 mM of (S)-phenyllactic acid (> 94% ee) and 95 mM of (S)-2-hydroxy-4-phenylbutanoic acid (> 94% ee) were produced in quantitative yields from 100 mM of phenylpyruvate and 2-oxo-4-phenylbutanoic acid.     

Chirality of the hydrogen transfer to the coenzyme catalyzed by ribitol dehydrogenase from Klebsiella pneumoniae and D-mannitol 1-phosphate dehydrogenase from Escherichia coli.

The stereochemistry of the hydrogen transfer to NAD catalyzed by ribitol dehydrogenase (ribitol:NAD 2-oxidoreductase, EC 1.1.1.56) from Klebsiella pneumoniae and D-mannitol-1-phosphate dehydrogenase (D-mannitol-1-phosphate:NAD 2-oxidoreductase, EC 1.1.1.17) from Escherichia coli was investigated. [4-3H]NAD was enzymatically reduced with nonlabelled ribitol in the presence of ribitol dehydrogenase and with nonlabelled D-mannitol 1-phosphate and D-mannitol 1-phosphate dehydrogenase, respectively. In both cases the [4-3H]-NADH produced was isolated and the chirality at the C-4 position determined. It was found that after the transfer of hydride, the label was in both reactions exclusively confined to the (4R) position of the newly formed [4-3H]NADH. In order to explain these results, the hydrogen transferred from the nonlabelled substrates to [4-3H]NAD must have entered the (4S) position of the nicotinamide ring. These data indicate for both investigated inducible dehydrogenases a classification as B or (S) type enzymes. Ribitol also can be dehydrogenated by the constitutive A-type L-iditol dehydrogenase (L-iditol:NAD 5-oxidoreductase, EC 1.1.1.14) from sheep liver. When L-iditol dehydrogenase utilizes ribitol as hydrogen donor, the same A-type classification for this oxidoreductase, as expected, holds true. For the first time, opposite chirality of hydrogen transfer to NAD in one organic reaction--ribitol + NAD = D-ribu + NADH + H--is observed when two different dehydrogenases, the inducible ribitol dehydrogenase from K. pneumoniae and the constitutive L-iditol dehydrogenase from sheep liver, are used as enzymes. This result contradicts the previous generalization that the chirality of hydrogen transfer to the coenzyme for the same reaction is independent of the source of the catalyzing enzyme.     

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.     

Effect of triamcinolone administration on content of flavins in rabbit liver.

Triamcinoline acetonide (10 mg per kg of body weight a day) was administered to rabbit fed on a laboratory chow diet. The content of flavins in liver but not in kidney, muscle and brain started to decrease 24 h after a single dose. The activities of enzymes in the liver were determined: the activities of pyruvate dehydrogenase complex, lipoamide dehydrogenase (NADH:lipoamide oxidoreductase EC 1.6.4.3), NADH dehydrogenase (NADH : (acceptor) oxidoreductase EC 1.6.99.3) and D-amino acid oxidase (D-amino acid: oxygen oxidoreductase (deaminating) EC 1.4.3.3) were decreased but those of succinate dehydrogenase (succinate : (acceptor) oxidoreductase EC 1.3.99.1) and xanthine oxidase (xanthine : oxygen oxidoreductase EC 1.2.3.2) remained unchanged. The activities of enzymes in the kidney, however, remained unchanged except the decrease in the activity of pyruvate dehydrogenase complex.     

Investigating the stereochemistry of binding to HIV-1 protease with inhibitors containing isomers of 4-amino-3-hydroxy-5-phenylpentanoic acid

A series of inhibitors containing all possible isomers of 4-amino-3-hydroxy-5-phenylpentanoic acid was synthesized and tested for inhibition of HIV-1 protease. Incorporation of the (3S,4S) isomer of the t-butyloxycarbonyl protected amino acid into the sequence Glu-Phe resulted in a potent inhibitor of HIV-1 protease (Ki = 63 nM). This inhibitor is at least 47-times more potent than the inhibitors containing other isomers of 4-amino-3-hydroxy-5-phenylpentanoic acid, indicating that the (3S,4S) isomer is the preferred isomer for binding to HIV-1 protease.     

