Purification and properties of NADP(+)-dependent glycerol dehydrogenases from Aspergillus nidulans and A. niger

Glycerol dehydrogenase, NADP(+)-specific (EC 1.1.1.72), was purified from mycelium of Aspergillus nidulans and Aspergillus niger using different purification procedures. Both enzymes had an Mr of approximately 38,000 and were immunologically cross-reactive, but had different amino acid compositions and isoelectric points. For both enzymes, the substrate specificity was limited to glycerol and erythritol for the oxidative reaction and to dihydroxyacetone (DHA), diacetyl, methylglyoxal, erythrose and D-glyceraldehyde for the reductive reaction. The A. nidulans enzyme had a turnover number twice that of the A. niger enzyme at pH 6.0, whereas inhibition by NADP+ was less (Ki = 45 microM vs 13 microM). It is proposed that both enzymes catalyse in vivo the reduction of DHA to glycerol and that they are regulated by the anabolic reduction charge.     

Glycerol metabolism in the methylotrophic yeast Hansenula polymorpha: phosphorylation as the initial step

In hansenula polymorpha glycerol is metabolized via glycerol kinase and NAD(P)-independent glycerol-3-phosphate (G3P) dehydrogenase, enzymes which hitherto were reported to be absent in this methylotrophic yeast. Activity of glycerol kinase was readily detectable when cell-free extracts were incubated at pH 7–8 with glycerol/ATP/Mg²⁺ and a discontinuous assay for G3P formation was used. This glycerol kinase activity could be separated from dihydroxyacetone (DHA) kinase activity by ion exchange chromatography. Glycerol kinase showed relatively low affinities for glycerol (apparent K _m=1.0 mM) and ATP (apparent K _m=0.5 mM) and was not active with other substrates tested. No inhibition by fructose-1,6-bisphosphate (FBP) was observed. Both NAD-dependent and NAD(P)-independent G3P dehydrogenases were present. The latter enzyme could be assayed with PMS/MTT and cosedimented with the mitochondrial fraction. Glucose partly repressed synthesis of glycerol kinase and NAD(P)-independent G3P dehydrogenase, but compared to several other non-repressing carbon sources no clear induction of these enzymes by glycerol was apparent. Amongst glycerolnegative mutants of H. polymorpha strain 17B (a DHA kinase-negative mutant), strains blocked in either glycerol kinase or membrane-bound G3P dehydrogenase were identified. Crosses between representatives of the latter mutants and wild type resulted in the isolation of, amongst others, segregants which had regained DHA kinase but were still blocked in the membrane-bound G3P dehydrogenase. These strains, employing the oxidative pathway, were only able to grow very slowly in glycerol mineral medium.Abbreviations DHA dihydroxyacetone - G3P glycerol-3-phosphate - EMS ethyl methanesulphonate - MTT 3-(4,5-dimethyl-thiazolyl-2)-2,5-diphenyl tetrazolium bromide - PMS phenazine methosulphate - FBP fructose-1,6-bisphosphate     

NAD(+)-dependent isocitrate dehydrogenase. Cloning, nucleotide sequence, and disruption of the IDH2 gene from Saccharomyces cerevisiae

NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae is composed of two nonidentical subunits, designated IDH1 (Mr approximately 40,000) and IDH2 (Mr approximately 39,000). We have isolated and characterized a yeast genomic clone containing the IDH2 gene. The amino acid sequence deduced from the gene indicates that IDH2 is synthesized as a precursor of 369 amino acids (Mr 39,694) and is processed upon mitochondrial import to yield a mature protein of 354 amino acids (Mr 37,755). Amino acid sequence comparison between S. cerevisiae IDH2 and S. cerevisiae NADP(+)-dependent isocitrate dehydrogenase shows no significant sequence identity, whereas comparison of IDH2 and Escherichia coli NADP(+)-dependent isocitrate dehydrogenase reveals a 33% sequence identity. To confirm the identity of the IDH2 gene and examine the relationship between IDH1 and IDH2, the IDH2 gene was disrupted by genomic replacement in a haploid yeast strain. The disruption strain expressed no detectable IDH2, as determined by Western blot analysis, and was found to lack NAD(+)-dependent isocitrate dehydrogenase activity, indicating that IDH2 is essential for a functional enzyme. Overexpression of IDH2, however, did not result in increased NAD(+)-dependent isocitrate dehydrogenase activity, suggesting that both IDH1 and IDH2 subunits are required for catalytic activity. The disruption strain was unable to utilize acetate as a carbon source and exhibited a 2-fold slower growth rate than wild type strains on glycerol or lactate. This growth phenotype is consistent with NAD(+)-dependent isocitrate dehydrogenase performing an essential role in the oxidative function of the citric acid cycle.     

Regulation of methanol metabolism in the yeast Hansenula polymorpha

A study of enzyme profiles in Hansenula polymorpha grown on various carbon substrates revealed that the synthesis of the methanol dissimilatory and assimilatory enzymes is regulated in the same way, namely by catabolite repression and induction by methanol. Mutants of H. polymorpha blocked in dihydroxyacetone (DHA) synthase (strain 70 M) or DHA kinase (strain 17 B) were unable to grow on methanol which confirmed the important role attributed to these enzymes in the biosynthetic xylulose monophosphate (XuMP) cycle. Both mutant strains were still able to metabolize methanol. In the DNA kinase-negative strain 17 B this resulted in accumulation of DHA. Although DHA kinase is thought to be involved in DHA and glycerol metabolism in methylotrophic yeasts, strain 17 B was still able to grow on glycerol at a rate similar to that of the wild type. DHA on the other hand only supported slow growth of this mutant when relatively high concentrations of this compound were provided in the medium. This slow but definite growth of strain 17 B on DHA was not based on the reversible DHA synthase reaction but on conversion of DHA into glycerol, a reaction catalyzed by DNA reductase. The subsequent metabolism of glycerol in strain 17 B and in wild type H. polymorpha, however, remains to be elucidated.Abbreviations XuMP xylulose monophosphate - DHA dihydroxyacetone - EMS ethyl methanesulphonate     

快原子轰击质谱技术结合酶学方法鉴定黑曲霉1,6—缩水—β—D—吡喃葡萄糖激酶的诱导合成

1,6－缩水－β－D－吡喃葡萄糖是纤维素类物质热解的主要产物,黑曲霉突变株CBX－209能较好地利用该糖作为唯一的碳源和能源生长并产生有用的代谢产物柠檬酸,其效率与利用葡萄糖大致相当。利用葡萄糖氧化酶和竦根过氧化物酶复合系统测定证明该菌株不存在1,6－缩水－β－D－吡喃葡萄糖水解酶,采用快原子轰击质谱技术结合6－磷酸葡萄糖脱氢酶系统进行测定,结果表明经（NH4）2SO4沉淀或阴离子交换层析析处理后的无细胞提取液在加入ATP和Mg^2 条件下能直接催化1,6－缩水－β－D－吡喃葡萄糖合成6－磷酸葡萄糖,证明黑曲霉突变株中存在一个新酶,即1,6－缩水－β－D－吡喃葡萄糖激酶。该酶为诱导酶。     

Dihydroxyacetone kinase from a methylotrophic yeast,Hansenula polymorpha CBS 4732

Dihydroxyacetone (DHA) kinase was purified to electrophoretic homogeneity from methanol-grown Hansenula polymorpha CBS 4732. The enzyme was a dimer with a molecular weight of 150,000, and had an isoelectric point of 4.9. The enzyme was active toward DHA, and D- and L-glyceraldehydes as phosphorylation acceptors, and only ATP served as a donor. ADP inhibited the enzyme at a physiological concentration. Magnesium ion was essential for the activity and stability. Some other divalent cations can substitute in part the magnesium ion. The DHA kinases found in cells grown on methanol and glycerol were immunologically identical, but were different from those of other methylotrophic yeasts as shown by immunotitration. A mutant (204D) derived from the yeast, which could not grow on methanol or DHA but could so on glycerol, was deficient in DHA kinase. Glycerol kinase activity was found in glycerol-grown 204D cells as well as the parent strain.Abbreviation DHA dihydroxyacetone     

