重组大肠杆菌发酵生产谷胱甘肽的氨基酸添加策略优化

对表达双功能谷胱甘肽合成酶的重组大肠杆菌发酵生产谷胱甘肽(Glutathione,GSH)进行氨基酸添加策略优化,结果表明:基本培养基中未添加氨基酸时GSH产量为0.81 g/L;诱导2 h后添加17 mmol/L半胱氨酸GSH产量为1.16 g/L,比不加氨基酸提高43%;添加17 mmol/L的3种前体氨基酸,GSH产量达到3.86 g/L,比只添加半胱氨酸提高2.33倍;进一步提高3种氨基酸添加量至25 mmol/L,GSH产量可达4.64 g/L,比不添加氨基酸提高4.73倍,总生产强度高达317.8 mg/(L·h),半胱氨酸转化为谷胱甘肽达到0.60 mol/mol;考察氨基酸添加模式发现一次性添加25 mmol/L氨基酸较恒速流加模式生产速率提高了29.8%。后续在50 L罐放大生产GSH,产量为4.31 g/L,总生产强度达到310.1 mg/(L·h),为工业化放大生产GSH奠定了基础。     

培养条件对离体丹参根苯丙氨酸解氨酶和多酚氧化酶活性的影响

以2年生丹参离体根为材料,研究了反应液pH、反应时间和材料预培养时间以及苯丙氨酸、肉桂酸和阿魏酸溶液处理对根中苯丙氨酸解氨酶(PAL)和多酚氧化酶(PPO)活性的影响.结果表明:(1)PPO和PAL的最适反应pH分别为6.0和8.8,反应时间分别为30 min和60 min,最适预培养时间为10~12 h.(2)苯丙氨酸处理能抑制PAL活性,且在0.062 5 mmol·L-1时抑制作用最大,但随浓度增加无规律性变化;浓度低于1.0 mmol·L-1的苯丙氨酸处理能提高PPO的活性,且在0.062 5 mmol·L-1时促进作用最强.(3)不同浓度肉桂酸均能抑制PAL活性,并在0.125 mmol·L-1时抑制作用最大,且低浓度(≤0.125 mmol·L-1)的影响比高浓度(≥0.25 mmol·L-1)更大;低浓度肉桂酸(≤0.25 mmol·L-1)处理能提高PPO的活性并在0.25 mmol·L-1时达最大值,而在0.25~2.0 mmol·L-1浓度范围内肉桂酸对PPO活性的抑制作用随浓度的升高而增强.(4)阿魏酸对PAL表现出产物反馈抑制作用,并在0.125 mmol·L-1时抑制作用最大,但对PPO的活性有促进作用,且在0.5 mmol·L-1时PPO活性最高.可见,离体丹参根的苯丙氨酸解氨酶和多酚氧化酶活性测定有其适宜的pH、反应时间和与培养时间,苯丙氨酸、肉桂酸和阿魏酸溶液对2种酶活性的影响不同且浓度间有差异.     

外源钙离子对小麦幼苗氮素代谢的影响

以普通小麦豫麦34为材料,研究了不同浓度的外源Ca2 对小麦幼苗氮素代谢的影响.在小麦第一片叶完全展开后,开始外源Ca2 处理,设0 (对照)、2、4 mmol · L-1 和8 mmol · L-1 4个Ca2 浓度梯度.处理5d后,测定氮同化酶活性、氮同化量及其它相关代谢物含量.结果表明,小麦幼苗叶片中硝酸还原酶(NR)和谷氨酰胺合成酶(GS)在2 mmol · L-1 Ca2 处理下活性比对照有显著增加,4 mmol · L-1 Ca2 处理的NR活性增加明显,但GS活性增加不显著;8 mmol · L-1 Ca2 处理下NR和GS活性比对照均明显降低.谷氨酸脱氢酶(NADH-GDH)活性在2 mmol · L-1 Ca2 处理下活性增加不明显,而在4、8 mmol · L-1 Ca2 处理下活性显著增加.小麦幼苗氮同化量以4 mmol · L-1处理最大,2 mmol · L-1处理与4 mmol · L-1之间差异不显著;Ca2 浓度为8 mmol · L-1时,氮素同化量明显降低.结果揭示了小麦幼苗不同氮同化途径对Ca2 的响应不同,GS途径比GDH途径对小麦氮素同化量的增加作用更大;4 mmol · L-1对小麦幼苗的氮素利用可能是最有效的Ca2 浓度.     

