高等植物的3-羟基-3-甲基戊二酰辅酶A还原酶

介绍了植物3-羟基-3-甲基戊二酰辅酶A还原酶(HMGR)的结构和调控,并简略讨论了HMGR调控与植物类异戊二烯途径的关系.     

Overexpressing 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase (HMGR) in the Lactococcal Mevalonate Pathway for Heterologous Plant Sesquiterpene Production

Isoprenoids are a large and diverse group of metabolites with interesting properties such as flavour, fragrance and therapeutic properties. They are produced via two pathways, the mevalonate pathway or the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway. While plants are the richest source of isoprenoids, they are not the most efficient producers. Escherichia coli and yeasts have been extensively studied as heterologous hosts for plant isoprenoids production. In the current study, we describe the usage of the food grade Lactococcus lactis as a potential heterologous host for the production of sesquiterpenes from a local herbaceous Malaysian plant, Persicaria minor (synonym Polygonum minus). A sesquiterpene synthase gene from P. minor was successfully cloned and expressed in L. lactis. The expressed protein was identified to be a β-sesquiphellandrene synthase as it was demonstrated to be functional in producing β-sesquiphellandrene at 85.4% of the total sesquiterpenes produced based on in vitro enzymatic assays. The recombinant L. lactis strain developed in this study was also capable of producing β-sesquiphellandrene in vivo without exogenous substrates supplementation. In addition, overexpression of the strain’s endogenous 3-hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR), an established rate-limiting enzyme in the eukaryotic mevalonate pathway, increased the production level of β-sesquiphellandrene by 1.25–1.60 fold. The highest amount achieved was 33 nM at 2 h post-induction.     

The Role of the 3-Hydroxy 3-Methylglutaryl Coenzyme A Reductase Cytosolic Domain in Karmellae Biogenesis

In all cells examined, specific endoplasmic reticulum (ER) membrane arrays are induced in response to increased levels of the ER membrane protein 3-hydroxy 3-methylglutaryl coenzyme A (HMG-CoA) reductase. In yeast, expression of Hmg1p, one of two yeast HMG-CoA reductase isozymes, induces assembly of nuclear-associated ER stacks called karmellae. Understanding the features of HMG-CoA reductase that signal karmellae biogenesis would provide useful insights into the regulation of membrane biogenesis. The HMG-CoA reductase protein consists of two domains, a multitopic membrane domain and a cytosolic catalytic domain. Previous studies had indicated that the HMG-CoA reductase membrane domain was exclusively responsible for generation of ER membrane proliferations. Surprisingly, we discovered that this conclusion was incorrect: sequences at the carboxyl terminus of HMG-CoA reductase can profoundly affect karmellae biogenesis. Specifically, truncations of Hmg1p that removed or shortened the carboxyl terminus were unable to induce karmellae assembly. This result indicated that the membrane domain of Hmg1p was not sufficient to signal for karmellae assembly. Using beta-galactosidase fusions, we demonstrated that the carboxyl terminus was unlikely to simply serve as an oligomerization domain. Our working hypothesis is that a truncated or misfolded cytosolic domain prevents proper signaling for karmellae by interfering with the required tertiary structure of the membrane domain.     

Enhancement of Ganoderic Acid Accumulation by Overexpression of an N-Terminally Truncated 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Gene in the Basidiomycete Ganoderma lucidum

Ganoderic acids produced by Ganoderma lucidum, a well-known traditional Chinese medicinal mushroom, exhibit antitumor and antimetastasis activities. Genetic modification of G. lucidum is difficult but critical for the enhancement of cellular accumulation of ganoderic acids. In this study, a homologous genetic transformation system for G. lucidum was developed for the first time using mutated sdhB, encoding the iron-sulfur protein subunit of succinate dehydrogenase, as a selection marker. The truncated G. lucidum gene encoding the catalytic domain of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) was overexpressed by using the Agrobacterium tumefaciens-mediated transformation system. The results showed that the mutated sdhB successfully conferred carboxin resistance upon transformation. Most of the integrated transfer DNA (T-DNA) appeared as a single copy in the genome. Moreover, deregulated constitutive overexpression of the HMGR gene led to a 2-fold increase in ganoderic acid content. It also increased the accumulation of intermediates (squalene and lanosterol) and the upregulation of downstream genes such as those of farnesyl pyrophosphate synthase, squalene synthase, and lanosterol synthase. This study demonstrates that transgenic basidiomycete G. lucidum is a promising system to achieve metabolic engineering of the ganoderic acid pathway.     