Glycerol catabolism in Aspergillus nidulans

Glycerol is catabolized in Aspergillus nidulans by glycerol kinase and a mitochondrial FAD-dependent sn-glycerol 3-phosphate dehydrogenase. The levels of both enzymes are controlled by carbon catabolite repression and by specific induction. Biochemical and genetical analyses show that dihydroxyacetone and D-glyceraldehyde are converted into glycerol and then catabolized by the same pathway. D-Glyceraldehyde can be reduced by NADP(+)-dependent glycerol dehydrogenase or by alcohol dehydrogenase I, while dihydroxyacetone is only reduced by the first enzyme. Three new glycerol non-utilizing mutants have been found. These three mutations define three hitherto unknown loci, glcE, glcF and glcG. The mutation in glcG leads to a greatly decreased sn-glycerol-3-phosphate dehydrogenase activity.     

Phosphorylation of glycerol and dihydroxyacetone in Acetobacter xylinum and its possible regulatory role.

Extracts of Acetobacter xylinum catalyze the phosphorylation of glycerol and dihydroxyacetone (DHA) by adenosine 5'-triphosphate (ATP) to form, respectively, L-alpha-glycerophosphate and DHA phosphate. The ability to promote phosphorylation of glycerol and DHA was higher in glycerol-grown cells than in glucose- or succinate-grown cells. The activity of glycerol kinase in extracts is compatible with the overall rate of glycerol oxidation in vivo. The glycerol-DHA kinase has been purified 210-fold from extracts, and its molecular weight was determined to be 50,000 by gel filtration. The glycerol kinase to DHA kinase activity ratio remained essentially constant at 1.6 at all stages of purification. The optimal pH for both reactions was 8.4 to 9.2. Reaction rates with the purified enzyme were hyperbolic functions of glycerol, DHA, and ATP. The Km for glycerol is 0.5 mM and that for DHA is 5 mM; both are independent of the ATP concentration. The Km for ATP in both kinase reactions is 0.5 mM and is independent of glycerol and DHA concentrations. Glycerol and DHA are competitive substrates with Ki values equal to their respective Km values as substrates. D-Glyceraldehyde and l-Glyceraldehyde were not phosphorylated and did not inhibit the enzyme. Among the nucleotide triphosphates tested, only ATP was active as the phosphoryl group donor. Fructose diphosphate (FDP) inhibited both kinase activities competitively with respect to ATP (Ki= 0.02 mM) and noncompetitively with respect to glycerol and DHA. Adenosine 5'-diphosphate (ADP) and adenosine 5'-monophosphate (AMP) inhibited both enzymic activities competitively with respect to ATP (Ki (ADP) = 0.4 mM; Ki (AMP) =0.25 mM). A. xylinum cells with a high FDP content did not grow on glycerol. Depletion of cellular FDP by starvation enabled rapid growth on glycerol. It is concluded that a single enzyme from A. xylinum is responsible for the phosphorylation of both glycerol and DHA. This as well as the sensitivity of the enzyme to inhibition by FDP and AMP suggest that it has a regulatory role in glycerol metabolism.     

