Characterization of human DHRS4: an inducible short-chain dehydrogenase/reductase enzyme with 3beta-hydroxysteroid dehydrogenase activity

Human DHRS4 is a peroxisomal member of the short-chain dehydrogenase/reductase superfamily, but its enzymatic properties, except for displaying NADP(H)-dependent retinol dehydrogenase/reductase activity, are unknown. We show that the human enzyme, a tetramer composed of 27 kDa subunits, is inactivated at low temperature without dissociation into subunits. The cold inactivation was prevented by a mutation of Thr177 with the corresponding residue, Asn, in cold-stable pig DHRS4, where this residue is hydrogen-bonded to Asn165 in a substrate-binding loop of other subunit. Human DHRS4 reduced various aromatic ketones and α-dicarbonyl compounds including cytotoxic 9,10-phenanthrenequinone. The overexpression of the peroxisomal enzyme in cultured cells did not increase the cytotoxicity of 9,10-phenanthrenequinone. While its activity towards all-trans-retinal was low, human DHRS4 efficiently reduced 3-keto-C₁₉/C₂₁-steroids into 3β-hydroxysteroids. The stereospecific conversion to 3β-hydroxysteroids was observed in endothelial cells transfected with vectors expressing the enzyme. The mRNA for the enzyme was ubiquitously expressed in human tissues and several cancer cells, and the enzyme in HepG2 cells was induced by peroxisome-proliferator-activated receptor α ligands. The results suggest a novel mechanism of cold inactivation and role of the inducible human DHRS4 in 3β-hydroxysteroid synthesis and xenobiotic carbonyl metabolism.     

Structural basis for chiral substrate recognition by two 2,3-butanediol dehydrogenases

2,3-Butanediol dehydrogenase (BDH) catalyzes the NAD-dependent redox reaction between acetoin and 2,3-butanediol. There are three types of homologous BDH, each stereospecific for both substrate and product. To establish how these homologous enzymes possess differential stereospecificities, we determined the crystal structure of l-BDH with a bound inhibitor at 2.0 Å. Comparison with the inhibitor binding mode of meso-BDH highlights the role of a hydrogen-bond from a conserved Trp residue¹⁹². Site-directed mutagenesis of three active site residues of meso-BDH, including Trp¹⁹⁰, which corresponds to Trp¹⁹² of l-BDH, converted its stereospecificity to that of l-BDH. This result confirms the importance of conserved residues in modifying the stereospecificity of homologous enzymes.     

Identification and characterization of retinoid-active short-chain dehydrogenases/reductases in Drosophila melanogaster

Background

In chordates, retinoid metabolism is an important target of short-chain dehydrogenases/reductases (SDRs). It is not known whether SDRs play a role in retinoid metabolism of protostomes, such as Drosophila melanogaster.

Methods

Drosophila genome was searched for genes encoding proteins with ∼ 50% identity to human retinol dehydrogenase 12 (RDH12). The corresponding proteins were expressed in Sf9 cells and biochemically characterized. Their phylogenetic relationships were analyzed using PHYLIP software.

Results

A total of six Drosophila SDR genes were identified. Five of these genes are clustered on chromosome 2 and one is located on chromosome X. The deduced proteins are 300 to 406 amino acids long and are associated with microsomal membranes. They recognize all-trans-retinaldehyde and all-trans-3-hydroxyretinaldehyde as substrates and prefer NADPH as a cofactor. Phylogenetically, Drosophila SDRs belong to the same branch of the SDR superfamily as human RDH12, indicating a common ancestry early in bilaterian evolution, before a protostome–deuterostome split.

Conclusions

Similarities in the substrate and cofactor specificities of Drosophila versus human SDRs suggest conservation of their function in retinoid metabolism throughout protostome and deuterostome phyla.

General significance

The discovery of Drosophila retinaldehyde reductases sheds new light on the conversion of β-carotene and zeaxantine to visual pigment and provides a better understanding of the evolutionary roots of retinoid-active SDRs.     