高产谷胱甘肽酿酒酵母的选育及发酵工艺的研究

以酿酒酵母SC-001为出发菌株,利用紫外诱变结合乙硫氨酸和氯化锌的底物选择筛选到1株有较高GSH产量的酿酒酵母菌株SC-001-24,其GSH产量为743 mg/L,比出发菌株提高了22%。进一步在10 L发酵罐中考察了不同发酵模式和前体氨基酸补加方式对酿酒酵母产GSH的影响,确定最佳发酵模式为补料分批发酵,最佳前体氨基酸补加策略为:初始培养基加入41 mmol/L半胱氨酸,发酵42 h后一次性补加半胱氨酸246 mmol,甘氨酸160 mmol,谷氨酸82 mmol。在最佳发酵条件下GSH产量达到1 280.4 mg/L。     

不同甘氨酸浓度对无菌水培番茄幼苗生长和氮代谢的影响

植物不但能吸收矿质氮(NH+4-N、NO-3-N),而且也能直接吸收有机态氮,如氨基酸、小分子蛋白质等.为探讨有机态氮浓度对番茄幼苗生长和氮代谢的影响,无菌水培条件下采用2个番茄品种(申粉918、沪樱932)设置4种不同浓度(0、1.5、3.0、6 0mmol·L-1)的甘氨酸态氮(Gly-N),研究了番茄幼苗干物质重、吸氮量、氮代谢相关产物和氮代谢关键酶活性.结果表明,无菌水培条件下,随营养液中Gly浓度的增加,番茄植株干物质重、总氮量、地上部和根系游离氨基酸、可溶性蛋白、地上部可溶性糖含量增加.与无氮对照相比,各处理均显著降低了番茄地上部淀粉含量(P<0.05),而Gly浓度对根系淀粉含量无显著影响.随营养液中Gly浓度的增加,番茄地上部和根系的硝酸还原酶(NR)、谷氨酸脱氢酶(NADH-GDH)、丙转氨酶(GPT)和谷草转氨酶(GOT)活性均提高.无氮对照的NR活性与1.5 mmol·L-1 Gly处理之间差异不显著,而与3.0 mmol·L-1和6.0 mmol·L-1 Gly两处理之间差异显著(P<0.05);1.5 mmol·L-1 Gly和3.0 mmol·L-1 Gly两个处理之间的地上部NADH-GDH、GPT和GOT活性差异不显著.Gly浓度与番茄植株干物质重、总氮量呈显著正相关(R2>0.905* *),这表明两个番茄品种均能直接吸收利用甘氨酸.沪樱932吸收Gly的能力显著大于申粉918(P<0.05).因此,Gly-N可以成为番茄生长的良好氮源,其生理效应受Gly浓度的影响;不同品种番茄对Gly的吸收利用能力不同.     

柱状田头菇菌丝对镉胁迫的抗氧化响应

研究了不同浓度Cd处理对柱状田头菇菌丝抗氧化酶及谷胱甘肽含量的影响.结果表明,在低浓度范围内随着Cd处理浓度的增加,菌丝抗氧化酶的活力上升,过氧化氢酶(CAT)与超氧化物歧化酶(SOD)的活性分别在Cd浓度为0.1和0.4mmol·L-1时达最大值;过氧化物酶(POD)、谷胱甘肽还原酶(GR)和脂氧合酶(LOX)的活性在Cd浓度为0.2mmol·L-1时达到峰值.而在高Cd浓度处理时,柱状田头菇菌丝抗氧化酶系(POD、CAT、SOD等)显著受到抑制.0.4~1.6mmol·L-1Cd处理可显著提高菌丝体内还原型谷胱甘肽(GSH)水平,却不影响氧化型谷胱甘肽(GSSG)含量.在整个试验过程中,均未检测到抗坏血酸及抗坏血酸过氧化物酶(APX)的活性.用聚丙烯酰胺凝胶电泳分析Cd胁迫下柱状田头菇菌丝抗氧化酶的同工酶谱发现,0.1~0.8mmol·L-1Cd处理可诱导过氧化物酶(POD)、酯酶(EST)和脂氧合酶(LOX)新同工酶的表达,提高组成型过氧化氢酶(CAT)、超氧化物歧化酶(SOD)同工酶的表达强度;1.6mmol·L-1Cd处理显著抑制POD、CAT、SOD等的表达.     