The subcellular distribution and properties of aldehyde dehydrogenases in rat liver

1. Kinetic experiments suggested the possible existence of at least two different NAD(+)-dependent aldehyde dehydrogenases in rat liver. Distribution studies showed that one enzyme, designated enzyme I, was exclusively localized in the mitochondria and that another enzyme, designated enzyme II, was localized in both the mitochondria and the microsomal fraction. 2. A NADP(+)-dependent enzyme was also found in the mitochondria and the microsomal fraction and it is suggested that this enzyme is identical with enzyme II. 3. The K(m) for acetaldehyde was apparently less than 10mum for enzyme I and 0.9-1.7mm for enzyme II. The K(m) for NAD(+) was similar for both enzymes (20-30mum). The K(m) for NADP(+) was 2-3mm and for acetaldehyde 0.5-0.7mm for the NADP(+)-dependent activity. 4. The NAD(+)-dependent enzymes show pH optima between 9 and 10. The highest activity was found in pyrophosphate buffer for both enzymes. In phosphate buffer there was a striking difference in activity between the two enzymes. Compared with the activity in pyrophosphate buffer, the activity of enzyme II was uninfluenced, whereas the activity of enzyme I was very low. 5. The results are compared with those of earlier investigations on the distribution of aldehyde dehydrogenase and with the results from purified enzymes from different sources.     

黑曲霉纤维素酶突变子T-DNA插入位点的遗传分析及性质鉴定

采用羧甲基纤维素钠筛选培养基,对黑曲霉(Aspergillus niger)T-DNA突变子文库进行筛选,分离到一株纤维素酶分泌水平较低的菌株AN-108,为野生型菌株的83.3%。进一步测定该突变子固体发酵的纤维素酶活力,与野生型菌株相比没有明显差别,推测与固体发酵培养基中含有的天然糖类有关。在添加不同糖类的CMC-Na平板上培养该突变子,菌落周围均出现较明显的水解圈,结果显示糖类可能作为诱导物克服突变带来的影响。为了确定突变子AN-108中何种基因被阻断,采用反向PCR方法分析了T-DNA插入位点的序列,获得序列经过比对分析发现,该序列与黑曲霉An14g03730同源程度达90%,编码富含脯氨酸蛋白(proline-rich protein,PRP)。     

L-xylo-3-hexulose reductase is the missing link in the oxidoreductive pathway for D-galactose catabolism in filamentous fungi

In addition to the well established Leloir pathway for the catabolism of d-galactose in fungi, the oxidoreductive pathway has been recently identified. In this oxidoreductive pathway, D-galactose is converted via a series of NADPH-dependent reductions and NAD(+)-dependent oxidations into D-fructose. The pathway intermediates include galactitol, L-xylo-3-hexulose, and d-sorbitol. This study identified the missing link in the pathway, the L-xylo-3-hexulose reductase that catalyzes the conversion of L-xylo-3-hexulose to D-sorbitol. In Trichoderma reesei (Hypocrea jecorina) and Aspergillus niger, we identified the genes lxr4 and xhrA, respectively, that encode the l-xylo-3-hexulose reductases. The deletion of these genes resulted in no growth on galactitol and in reduced growth on D-galactose. The LXR4 was heterologously expressed, and the purified protein showed high specificity for L-xylo-3-hexulose with a K(m) = 2.0 ± 0.5 mm and a V(max) = 5.5 ± 1.0 units/mg. We also confirmed that the product of the LXR4 reaction is D-sorbitol.     

Long-chain alcohol and aldehyde dehydrogenase activities in Acinetobacter calcoaceticus strain HO1-N.

Three alcohol dehydrogenases have been identified in Acinetobacter calcoaceticus sp. strain HO1-N: an NAD(+)-dependent enzyme and two NADP(+)-dependent enzymes. One of the NADP(+)-dependent alcohol dehydrogenases was partially purified and was specific for long-chain substrates. With tetradecanol as substrate an apparent Km value of 5.2 microM was calculated. This enzyme has a pI of 4.5 and a molecular mass of 144 kDa. All three alcohol dehydrogenases were constitutively expressed. Three aldehyde dehydrogenases were also identified: an NAD(+)-dependent enzyme, an NADP(+)-dependent enzyme and one which was nucleotide independent. The NAD(+)-dependent enzyme represented only 2% of the total activity and was not studied further. The NADP(+)-dependent enzyme was strongly induced by growth of cells on alkanes and was associated with hydrocarbon vesicles. With tetradecanal as substrate an apparent Km value of 0.2 microM was calculated. The nucleotide-independent aldehyde dehydrogenase could use either Würster's Blue or phenazine methosulphate (PMS) as an artificial electron acceptor. This enzyme represents approximately 80% of the total long-chain aldehyde oxidizing activity within the cell when the enzymes were induced by growing the cells on hexadecane. It is particulate but can be solubilized using Triton X-100. The enzyme has an apparent Km of 0.36 mM for decanal.     