Human carbonyl reductase 4 is a mitochondrial NADPH-dependent quinone reductase

A protein encoded in the gene Cbr4 on human chromosome 4q32.3 belongs to the short-chain dehydrogenase/reductase family. Contrary to the functional annotation as carbonyl reductase 4 (CBR4), we show that the recombinant tetrameric protein, composed of 25-kDa subunits, exhibits NADPH-dependent reductase activity for o- and p-quinones, but not for other aldehydes and ketones. The enzyme was insensitive to dicumarol and quercetin, potent inhibitors of cytosolic quinone reductases. The 25-kDa CBR4 was detected in human liver, kidney and cell lines on Western blotting using anti-CBR4 antibodies. The overexpression of CBR4 in bovine endothelial cells reveals that the enzyme has a non-cleavable mitochondrial targeting signal. We further demonstrate that the in vitro quinone reduction by CBR4 generates superoxide through the redox cycling, and suggest that the enzyme may be involved in the induction of apoptosis by cytotoxic 9,10-phenanthrenequinone.     

Molecular determinants for the stereospecific reduction of 3-ketosteroids and reactivity towards all-trans-retinal of a short-chain dehydrogenase/reductase (DHRS4)

DHRS4, a member of the short-chain dehydrogenase/reductase superfamily, reduces all-trans-retinal and xenobiotic carbonyl compounds. Human DHRS4 differs from other animal enzymes in kinetic constants for the substrates, particularly in its low reactivity to retinoids. We have found that pig, rabbit and dog DHRS4s reduce benzil and 3-ketosteroids into S-benzoin and 3α-hydroxysteroids, respectively, in contrast to the stereoselectivity of human DHRS4 which produces R-benzoin and 3β-hydroxysteroids. Among substrate-binding residues predicted from the crystal structure of pig DHRS4, F158 and L161 in the animal DHRS4 are serine and phenylalanine, respectively, in the human enzyme. Double mutation (F158S/L161F) of pig DHRS4 led to an effective switch of its substrate affinity and stereochemistry into those similar to human DHRS4. The roles of the two residues in determining the stereospecificity in 3-ketosteroid reduction were confirmed by reverse mutation (S158F/F161L) in the human enzyme. The stereochemical control was evaluated by comparison of the 3D models of pig wild-type and mutant DHRS4s with the modeled substrates. Additional mutation of T177N into the human S158F/F161L mutant resulted in almost complete kinetic conversion into a pig DHRS4-type form, suggesting a role of N177 in forming the substrate-binding cavity through an intersubunit interaction in pig and other animal DHRS4s, and explaining why the human enzyme shows low reactivity towards retinoids.     

The Crystal Structure of SDR-Type Pyridoxal 4-Dehydrogenase of Mesorhizobium loti

Pyridoxal 4-dehydrogenase catalyzes the irreversible oxidation of pyridoxal to 4-pyridoxolactone and is involved in degradation pathway I of pyridoxine, a vitamin B₆ compound. Its crystal structure was elucidated for the first time. Molecular replacement with (S)-1-phenylthanol dehydrogenase (PDB code 2EW8) was adopted to determine the tertiary structure of the NAD⁺-bound enzyme.     

A novel NADP+-dependent L-1-amino-2-propanol dehydrogenase from Rhodococcus erythropolis MAK154: a promising enzyme for the production of double chiral aminoalcohols

AIM: A novel NADP(+)-dependent L-1-amino-2-propanol dehydrogenase was isolated from Rhodococcus erythropolis MAK154, and characterized. METHODS AND RESULTS: The enzyme was inducibly produced on cultivation with aminoalcohols such as 1-amino-2-propanol, 1-amino-2-butanol and 2-aminocyclohexanol. The enzyme catalyses the NADP(+)-dependent oxidation of several aminoalcohols, and also the NADPH-dependent asymmetric reduction of an aminoketone compound to a double chiral aminoalcohol, d-pseudoephedrine. Amino acid sequence analysis showed that the enzyme might belong to the short-chain dehydrogenase/reductase family. CONCLUSIONS: NADP(+)-dependent L-1-amino-2-propanol dehydrogenase isolated from R. erythropolis MAK154 reversibly catalysed dehydrogenation of aminoalcohols, and exhibited a unique sterospecifity for the reduction reaction. SIGNIFICANCE AND IMPACT OF THE STUDY: The enzyme is a promising catalyst for the production of double chiral compound, d-pseudoephedrine, from prochiral substrate.     