外源抗坏血酸对姜离体叶片光抑制和抗氧化酶活性的影响

以莱芜生姜(Zingiber of ficinale Rosc.)离体叶片为试验材料,研究了不同浓度抗坏血酸(AsA)对强光照射下姜离体叶片光抑制及抗氧化酶活性的影响.结果显示,在强光条件下,姜离体叶片的Fv/Fm、qp和qn随强光照射时间增加呈下降趋势.在照光10～30 min,较低浓度AsA(10、50、100 mmol·L-1)处理的Fv/Fm高于CK和150 mmol·L-1 AsA处理.10 mmol·L-1AsA处理的叶片qp值始终高于对照,所有AsA处理qN都高于对照.AsA处理叶片的超氧化物岐化酶(SOD)和脱氢抗坏血酸还原酶(DHAR)活性高于对照.强光照射30 min时,10、50、100 mmol·L-1AsA处理的过氧化物酶(POD)和抗坏血酸过氧化物酶(APX)活性高于对照和高浓度的AsA处理(150 mmol·L-1),尤以10 mmol·L-1 AsA处理最佳,Fv/Fm和qp分别比对照高7.22%、15.88%,qN和抗氧化酶活性也维持较高水平.研究表明外源AsA对强光引起的姜叶片光抑制有一定的保护作用.     

NaCl胁迫对西瓜幼苗生长和光合气体交换参数的影响

以早春红玉品种为材料,采用营养液水培法,研究了不同浓度NaCl胁迫对西瓜幼苗生长、叶片光合气体交换参数、质膜透性和脯氨酸含量的影响.结果表明:25 mmol·L-1NaCl处理9 d后对西瓜幼苗生长有促进作用,>75 mmol·L-1NaCl处理则显著抑制幼苗生长;NaCl处理显著提高了叶片光合色素含量,并在100 mmol·L-1NaCl处理下达到最大;叶片净光合速率、气孔导度和蒸腾速率均随NaCl浓度提高而显著降低;胞间CO2浓度随NaCl浓度提高呈先降低后升高的趋势,在75 mmol·L-1NaCl处理下降到最小;气孔限制值随NaCl浓度提高而增加,在75 mmol·L-1NaCl处理下达到最大值后趋于稳定;水分利用率随NaCl浓度提高呈先增加后降低的趋势,在75 mmol·L-1NaCl处理下达到最大;叶片质膜透性和脯氨酸含量均随NaCl浓度提高而显著增加.结果说明:NaCl胁迫显著抑制了西瓜叶片光合作用,且低浓度处理下光合速率降低的主要原因是气孔因素限制,高浓度胁迫下则转变为非气孔因素限制.     

燕麦幼苗对盐胁迫的响应及过氧化氢对响应的调节

为了探讨‘定莜6号’燕麦对盐胁迫的生理生化响应及H2O2的调节作用,采用水培方法,研究外源H2O2对盐胁迫下燕麦活性氧代谢、渗透溶质积累和Na+、K+平衡的影响。结果表明:小于100 mmol·L-1Na Cl未对‘定莜6号’幼苗的生长造成明显影响,150 mmol·L-1及以上浓度Na Cl使幼苗干重和叶片K+/Na+显著降低,O2-·产生速率、H2O2、丙二醛(MDA)、可溶性蛋白质和脯氨酸含量及过氧化氢酶(CAT)、质膜H+-ATP酶活性明显提高,但抗坏血酸(ASA)和谷胱甘肽(GSH)含量变化不大;外施5μmol·L-1H2O2可显著缓解150 mmol·L-1Na Cl胁迫对燕麦幼苗生长的抑制作用,使燕麦叶片超氧化物歧化酶(SOD)、抗坏血酸过氧化物酶(APX)和CAT活性及H2O2、GSH含量明显提高,O2-·产生速率和MDA含量显著降低;5μmol·L-1H2O2还提高了150 mmol·L-1Na Cl胁迫下燕麦叶片可溶性蛋白质、可溶性糖、有机酸和脯氨酸含量及质膜H+-ATP酶活性和K+/Na+,降低了游离氨基酸含量;表明外源H2O2可调控燕麦幼苗活性氧代谢和渗透溶质积累,维持K+、Na+平衡,从而增强耐盐性。     