Threonine 11 of human NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase may interact with NAD(+) during catalysis

NAD(+)-dependent 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a member of the short-chain dehydrogenase family, catalyzes the first step in the catabolic pathway of the prostaglandins. This enzyme oxidizes the 15-hydroxyl group of prostaglandins to produce 15-keto metabolites which are usually biologically inactive. A relatively conserved threonine residue corresponding to threonine 11 of 15-PGDH is proposed to be involved in the interaction with NAD(+). Site-directed mutagenesis was used to examine the important role of this residue. Threonine 11 was changed to alanine (T11A), cysteine (T11C), serine (T11S) or tyrosine (T11Y) and the mutant proteins were expressed in E. coli. Western-blot analysis showed that the expression levels of mutant proteins were comparable to that of the wild-type enzyme. Mutants T11A, T11C and T11Y were found to be inactive. Mutant T11S still retained substantial activity and the K(m) value for prostaglandin E(2) (PGE(2)) was similar to the wild-type enzyme; however, the K(m) value for NAD(+) was increased over 23-fold. These results suggest that threonine 11 may be involved in the interaction with NAD(+) either directly or indirectly and contributes to the full catalytic activity of 15-PGDH.     

Cloning and characterization of the gene encoding the IDH1 subunit of NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae.

NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae is composed of two nonidentical subunits, designated IDH1 and IDH2. The gene encoding IDH2 was previously cloned and sequenced (Cupp, J.R., and McAlister-Henn, L. (1991) J. Biol. Chem. 266, 22199-22205), and in this paper we describe the isolation of a yeast genomic clone containing the IDH1 gene. A fragment of the IDH1 gene was amplified by the polymerase chain reaction method utilizing degenerate oligonucleotides based on tryptic peptide sequences of the purified subunit; this fragment was used to isolate a full length IDH1 clone. The nucleotide sequence of the IDH1 coding region was determined and encodes a 360-residue polypeptide including an 11-residue mitochondrial targeting presequence. Amino acid sequence comparison between IDH1 and IDH2 reveals a 42% sequence identity, and both IDH1 and IDH2 show approximately 32% identity to Escherichia coli NAD(P)(+)-dependent isocitrate dehydrogenase. To examine the function of the IDH1 subunit and to determine the metabolic role of NAD(+)-dependent isocitrate dehydrogenase the IDH1 gene was disrupted in a wild type haploid yeast strain and in a haploid strain lacking IDH2. The IDH1 disruption strains expressed no detectable IDH1 as determined by Western blot analysis, and these strains were found to lack NAD(+)-dependent isocitrate dehydrogenase activity indicating that IDH1 is essential for a functional enzyme. Over-expression of IDH1 in a strain containing IDH2 restored wild type activity but did not result in increased levels of activity, suggesting that both IDH1 and IDH2 are required for a functional enzyme. Growth phenotype analysis of the IDH1 disruption strains revealed that they grew at a reduced rate on the nonfermentable carbon sources examined (glycerol, lactate, and acetate), consistent with NAD(+)-dependent isocitrate dehydrogenase performing a critical role in oxidative function of the citric acid cycle. In addition, the IDH1 disruption strains grew at wild type rates in the absence of glutamate, indicating that these strains are not glutamate auxotrophs.     