Studies on the Formation and the Degradation of Glucose 1,6-Bisphosphate in the Beef Liver Homogenate

Four kinds of the enzyme reactions have been reported for the synthesis of Glc-1,6-P₂. However, any activity of Glc-1-P dismutase and phosphoglucokinase was not observed in the beef liver homogenate. When the liver homogenate was incubated with Glc-1-P and Fru-1,6-P₂, a significant amount of Glc-1,6-P₂ was formed. The Glc-1,6-P₂ synthesis activity from Glc-1-P and Fru-1,6-P₂ was caused by the action of phosphoglucomutase present in the liver homogenate. The most remarkable activity for Glc-1,6-P₂ synthesis was observed when the homogenate was incubated with Glc-1-P and glycerate-1,3-P₂. The Glc-1,6-P₂ synthesis activity from Glc-1-P and glycerate-1,3-P₂ was separated from the major peak of phosphoglucomutase activity by DEAE-Sephadex chromatography. The peak of Glc-1,6-P₂ synthesis activity, however, still retained phosphoglucomutase activity.

Glc-1,6-P₂ phosphatase activity was mainly observed in the mitochondria and microsome fraction. The properties of Glc-1,6-P₂ phosphatase were differentiated from those of acid phosphatase and Glc-6-P phosphatase.     

葡萄糖酸氧化杆菌木糖醇脱氢酶基因的克隆与表达

【目的】获得葡萄糖酸氧化杆菌(Gluconobacter oxydans CGMCC 1.637)的木糖醇脱氢酶基因,研究其酶学性质及碳源特别是D-阿拉伯醇和木糖醇对该酶活性的影响。【方法】通过已报道序列的木糖醇脱氢酶的保守区设计引物,用聚合酶链式反应(polymerase chain reaction,PCR)扩增获得目的基因片段。根据获得的片段序列设计引物克隆目的基因的5’和3’片段,将所获得的片段拼接,获得完整的木糖醇脱氢酶基因。通过构建工程菌获得重组蛋白,并利用氧化还原反应测定重组酶的活性。用含不同碳源的培养基培养G.oxydans CGMCC 1.637,并测定其破胞上清液木糖醇脱氢酶氧化木糖醇的活性;用不同碳源培养的G.oxydans CGMCC 1.637转化木酮糖,用高效液相色谱法测定木糖醇的产量。【结果】获得一个新的798bp的木糖醇脱氢酶基因,所编码的木糖醇脱氢酶含265个氨基酸,属于短链脱氢酶家族。酶学性质研究发现,该木糖醇脱氢酶催化木糖醇氧化的最适合条件为35℃、pH 10.0,最高活性为23.27 U/mg,催化木酮糖还原为木糖醇的最适条件为30℃、pH 6.0。最高活性为255.55 U/mg;该木糖醇脱氢酶的对木糖醇的Km和Vmax分别为78.97 mmol/L和40.17 U/mg。碳源诱导实验表明,d-山梨醇对G.oxydans CGMCC 1.637木糖醇脱氢酶的活性有明显的促进作用,而葡萄糖、果糖、木糖、木糖醇、D-阿拉伯醇对木糖醇脱氢酶活性有明显的抑制作用。而在转化实验中,用d-甘露糖培养的G.oxydans CGMCC 1.637的转化能力明显高于其他碳源培养的G.oxydans CGMCC 1.637的转化能力,其中,用阿拉伯醇培养的G.oxydans CGMCC 1.637的转化能力最低,仅为对照的35%。【结论】克隆自G.oxydans CGMCC 1.637的木糖醇脱氢酶基因是一个新的基因,用阿拉伯醇培养的G.oxydans CGMCC 1.637破胞液木糖醇脱氢酶活性低;且阿拉伯醇对G.oxydans CGMCC 1.637木酮糖的还原能力具有抑制作用。     