温度对中华鲟幼鱼血液生化指标的影响

分别在15℃、20℃、25℃和30℃水温养殖中华鲟幼鱼66d,研究温度对中华鲟幼鱼8项血液生化指标的影响。结果表明:中华鲟幼鱼血液中能源物质总蛋白、白蛋白、血糖、总胆固醇和高密度脂蛋白浓度变化规律相似,均呈现先升高后降低的趋势,而且均在20℃时达到最高值,分别为15.98g·L-1、5.6g·L-1、4.85mmol·L-1、2.44mmol·L-1和0.61U.L-1;在30℃时血液总蛋白、白蛋白、血糖、总胆固醇和甘油三酯浓度达到最低值,分别为8.93g·L-1、3.1g·L-1、2.26mmol·L-1、1.34mmol·L-1和3.35mmol·L-1;代谢产物总胆红素浓度呈现先降低后升高的趋势,在15℃组最高,在25℃组最低,分别为2.50±0.84和1.65±0.10mmol·L-1;尿素浓度随着温度升高而下降,在30℃时最低,为0.54±0.36mmol·L-1,显著低于15℃组和20℃组。综合不同温度组中华鲟幼鱼血液生化指标的变动规律表明,20℃时中华鲟幼鱼能量支出较少,是其较适宜的生长温度。     

FosB, a cysteine-dependent fosfomycin resistance protein under the control of sigma(W), an extracytoplasmic-function sigma factor in Bacillus subtilis

We demonstrate that the Bacillus subtilis fosB(yndN) gene encodes a fosfomycin resistance protein. Expression of fosB requires sigma(W), and both fosB and sigW mutants are fosfomycin sensitive. FosB is a metallothiol transferase related to the FosA class of Mn(2+)-dependent glutathione transferases but with a preference for Mg(2+) and L-cysteine as cofactors.     

Enhancement of glutathione production by altering adenosine metabolism of Escherichia coli in a coupled ATP regeneration system with Saccharomyces cerevisiae

Aims: Adenosine triphosphate (ATP) during the enzymatic production of glutathione is necessary. In this study, our aims were to investigate the reason for low glutathione production in Escherichia coli coupled with an ATP regeneration system and to develop a new strategy to improve the system. Methods and Results: Glutathione can be synthesized by enzymatic methods in the presence of ATP and three precursor amino acids (l ‐glutamic acid, l ‐cysteine and glycine). In this study, glutathione was produced from E. coli JM109 (pBV03) coupled with an ATP regeneration system, by using glycolytic pathway of Saccharomyces cerevisiae WSH2 as ATP regenerator from adenosine and glucose. In the coupled system, adenosine used for ATP regeneration by S. cerevisiae WSH2 was transformed into hypoxanthine irreversibly by E. coli JM109 (pBV03). As a consequence, S. cerevisiae WSH2 could not obtain enough adenosine for ATP regeneration in the glycolytic pathway in spite of consuming 400 mmol l^?1 glucose within 1 h. By adding adenosine deaminase inhibitor to block the metabolism from adenosine to hypoxanthine, glutathione production (8·92 mmol l^?1) enhanced 2·74‐fold in the coupled system. Conclusions: This unusual phenomenon that adenosine was transformed into hypoxanthine irreversibly by E. coli JM109 (pBV03) revealed that less glutathione production in the coupled ATP regeneration system was because of the poor efficiency of ATP generation. Significance and Impact of the Study: The results presented here provide a strategy to improve the efficiency of the coupled ATP regeneration system for enhancing glutathione production. The application potential can be microbial processes where ATP is needed.     

Lysine biosynthesis in Saccharomyces. Conversion of α-aminoadipate into α-aminoadipic δ-semialdehyde

The reduction of alpha-aminoadipate to alpha-aminoadipic delta-semialdehyde by a cell-free extract of Saccharomyces is shown to be a three-step process. First the amino acid reacts with ATP to form an adenylyl derivative. Then the adenylyl derivative of alpha-aminoadipate is reduced in the presence of NADPH. In the third step the reduced adenylyl derivative of the amino acid is cleaved to form alpha-aminoadipic delta-semialdehyde. The presence of Mg(2+) is necessary for the first and second steps. The third step does not need any cofactors. The product of the first step was isolated by chromatography after incubating the cell-free extract of Saccharomyces with alpha-aminoadipate, ATP and Mg(2+). The isolated product was identified as an adenylyl derivative of alpha-aminoadipate and could be converted into alpha-aminoadipic delta-semialdehyde under the stated experimental conditions. The product of the second step was too unstable to be identified.     