Clinical Characteristics of Anti-3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Antibodies in Chinese Patients with Idiopathic Inflammatory Myopathies

Objective

The objective of this study was to detect the prevalence of anti-3-hydroxyl-3- methylglutaryl coenzyme A reductase (anti-HMGCR) antibodies in Chinese patients with idiopathic inflammatory myopathies (IIMs), and to analyze the clinical features of the antibody-positive IIM patients.

Methods

The presence of anti-HMGCR antibodies was detected in 405 patients with IIMs, 90 healthy controls, and 221 patients with other rheumatic diseases by using an ELISA kit. Clinical data from anti-HMGCR antibody-positive and -negative patients were compared. Long-term follow-up of the anti-HMGCR antibody-positive patients was conducted to evaluate the role of anti-HMGCR antibody in IIM disease prognosis.

Results

Of the 405 IIM patients, 22 (5.4%) were found to carry the anti-HMGCR antibody. These IIM patients were predominantly female (73%), and only 3 anti-HMGCR antibody-positive patients with IIM were exposure to statins. Most patients experienced progressive onset, and presented with muscular weakness. Dysphagia was observed in half of the patients (p < 0.01), and 15% of these patients experienced the complication of interstitial lung disease (ILD) (p > 0.05). Mean creatine kinase (CK) levels were higher in antibody-positive patients than in antibody-negative patients (p < 0.05). Muscle biopsies were available from 12 anti-HMGCR antibody-positive patients, eight who experienced myofiber necrosis and showed very little or no evidence of inflammatory cell infiltrates in their muscle biopsies. Of these eleven patients who were followed-up 2.5- to 29-month, 73% experienced improvement after treatment. A cross-sectional study showed that anti-HMGCR antibody levels were significantly associated with CK levels (r = 0.486, p = 0.026) as well as with Myositis Disease Activity Assessment (MYOACT) scores (r = -0.67, p = 0.003) during the initial visit. However, changes in serum anti-HMGCR antibody levels did not correlate with changes in CK levels, Manual Muscle Testing 8 (MMT-8) scores or MYOACT scores in long-term follow-up.

Conclusion

The major clinical features of anti-HMGCR antibody-positive Chinese IIM patients were muscle weakness and dysphagia, which were seen in patients with and without statin exposure. This subtype of patients were responsive to immunosuppressive treatment and received good prognoses after treatment, but serum levels of the anti-HMGCR antibody do not correlate with disease activity.     

The SUD1 Gene Encodes a Putative E3 Ubiquitin Ligase and Is a Positive Regulator of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity in Arabidopsis

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the major rate-limiting step of the mevalonic acid (MVA) pathway from which sterols and other isoprenoids are synthesized. In contrast with our extensive knowledge of the regulation of HMGR in yeast and animals, little is known about this process in plants. To identify regulatory components of the MVA pathway in plants, we performed a genetic screen for second-site suppressor mutations of the Arabidopsis thaliana highly drought-sensitive drought hypersensitive2 (dry2) mutant that shows decreased squalene epoxidase activity. We show that mutations in SUPPRESSOR OF DRY2 DEFECTS1 (SUD1) gene recover most developmental defects in dry2 through changes in HMGR activity. SUD1 encodes a putative E3 ubiquitin ligase that shows sequence and structural similarity to yeast Degradation of α factor (Doα10) and human TEB4, components of the endoplasmic reticulum–associated degradation C (ERAD-C) pathway. While in yeast and animals, the alternative ERAD-L/ERAD-M pathway regulates HMGR activity by controlling protein stability, SUD1 regulates HMGR activity without apparent changes in protein content. These results highlight similarities, as well as important mechanistic differences, among the components involved in HMGR regulation in plants, yeast, and animals.     