沙丘芦苇胸腺嘧啶二磷酸葡萄糖脱水酶的序列分析与结构预测

目的：用生物信息学方法对沙丘芦苇胸腺嘧啶二磷酸葡萄糖脱水酶（PcTGD）的序列进行分析与结构预测,为后续研究提供参考。方法：以GOR法预测了PcTGD的二级结构,以ProtScale分析它的疏水性,最终通过现有的序列同源性比较,用MODELLER预测了PcTGD的三维结构。结果：PcTGD具有高度保守的活性中心Tyr＊＊＊Lys和N末端的Gly＊Gly＊＊Gly的高度保守结构。结论：PcTGD属于一个短链脱氢酶／还原酶家族。     

Purification and Characterization of L-2,3-Butanediol Dehydrogenase of Brevibacterium saccharolyticum C-1012 Expressed in Escherichia coli

The L-2,3-butanediol dehydrogenase produced in E. coli JM109/pLBD2-CTC was purified by 5 steps. The molecular mass of this enzyme was estimated at 110 kDa and the subunit was mesured to be 30 kDa. The L-BDH had some differences from the BDHs from other sources in substrate specificity, pI value, pH stability, effects of divalent cations, and organic acids.     

The role of NADPH in the regulation of glucose-6-phosphate and 6-phosphogluconate dehydrogenases in rat adipose tissue

Summary Previous studies examining the regulation of the synthesis of G6PDH and 6PGDH in rat liver and adipose tissue have focused on the induction of these enzymes by different diets and some hormones. In rat liver these enzymatic activities seem to be regulated by a mechanism involving changes in the NADPH requirements. In this paper we have studied the effect of changes in the flux through different NADPH-consuming pathways on G6PDH and 6PGDH levels in adipose tissue and on the NADPH/NADP ratio. The results show that: I) an increase in the consumption of NADPH, caused by the activation of either fatty acid synthesis or detoxification systems which consume NADPH, is paralleled by an increase in the levels of these enzymes; II) when the increase in consumption of NADPH is prevented, the G6PDH and 6PGDH levels do not change.Abbreviations G6PDH Glucose-6-Phosphate Dehydrogenase - 6PGDH 6-Phosphogluconate Dehydrogenase - GR Glutathione Reductase - ME Malic Enzyme - tBHP t-Butyl Hydroperoxide - NF Nitrofurantoin - CumOOH Cumene Hydroperoxide     

Characterization of rat and mouse NAD+-dependent 3alpha/17beta/20alpha-hydroxysteroid dehydrogenases and identification of substrate specificity determinants by site-directed mutagenesis

In this study, we characterized rat and mouse aldo-keto reductases (AKR1C16 and AKR1C13, respectively) with 92% sequence identity. The recombinant enzymes oxidized non-steroidal alcohols using NAD⁺ as the preferred coenzyme, and showed low 3α/17β/20α-hydroxysteroid dehydrogenase (HSD) activities. The substrate specificity differs from that of rat NAD⁺-dependent 3α-HSD (AKR1C17) that shares 95% sequence identity with AKR1C16. To elucidate the residues determining the substrate specificity of the enzymes, we performed site-directed mutagenesis of Tyr24, Asp128 and Phe129 of AKR1C16 with the corresponding residues (Ser, Tyr and Leu, respectively) of AKR1C17. The double mutation (Asp128/Tyr-Phe129/Leu) had few effects on the substrate specificity, while the Tyr24/Ser mutant showed only 3α-HSD activity, and the triple mutation of the three residues produced an enzyme that had almost the same properties as AKR1C17. The importance of the residue 24 for substrate recognition was verified by the mutagenesis of Ser24/Tyr of AKR1C17 which resulted in a decrease in 3α-HSD activity and appearance of 17β- and 20α-HSD activities. AKR1C16 is also 92% identical with rat NAD⁺-dependent 17β-HSD (AKR1C24), which possesses Tyr24. The replacement of Asp128, Phe129 and Ser137 of AKR1C16 with the corresponding residues (Glu, Ser and Phe, respectively) of AKR1C24 increased the catalytic efficiency for 17β- and 20α-hydroxysteroids.