Fatty acid utilization during development of the rat

The effects of dimethyl sulphoxide and glycerol on ox brain microsomal Na(+)+K(+)-stimulated adenosine triphosphatase (EC 3.6.1.3), K(+)-stimulated p-nitrophenyl phosphatase and K(+)-dependent muscle pyruvate kinase (EC 2.7.1.40) were studied. Dimethyl sulphoxide at concentrations below 20% (v/v) was found to stimulate the p-nitrophenyl phosphatase and pyruvate kinase by increasing their affinity for K(+) but to inhibit the Na(+)+K(+)-stimulated adenosine triphosphatase. The latter enzyme activity was also inhibited by glycerol, which like dimethyl sulphoxide, stimulated the K(+)-activated p-nitrophenyl phosphatase at a wide range of concentrations. The solvent effects were promptly reversed by dilution. Similarity was found between glycerol and dimethyl sulphoxide, on one hand, and ATP, on the other, in their stimulatory effect and their ability to increase the ouabain- and oligomycin-sensitivity of the K(+)-stimulated p-nitrophenyl phosphatase. However, only the solvents, not the ATP, increased the binding of K(+) by the microsomes. From the above findings it is suggested that solvents may act on K(+)-dependent enzymes by altering the state of solvation of the activating cation as well as by changing the enzyme structure.     

Purification and some properties of NAD-degrading purine nucleosidase from Aspergillus niger.

An enzyme which degrades NAD at the adenine-ribose linkage has been purified from the mycelial extract of Aspergillus niger. NADP, deamido-NAD, and purine nucleosides and nucleotides were also susceptible to the hydrolytic cleavage. Pyrimidine- and nicotinamide-ribose linkages were not attacked. The substrate specificity showed that the enzyme may be classified as a N-ribosyl-purine ribohydrolase (EC 3.2.2.1). The enzyme had a maximum activity in the pH range of 4.0-4.5 toward NAD. The Km values for NAD, 5'-AMP, and inosine were 3.0, 2.9 and 1.6mM respectively.     

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

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

Purification and properties of dihydroxyacetone kinase from Klebsiella pneumoniae.

Dihydroxyacetone (DHA) kinase of Klebsiella pneumoniae, a gene product of the dha regulon responsible for fermentative dissimilation of glycerol and DHA, was purified 120-fold to a final specific activity of 10 mumol X min-1 X mg of protein-1 at 30 degrees C. The enzyme, a dimer of a 53,000 +/- 5,000-dalton polypeptide, is highly specific for DHA (Km, ca.4 microM). Glycerol is not a substrate at 1 mM and is not an inhibitor even at 100 mM. The enzyme is not inhibited by 5 mM fructose-1,6-diphosphate. Ca2+ gives a higher enzyme activity than Mg2+ as a cationic cofactor. Escherichia coli glycerol kinase acts on both glycerol and DHA and is allosterically inhibited by fructose-1,6-diphosphate. Antibodies raised against E. coli glycerol kinase cross-reacted with K. pneumoniae glycerol kinase but not with K. pneumoniae DHA kinase.     

基于比速率及代谢流的黑曲霉突变株和野生株分析

黑曲霉具备优异的外源蛋白表达和分泌能力,从而被广泛应用于工业酶制剂的生产。通过研究黑曲霉突变株和野生株在相同培养条件下生理参数和代谢流的差异,确定了黑曲霉合成糖化酶过程中的限制性因素。宏观动力学分析发现,较之野生株,突变株具有较高的最大比生长速率,并且副产物得率降低了90%,底物利用率提高了近30%,表明突变株与野生株在碳源分配和产物转化率上具有明显的差异。利用流平衡分析（FBA）计算胞内代谢通量分布,发现还原力和核糖的供应水平是限制菌体合成的主要因素,而前体氨基酸是合成糖化酶最主要的限制性因素。这些研究结果为后续发酵工艺优化和菌株基因改造提供了有益的思路。