Purification and Properties of an Exocellular γ-Glutamyl Arylamidase from Bacillus sp. Strain No. 12

An exocellular γ-glutamyl arylamide-hydrolyzing enzyme was produced by a Bacillus sp. in L-glutamate-containing medium. This enzyme was a tetrameric simple protein composed of two heavy subunits (Mr 56,000) and two light subunits (Mr 46,000). It hydrolyzed γ-amido, acyl and aryl bonds in L- and D-glutamyl compounds, and the activity on L-glutamic acid γ-p-nitroanilide was inhibited by the addition of glutamate and γ-glutamyl compounds but not by α-glutamyl compounds. The activity was stimulated by various dipeptides but not by free amino acids, L-Alanine, glycine, L-serine and L-cysteine inhibited the enzyme competitively. Addition of hy-droxylamine had no effect on the activity.     

Amino acid specificity of the Na+/alanine cotransporter in pancreatic acinar cells

The method of tight-seal whole-cell recording was used to study the amino-acid specificity of the Na+/alanine cotransporter in pancreatic acinar cells. Single cells or small clusters of electrically coupled cells were obtained by enzymatic dissociation of mouse pancreas. Inward currents were measured under 'zero-trans' conditions, i.e., at finite concentrations of Na+ and amino acid at the extracellular side and vanishing concentrations at the cytoplasmic side. The cotransporter, which corresponds to 'system A', as previously defined in the literature, was found to exhibit a wide tolerance to neutral amino acids (L-cysteine, L-serine, L-alanine, glycine, L-phenylalanine). Competition experiments with 2-methylaminoisobutyric acid (MeAIB) indicate that for glycine a second electrogenic transport system exists in pancreatic acinar cells.     

A novel metal-activated L-serine O-acetyltransferase from Thermus thermophilus HB8

L-Cysteine is an important amino acid in terms of its industrial applications. The biosynthesis of L-cysteine in enteric bacteria is regulated through the feedback inhibition by L-cysteine of L-serine O-acetyltransferase (SAT), a key enzyme in L-cysteine biosynthesis. We recently found that L-cysteine is overproduced in Escherichia coli strains expressing a gene encoding feedback inhibition-insensitive SAT. Further improvements in L-cysteine production are expected by the use of SAT with high stability. We report here the sat1 gene encoding SAT of an extreme thermophile, Thermus thermophilus HB8. The sat1 gene was cloned and overexpressed in E. coli cells based on the genome sequence in T. thermophilus HB8. The predicted amino acid sequence consists of 295 amino acids and is homologous to other O-acetyltransferase members. In particular, the carboxyl-terminal region shares approximately 30% identities with SATs found in bacteria and plants, despite showing only about 15% identity in the overall sequence. Enzymatic analysis and an atomic absorption study of the purified recombinant proteins revealed that the enzyme is highly activated by Co(2+) or Ni(2+), and contains Zn(2+) and Fe(2+). These results indicate that the T. thermophilus SAT is a novel type of enzyme different from other members of this protein family.     

Transport ofl-cysteine by rat renal brush border membrane vesicles

Brush border membranes were isolated from rat renal cortex by a divalent cation precipitation method. L-35S-cysteine uptake into the vesicles was measured by a rapid filtration method. Only minimal binding of the amino acid to the vesicles was observed. Sodium stimulates L-cysteine uptake specifically. Anion replacement experiments, experiments in the presence of potassium/valinomycin-induced diffusion potential as well as experiments with a potential-sensitive fluorescent dye document an electrogenic sodium-dependent uptake mechanism for L-cysteine. Tracer replacement experiments as well as the fluorescence experiments indicate a preferential transport of L-cysteine. Transport of L-cysteine is inhibited by L-alanine and L-phenylalanine but not by L-glutamic acid and the L-basic amino acids. Initial, linear influx kinetics provide evidence for the existence of two transport sites. The results suggest (a) sodium-dependent mechanism(s) for L-cysteine shared by other neutral amino acids.     

Elevated glutathione production by adding precursor amino acids coupled with ATP in high cell density cultivation of Candida utilis

