灰葡萄孢菌AUR1基因内含子对肌醇磷脂酰神经酰胺合成酶表达的影响及致病性

【目的】AUR1编码的肌醇磷脂酰神经酰胺(IPC)合成酶是真菌鞘脂代谢的关键酶,在转录水平和翻译水平研究AUR1内含子对其基因表达的影响,以及AUR1内含子对相关致病因子的影响,为内含子调控基因表达的分子机制提供理论依据。【方法】实时定量PCR测定野生型灰葡萄孢菌(BcAUR1)和AUR1缺失115 bp内含子突变体(BcAUR1a)的mRNA表达量,高效液相层析测定IPC合成酶活性,分别采用辣根过氧化物酶法、邻苯三酚自氧化法、愈创木酚法和紫外分光光度法测定单位菌体的H2O2含量、超氧化物歧化酶(SOD)、过氧化物酶(POD)和过氧化氢酶(CAT)的酶活力。【结果】突变体BcAUR1a的IPC合成酶基因cDNA测序结果表明,IPC合成酶无氨基酸突变。实时定量PCR和高效液相层析的结果表明BcAUR1a的AUR1基因mRNA表达量和IPC合成酶活力比野生型BcAUR1分别增加了50.2%和14.16%。短梗霉素A(AbA)显著刺激BcAUR1 H2O2、SOD、POD和CAT的分泌,但对BcAUR1a的这几种物质的分泌无显著影响。【结论】突变体BcAUR1a的AUR1基因在转录和翻译水平上表达上调,AbA显著增强野生型灰葡萄孢菌致病力,但对突变体影响较小。突变体产生了对AbA的抗性,推测AUR1基因内含子在AUR1基因表达调控中起转录抑制子的作用。     

定向进化提高灰盖鬼伞过氧化物酶染织废水脱色效率

【目的】获得染织废水脱色能力增强的灰盖鬼伞过氧化物酶。【方法】使用基因合成及定点突变平台合成突变灰盖鬼伞过氧化物酶基因CIPmt4(I49S、V53A、M166F和M242I),并调整密码子至毕赤酵母偏好性。以CIPmt4为模板进行定向进化,经过三轮易错PCR和高通量筛选得到一个酶学性质显著改善的突变体(CIPmt5)。通过3D建模和分子动力学模拟分析蛋白的结构及热稳定性,并进一步研究CIPmt5和野生型CIP对刚果红、氨基黑、甲基橙、次甲基蓝、苯胺蓝、结晶紫、溴酚蓝共7种染料的脱色能力。【结果】序列分析显示该突变体积累了I49S、V53A、T121A、M166F和Y272F共5个氨基酸突变,与野生型灰盖鬼伞过氧化物酶相比,以ABTS为底物酶活性是野生型的2.01倍(24.44 U/mg),最适反应p H由5.0提高到6.5,最适反应温度由25°C提高到45°C。除次甲基蓝外对其它染料脱色的最适p H都往中、碱性方向偏移,脱色率普遍高于野生型。模型分析显示CIPmt5活性中心更开放,热稳定性增强。【结论】突变体酶CIPmt5能够更好地替代野生型灰盖鬼伞过氧化物酶应用于染织业染料脱色、化工废水和染织废水的生物修复。     

腾冲嗜热厌氧菌丙氨酸消旋酶底物通道氨基酸位点的功能

【目的】通过定点突变探究腾冲嗜热厌氧菌MB4中生物合成型丙氨酸消旋酶Tt Alr底物通道内氨基酸位点A172和S173的功能。【方法】利用定点突变PCR技术构建突变体,通过亲和层析法纯化酶蛋白,采用D-氨基酸氧化酶偶联法检测各突变蛋白的活性及其稳定性。【结果】通过定点突变PCR成功得到8个突变体,酶学特性分析发现,A172位点突变为丝氨酸(S)后酶蛋白的相对活性有所提升,但含有该位点突变的酶蛋白稳定性均大幅下降;S173位点突变为天门冬氨酸(D)后导致突变体蛋白的最适反应温度提升了15°C,半衰期大幅延长,但相对活性明显下降。【结论】丙氨酸消旋酶Tt Alr底物通道内A172和S173位点均是影响酶蛋白催化活性和稳定性的关键位点。     

理性设计提高酯合成催化反应脂肪酶的热稳定性

【背景】南极假丝酵母脂肪酶B (Candida antarctica lipase B,CALB)具有优异的酯合成活性,是在非水相催化中应用极为广泛的工业用酶。【目的】在保留CALB优秀催化性能的基础上,提高CALB的热稳定性。【方法】采用预测软件PoPMuSiC和FoldX计算CALB潜在热稳定性突变位点,并根据氨基酸残基的空间位置进一步筛选。利用重叠延伸PCR技术在基因calb中引入10个单点突变,于毕赤酵母GS115中表达。【结果】点突变A146G、A151P、L278M均能有效提高CALB的热稳定性。在单点突变的基础上,组合突变体A146G-L278M和A146G-L278M-A151P的热稳定性得到进一步提高。与野生型相比,突变体A146G-L278M和A146G-L278M-A151P的最适反应温度均提高了5°C,T_m值分别提高了3.3°C和4.2°C。此外,合成己酸乙酯的酶促反应动力学分析表明,相比于野生型,突变体A146G-L278M和A146G-L278M-A151P对己酸和乙醇均具有更高的亲和力,且对己酸的催化效率k_(catA)/K_(m A)是野生型的4.1倍。通过分子动力学模拟,从分子水平阐明了突变体A146G-L278M和A146G-L278M-A151P热稳定性提高的机制。【结论】本研究采用的理性设计策略对提高CALB的热稳定性是行之有效的,该策略可作为其他工业用酶提高热稳定性的参考。     