Occurrence of 3-Hydroxy-3-methyl-glutaryl Coenzyme A Reductase in Sweet Potato

Illumination of the dark-grown Euglena gracilis, both the wild-green type and a permanently bleached mutant, for 4 hr at 2,000 lux caused about 6-fold increase of the cellular content of total l-ascorbic acid. The increase was mainly due to an increase of reduced-form l-ascorbic acid. From the action spectrum only blue light was found to be effective for the increase. Darkening stopped the increase and reillumination started a renewed increase. The activity of l-gulono-γ-lactone dehydrogenase, catalyzing the last step of l-ascorbic acid biosynthesis, was also increased two fold by illumination for 2 hr, and was changed in parallel to that of the cellular content of l-ascorbic acid depending on the presence or absence of illumination. The augmentation of l-ascorbic acid formation was markedly inhibited by various inhibitors and uncouplers, but not by dichlorophenyldimethylurea. The results in sum suggest that the light-dependent increase of l-ascorbic acid formation in E. gracilis is not primarily associated with photosynthesis, but is apparently related to the adaptation of the dark-grown cells to the illuminated state.     

Detection of Eubacterial 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductases from Natural Populations of Actinomycetes

Three natural populations of actinomycetes were investigated by PCR for the presence of type I 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG CoA), a gene associated with isoprenoid biosynthesis. The populations were obtained from an agricultural site (69 isolates), a coastal salt marsh (220 isolates), and a desert soil (96 isolates). A set (34) of standard actinomycete reference strains were also investigated. The target gene was only detected in 5 of the 419 actinomycetes screened, which represented 4 from the coastal salt marsh and one reference strain. The isolates that contained the gene were taxonomically diverse (4 Streptomyces spp. and 1 Nocardia sp.). These results suggest that type I HMG CoA containing pathways are rare in actinomycetes and their distribution within actinomycetes populations is not random.     

3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase Activity in Ochromonas malhamensis: A System to Study the Relationship between Enzyme Activity and Rate of Steroid Biosynthesis

3-Hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the key regulatory enzyme of the isoprenoid pathway, was found to be predominantly microsomal in Ochromonas malhamensis, a chrysophytic alga. Detection of HMG-CoA reductase requires the presence of 1% bovine serum albumin during cell homogenization, and the activity is stimulated by the presence of Triton X-100. The enzyme has a pH optimum of 8.0 and an absolute requirement for NADPH. When grown in 10 micromolar mevinolin, a competitive inhibitor of HMG-CoA reductase, O. malhamensis shows a 10- to 15-fold increase in HMG-CoA reductase activity (after washing) with little or no effect on cell growth rate. Cultures can be maintained in 10 micromolar mevinolin for months. O. malhamensis produces a large amount (1% dry weight) of poriferasterol, a product of the isoprenoid pathway. The addition of 10 micromolar mevinolin initially blocked poriferasterol biosynthesis by >90%; within 2 days the rate of synthesis returned to normal levels. Immediately after mevinolin was washed from the 2-day culture, there was a transient 2.5-fold increase in the rate of poriferasterol biosynthesis. The rate of poriferasterol biosynthesis and the level of HMG-CoA reductase activity both fell to control levels within hours.     

Cholesterol Biosynthesis and Its Regulation in Dissociated Cell Cultures of Fetal Rat Brain: Developmental Changes and the Role of 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase

Primary cultures of cells dissociated from fetal rat brain were utilized to define the developmental changes in cholesterol biosynthesis and the role of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in the regulation of these changes. Cerebral hemispheres of fetal rats of 15-16 days of gestation were dissociated mechanically into single cells and grown in the surface-adhering system. Cholesterol biosynthesis, studied as the rate of incorporation of [14C]acetate into digitonin-precipitable sterols, was shown to exhibit two distinct increases in synthetic rates, a prominent increase after 6 days in culture and a smaller one after 14 days in culture. Parallel measurements of HMG-CoA reductase activity also demonstrated two discrete increases in enzymatic activity, and the quantitative and temporal aspects of these increases were virtually identical to those for cholesterol synthesis. These data indicate that cholesterol biosynthesis undergoes prominent alterations with maturation and suggest that these alterations are mediated by changes in HMG-CoA reductase activity. The timing of the initial prominent peak in both cholesterol biosynthesis and HMG-CoA reductase activity at 6 days was found to be the same as the timing of the peak in DNA synthesis, determined as the rate of incorporation of [3H]thymidine into DNA. The second, smaller peak in reductase activity and sterol biosynthesis at 14 days occurred at the time of the most rapid rise in activity of the oligodendroglial enzyme, 2':3'-cyclic nucleotide 3'-phosphohydrolase (CNP). These latter observations suggest an intimate relationship of the sterol biosynthetic pathway with cellular proliferation and with oligodendroglial differentiation in developing mammalian brain.     