One-step synthesis of 12-ketoursodeoxycholic acid from dehydrocholic acid using a multienzymatic system

12-ketoursodeoxycholic acid (12-keto-UDCA) is a key intermediate for the synthesis of ursodeoxycholic acid (UDCA), an important therapeutic agent for non-surgical treatment of human cholesterol gallstones and various liver diseases. The goal of this study is to develop a new enzymatic route for the synthesis 12-keto-UDCA based on a combination of NADPH-dependent 7β-hydroxysteroid dehydrogenase (7β-HSDH, EC 1.1.1.201) and NADH-dependent 3α-hydroxysteroid dehydrogenase (3α-HSDH, EC 1.1.1.50). In the presence of NADPH and NADH, the combination of these enzymes has the capacity to reduce the 3-carbonyl- and 7-carbonyl-groups of dehydrocholic acid (DHCA), forming 12-keto-UDCA in a single step. For cofactor regeneration, an engineered formate dehydrogenase, which is able to regenerate NADPH and NADH simultaneously, was used. All three enzymes were overexpressed in an engineered expression host Escherichia coli BL21(DE3)Δ7α-HSDH devoid of 7α-hydroxysteroid dehydrogenase, an enzyme indigenous to E. coli, in order to avoid formation of the undesired by-product 12-chenodeoxycholic acid in the reaction mixture. The stability of enzymes and reaction conditions such as pH value and substrate concentration were evaluated. No significant loss of activity was observed after 5 days under reaction condition. Under the optimal condition (10 mM of DHCA and pH 6), 99 % formation of 12-keto-UDCA with 91 % yield was observed.     

异常汉逊酵母BD102金属硫蛋白的分离纯化和鉴定

从异常汉逊酵母中分离出拮抗Cu²⁺、Cd²⁺等重金属、并经铜、镉诱导产生金属硫蛋白的异常汉逊酵母 (Hansenulaanomala)BD1 0 2。无细胞抽提液经SephadexG 50、DEAESepharoseCL 6B、SephadexG 2 5三次凝胶及阴离子交换柱层析分离纯化 ,Cu²⁺诱导得到Cu MTs两个亚型 ,Cd²⁺诱导得到Cd MT一个亚型。Mr分别约为 7kD和 7 5kD ,由 60和 61个氨基酸组成 ,其中半胱氨酸含量各为 6.8%和 1.0 %。每分子金属硫蛋白 (Cu MTs或Cd MT)可结合 4个铜或镉原子     

Effect of triamcinolone administration on content of flavins in rabbit liver

Triamcinoline acetonide (10 mg per kg of body weight a day) was administered to rabbit fed on a laboratory chow diet. The content of flavins in liver but not in kidney, muscle and brain started to decrease 24 h after a single dose. The activities of enzymes in the liver were determined: the activities of pyruvate dehydrogenase complex, lipoamide dehydrogenase (NADH : lipoamide oxidoreductase EC 1.6.4.3), NADH dehydrogenase (NADH : (acceptor) oxidoreductace EC 1.6.99.3) and -amino acid oxidase ( -amino acid : oxygen oxidoreductase (deaminating) EC 1.4.3.3) were decreased but those of succinate dehydrogenase (succinate : (acceptor) oxidoreductase EC 1.3.99.1) and xanthine oxidase (xanthine : oxygen oxidoreductase EC 1.2.3.2) remained unchanged. The activities of enzymes in the kidney, however, remained unchanged except the decrease in the activity of pyruvate dehydrogenase complex.     

Preparation of 12-ketochenodeoxycholic acid from cholic acid using coimmobilized 12α-hydroxysteroid dehydrogenase and glutamate dehydrogenase with NADP cycling at high efficiency

12-Ketochenodeoxycholic acid, an essential intermediate in the synthesis of chenodeoxycholic acid, has been enzymatically prepared from cholic acid. The specific oxidation of the 12α-hydroxyl group of cholic acid with NADP⁺ was catalysed by 12α-hydroxysteroid dehydrogenase (12α-hydroxysteroid: NAD⁺ oxidoreductase, EC 1.1.1.176), and the regeneration of NADP⁺ was obtained through the glutamate dehydrogenase (l-glutamate:NADP⁺ oxidoreductase, EC 1.4.1.4) catalysed reduction of α-ketoglutarate. The two enzymes were immobilized onto Sepharose CL-4B activated with tresyl chloride. The coimmobilized enzymes showed a cycling efficiency for the coenzyme similar to that of the free enzymes. High concentrations of cholic acid (up to 4%, w/v) were completely and specifically transformed into the 12-keto derivative using amounts of cofactor about 1600 times lower on a molar basis. The immobilized enzymes maintained 70% of the initial activity after 2 months of continuous use.     

Clostridium scindens baiCD and baiH genes encode stereo-specific 7α/7β-hydroxy-3-oxo-Δ4-cholenoic acid oxidoreductases