Aims: Three precursor amino acids and adenosine triphosphate ( ATP) are necessary for fermentative production of glutathione. In this study, our aims were to develop a strategy to enhance glutathione production by adding three precursor amino acids coupled with ATP in high cell density (HCD) cultivation of Candida utilis. Methods and Results: A high-glutathione yeast strain, C. utilis WSH 02-08, was used in this study. Whole fermentative process for glutathione production was divided into two phases of cell growth and glutathione synthesis. Cells concentration was increased by HCD cultivation. Meanwhile, intracellular glutathione content was enhanced by the addition of three precursor amino acids. Concentrations of three precursor amino acids added at stationary phase were optimized by response surface methodology. Moreover, the addition of ATP 15 h after the addition of the three amino acids can further enhance glutathione production. Based on aforementioned phenomenon, a strategy of adding three precursor amino acids coupled with ATP was developed to enhance glutathione production. Conclusion: Without the addition of three precursor amino acids and the ATP, a total glutathione of 1123 mg l⁻¹ was achieved after 60-h cultivation. In comparison, addition of three precursor amino acid counterparts resulted in a total glutathione of 1841 mg l⁻¹. Moreover, by adding amino acids combined with ATP, a total glutathione of 2043 mg l⁻¹ was achieved after 72-h cultivation, increased by 81·9% and 11%, respectively, as compared with the control and the one without ATP addition. Significance and Impact of the Study: This is the first report on investigating changes of the intracellular three precursor amino acids and ATP, and γ-glutamylcysteine synthase activity in HCD cultivation of C. utilis for glutathione production. A strategy of combining addition of three precursor amino acids with ATP was developed to enhance glutathione production in C. utilis.     

The effect of magnesium ion deprivation on the synthesis of mucopeptide and its precursors in Bacillus subtilis

1. Mg(2+) or Mn(2+) starvation causes suspensions of Bacillus subtilis strain W 23 to accumulate bound amino sugars that are soluble in trichloroacetic acid. 2. The presence of chloramphenicol or puromycin produces higher intracellular concentrations of amino sugars during Mg(2+) starvation, but neither compound can stimulate the accumulation when Mg(2+) is present. 3. The major component of the amino sugar fraction extracted from cells deprived of Mg(2+) is a nucleotide containing uridine, phosphorus, N-acetylmuramic acid, alanine, glutamic acid and alphain-diaminopimelic acid in the molar proportions of 1:2:1:3:1:1. This compound represents at least 80% of the bound N-acetylhexosamine extracted by trichloroacetic acid. 4. Studies of the binding of this nucleotide with vancomycin support the proposal that it is the mucopeptide precursor UDP-N-acetylmuramyl-l-alanyl-d-glutaminyl- alphain-diaminopimelyl-d-alanyl-d-alanine. 5. A method is described for the isolation of this material labelled with [(3)H]alphain-diaminopimelic acid. 6. When Mg(2+) is supplied to cells previously starved of Mg(2+), the accumulated pool of amino sugars rapidly decreases. 7. The biosynthesis of mucopeptide is inhibited by 35-50% under conditions of Mg(2+) starvation. The presence of EDTA increases this inhibition to 70%. The amount of N-acetylhexosamine that accumulates is balanced exactly by the associated fall in mucopeptide synthesis. 8. ;Chase' experiments show that the accumulated N-acetylhexosamine compound is utilized in mucopeptide synthesis.     

Synergistic substrate inhibition of ent-copalyl diphosphate synthase: a potential feed-forward inhibition mechanism limiting gibberellin metabolism

Gibberellins (GAs) or gibberellic acids are ubiquitous diterpenoid phytohormones required for many aspects of plant growth and development, including repression of photosynthetic pigment production (i.e. deetiolation) in the absence of light. The committed step in GA biosynthesis is catalyzed in plastids by ent-copalyl diphosphate synthase (CPS), whose substrate, (E,E,E,)-geranylgeranyl diphosphate (GGPP), is also a direct precursor of carotenoids and the phytol side chain of chlorophyll. Accordingly, during deetiolation, GA production is repressed, whereas flux toward these photosynthetic pigments through their common GGPP precursor is dramatically increased. How this is accomplished has been unclear because no mechanism for regulation of CPS activity has been reported. We present here kinetic analysis of recombinant pseudomature CPS from Arabidopsis (Arabidopsis thaliana; rAtCPS) demonstrating that Mg(2+) and GGPP exert synergistic substrate inhibition effects on CPS activity. These results suggest that GA metabolism may be limited by feed-forward inhibition of CPS; in particular, the effect of Mg(2+) because light induces increases in plastid Mg(2+) levels over a similar range as that observed here to affect rAtCPS activity. Notably, this effect is most pronounced in the GA-specific AtCPS because the corresponding activity of the resin acid biosynthetic enzyme abietadiene synthase is 100-fold less sensitive to [Mg(2+)]. Furthermore, Mg(2+) allosterically activates the plant porphobilinogen synthase involved in chlorophyll production. Hence, Mg(2+) may have a broad role in regulating plastidial metabolic flux during deetiolation. Finally, the observed synergistic substrate/feed-forward inhibition of CPS also seems to provide a novel example of direct regulation of enzymatic activity in hormone biosynthesis.