Dietary Modification of the Activation State of Intestinal 3-Hydroxy-3-methylglutaryl Coenzyme A (HMG-CoA) Reductase

To ascertain whether the phosphorylation-dephosphorylation reaction is actually involved in the in vivo regulation of intestinal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, dietary modulation of the activation state of the enzyme was studied in isolated epithelial cells of rats. Substitution of a sucrose-enriched semipurified diet for the commercial non-purified diet caused a significant increase in jejunal activity with a concomitant decrease in ileal activity. Jejunal activity increased without influencing the activation state whereas at the early stage of dietary manipulation, there was a rapid decrease in apparent activity compared to total activity in the ileum, hence the reduction of the activation state. These observations favor the view that the phosphorylation (inactivation) reaction is responsible for the regulation of intestinal HMG-CoA reductase in vivo. In contrast, dietary fat-dependent stimulation of jejunal reductase activity was mainly attributable to an increase in enzyme protein rather than in the level of the activation. The results suggest a complex controlling feature of the cholesterol synthesis in the intestine.     

Mechanism of Photoregulated Carotenogenesis in Rhodotorula minuta V. Photoinduction of 3-Hydroxy-3-Methyl Glutaryl Coenzyme A Reductase

The effect of light on the activity of 3-hydroxy-3-methylglutarylCoenzyme A (HMG-CoA) reductase in Rhodotorula minuta was studiedin cell-free extracts prepared from cells grown under variouslight conditions. HMG-CoA reductase activity in cells grown under continuous illuminationwas higher than that in cells grown in the dark, and dependedon the light intensity used during incubation. The relationshipbetween activity [A (nmol/mg-N/min)] and light intensity [I(erg/cm²/sec)] was expressed by the equation A=0.72 log I$0.80. Illumination at –1.5?C followed by dark incubation at26?C resulted in a rapid increase in HMG-CoA reductase activityimmediately after the beginning of incubation. This photoinducedHMG-CoA reductase activity was regulated by the light dose andfollowed the Roscoe-Bunsen reciprocity law. When cycloheximide was added immediately after the beginningof incubation in the dark, the increase in HMG-CoA reductaseactivity was completely inhibited. The inhibitory effect ofcycloheximide, however, gradually decreased with the delay ofthe addition. On the basis of these results we have postulated that the photoregulationof carotenogenesis in Rh. minuta results from the photoregulationof HMG-CoA reductase synthesis. (Received November 7, 1981; Accepted March 19, 1982)     

一种基因工程改造的NADH高亲和力木糖还原酶突变基因可促进酿酒酵母发酵木糖生成乙醇

在导入表达毕赤酵母(Pichia stipitis)木糖还原酶(xylose reductase,XR)和木糖醇脱氢酶(xylitol dehydrogenase,XDH)基因的重组酿酒酵母中,木糖还原酶活性主要依赖辅酶NADPH,木糖醇脱氢酶活性依赖辅酶 NAD+,两者的辅助因子不同导致细胞内电子氧化还原的不平衡,是造成木糖醇积累,影响木糖代谢和乙醇产量的主要原因之一.将经过基因工程改造获得的NADH高亲和力的木糖还原酶突变基因m1,与毕赤酵母木糖醇脱氢酶(PsXDH)基因xyl2共转染酿酒酵母AH109,以转染毕赤酵母木糖还原酶(PsXR)基因xyl1和xyl2重组质粒的酵母细胞为对照菌株,在SC/-Leu/-Trp营养缺陷型培养基中进行筛选,获得的阳性转化子分别命名为AH-M-XDH和AH-XR-XDH.重组酵母在限制氧通气条件下对木糖和葡萄糖进行共发酵摇瓶培养,HPLC检测发酵底物的消耗和代谢产物的产出情况.结果显示,与对照菌株AH-XR-XDH相比,AH-M-XDH的木糖利用率明显提高,乙醇得率增加了16%,木糖醇产生下降了41.4%.结果证实,通过基因工程改造的木糖代谢关键酶,可用于酿酒酵母发酵木糖生产乙醇,其能通过改善酿酒酵母细胞内氧化还原失衡的问题,提高木糖利用率和乙醇产率.