Secondary bile acids, formed by intestinal bacteria, are suggested to play a significant role in cancers of the gastrointestinal tract in humans. Bile acid 7α/β-dehydroxylation is carried out by a few species of intestinal clostridia which harbor a multi-gene bile acid inducible (bai) operon. Several genes encoding enzymes in this pathway have been cloned and characterized. However, no gene product(s) has yet been assigned to the production of 3-oxo-Δ⁴-cholenoic acid intermediates of cholic acid (CA), chenodeoxycholic acid (CDCA) or ursodeoxycholic acid (UDCA). We previously reported that the baiH gene encodes an NADH:flavin oxidoreductase (NADH:FOR); however, the role of this protein in bile acid 7-dehydroxylation is unclear. Homology searches and secondary structural alignments suggest this protein to be similar to flavoproteins which reduce α/β-unsaturated carbonyl compounds. The baiH gene product was expressed in Escherichia coli, purified and discovered to be a stereo-specific NAD(H)-dependent 7β-hydroxy-3-oxo-Δ⁴-cholenoic acid oxidoreductase. Additionally, high sequence similarity between the baiH and baiCD gene products suggests the baiCD gene may encode a 3-oxo-Δ⁴-cholenoic acid oxidoreductase specific for CDCA and CA. We tested this hypothesis using cell extracts prepared from E. coli overexpressing the baiCD gene and discovered that it encodes a stereo-specific NAD(H)-dependent 7α-hydroxy-3-oxo-Δ⁴-cholenoic acid oxidoreductase.     

Yeasts in Ensiled High-Moisture Corn

A total of 1,365 yeasts were selected from ensiled high-moisture corn at various stages in the ensiling process to determine the sequence and relative numbers of yeast species. The yeast species most frequently isolated from freshly harvested corn were Candida parapsilosis and C. intermedia; these two species were isolated infrequently after the third week of storage. Species of yeasts that predominate after the 12th day of storage were Hansenula anomala (66% of the isolates studied) and C. krusei (26% of the isolates studied). The preponderance of H. anomala and C. krusei in ensiled corn is believed to be associated with the ability of these two species to assimilate lactic acid.     

Study of the Hansenula anomala yeast flavocytochrome-b2--cytochrome-c complex 1. Characterization of fluorescent Zn(II)-substituted cytochrome c

Substitution of Fe2+ for the Zn2+ ion in Hansenula anomala cytochrome c provides a luminescent derivative suitable as a probe for the determination of the interaction of cytochrome c with H. anomala flavocytochrome b2; its light absorption and fluorescence properties have been characterized. H. anomala Zn-cytochrome c appears to be in the form of a stable though non-covalent dimer from molecular weight determinations performed using gel filtration, polyacrylamide gel electrophoresis under denaturing conditions, and ultracentrifugation methods. By contrast, metal-free porphyrin-cytochrome c, the precursor of Zn-cytochrome c obtained upon removal of iron from cytochrome c in cold anhydrous fluorhydric acid, had the same partition coefficient as native cytochrome c through conventional gel filtration. Significant conformational perturbations of H. anomala cytochrome c should therefore follow from Zn2+ incorporation into the porphyrin c moiety. Titrations at low ionic strength with native, tetrameric H. anomala flavocytochrome b2 in the lactate-reduced state showed a simple binding equilibrium (Kd = 0.1 microM at I = 0.03 M, 10 degrees C) with a stoichiometry of one Zn-cytochrome c dimer per protomer of flavocytochrome b2. Quenching of the Zn-porphyrin c fluorescence within this complex was much larger (43%) than reported by other authors using cytochrome c and flavocytochrome b2 from different sources.     

Structural basis for the kinetic differences between flavocytochromes b2 from the yeasts Hansenula anomala and Saccharomyces cerevisiae.

To understand the structural basis for the different catalytic behaviour of the flavocytochromes b2 from Saccharomyces cerevisiae and Hansenula anomala we have cloned and sequenced the gene encoding the latter. We have compared the amino acid sequences of the mature proteins in the context of the known crystal structure of S. cerevisiae flavocytochrome b2. Overall there is 60% sequence identity, but two surface loops in particular are strikingly different in primary structure and net charge.     

Stereospecificity of hydrogen transfer in the saccharopine dehydrogenase reaction.

The stereospecificity of hydrogen transfer in the synthesis of saccharopine from alpha-ketoglutarate and L-lysine catalyzed by saccharopine dehydrogenase (N5-(1,3-dicarboxypropyl)-L-lysine: NAD oxidoreductase (L-lysine-forming), EC 1.5.1.7) was examined by using [4A-3H]- and [4B-3H]NADH. The enzyme showed the A-stereospecificity. The NMR analysis of the saccharopine prepared with [4"A-2H]NADH revealed that the label was incorporated into the C-2 of the glutaryl moiety.     

Purification of 20α-hydroxysteroid oxidoreductase from bovine fetal erythrocytes

NADPH-dependent 20α-hydroxysteroid oxidoreductase (20α-HSD; EC 1.1.1.149) from bovine fetal erythrocytes was obtained for the first time free of hemoglobin by a new 2,500-fold purification scheme. This was achieved by a sequence of calcium phosphate gel adsorption, ammonium sulfate fractionation, and affinity chromatography. The present results lead us to believe that the NADPH-dependent 3β-hydroxysteroid oxidoreductase activity, which was co-purified with 20α-activity, may originate at the active site of 20α-HSD (2).