定点突变对酰胺水解酶DamH可溶性表达和酶活的影响

摘要:【目的】DamH是一种具有酯酶活性的酰胺水解酶,其非活性中心氨基酸残基的突变对重组酶可溶性表达和比酶活产生一定的影响。拟探索DamH的活性中心氨基酸残基构成,并对其非活性中心氨基酸残基突变对可溶性表达和比酶活的影响进行研究。【方法】通过重叠延伸的方法对DamH可能的活性中心氨基酸S149、E244和H274以及非活性中心氨基酸D165及N192进行定点突变,通过静息细胞测活验证了S149、E244和H274 在催化2-氯-N-(2’-甲基-6’-乙基苯基)乙酰胺(CMEPA)水解反应中的作用,通过Ni2+- NTA亲和层析对D165及N192突变子进行纯化,对突变株和野生型比酶活进行比较。【结果】研究表明S149A使DamH的CMEPA 水解酶活性下降为野生型的5%,E244A和H274A突变导致其失去活性;D165P和N192P突变影响到DamH的可溶性表达,表达量分别为野生型的28.2%和20.8%,突变子N192P、D165P比酶活分别为野生型比酶活的55.5%和49.7%。【结论】DamH催化酯类底物和芳基酰胺类底物可能共用同一活性中心S149、E244和H274,其两个α螺旋的转角处氨基酸侧链极性和刚性结构的改变对可溶性表达以及活性有很大的影响。     

长双歧杆菌N-乙酰氨基己糖1-位激酶的活性位点

【目的】研究长双歧杆菌(Bifidobacterium longum)JCM1217的N-乙酰氨基己糖1-位激酶(Nacetylhexosamine 1-kinase,Nah K)中对催化活性有影响的位点。【方法】利用点突变试剂盒,获得Nah K的4个位点的共10种单点突变体表达菌株。诱导表达并纯化野生型和突变体酶,用DNS法和NADH偶联的微孔板分光光度法检测野生型及突变体酶的最适p H和最适Mg~(2+)浓度,并测定酶促反应动力学参数。【结果】D208A、D208N、D208E和I24A四种突变体的催化活性几乎丧失。突变体H31A、H31V、F247A和I24V的最适p H由野生型的7.5变为7.0,突变体H31A和F247A的最适Mg~(2+)浓度由野生型的5 mmol/L变为10 mmol/L。反应动力学参数测定结果表明,突变体F247Y对底物Glc NAc/Gal NAc及ATP的催化活性均高于野生型。【结论】通过定点突变,确定了对Nah K催化活性有影响的4个位点,并且获得了一个催化效率提高的突变体(F247Y),为进一步对Nah K进行分子改造奠定了一定基础。     

灰葡萄孢菌AUR1基因真核表达载体的构建、表达及酶活性分析

为了研究灰葡萄孢菌肌糖磷脂酰神经酰胺合成酶(BcAUR1基因)的表达及酶活性,采用RT-PCR方法,利用含有FLAG标签以及BamH Ⅰ、Xho Ⅰ酶切位点的AUR1特异引物从灰葡萄孢菌中扩增得到BcAUR1基因.将BcA UR1基因与穿梭质粒pYES2重组,得到pYES2-BcAUR1质粒采用醋酸锂转化法导入酿酒酵母尿嘧啶突变菌株△yor1中,Western blotting检测肌糖磷脂酰神经酰胺(IPC)合成酶表达,HPLC检测IPC合成酶活力.结果显示pYE S2-BcA UR1在酿酒酵母尿嘧啶突变菌株△yorl中获得表达,pYES2-BcA UR1转化子IPC合成酶活性显著增高,比空载转化子约提高1倍.低浓度的AbA能够抑制空载pYES2酵母转化子生长,但pYES2-BcA UR1酵母转化子能抵抗AbA对菌体生长的抑制.     

红纹黄单胞菌a-氨基酸酯水解酶的克隆、序列分析及热稳定性提高

【目的】克隆红纹黄单胞菌α-氨基酸酯水解酶基因全序列,对序列进行生物信息学分析,并提高酶的热稳定性。【方法】利用多聚酶链式反应(PCR)克隆α-氨基酸酯水解酶基因全序列;应用生物信息学软件对获得的基因序列及编码的蛋白序列进行分析;通过同源建模,预测红纹黄单胞菌α-氨基酸酯水解酶的三维结构;通过定点突变替换氨基酸序列中高度柔性的位点,提高该酶的热稳定性。【结果】从红纹黄单胞菌(Xanthomonas rubrillineans)中扩增得到α-氨基酸酯水解酶基因aeh(GenBank登录号JF744990),核苷酸序列长度1 917 bp,编码638个氨基酸。序列比对和同源性分析显示,该酶与白纹黄单胞菌Xanthomonas albilineans str.GPE PC73的肽酶及地毯草黄单胞菌Xanthomonas axono-podis pv. citri str. 306的戊二酰-7-氨基头孢烷酸酰化酶氨基酸序列相似性最高,分别为91%和83%,系统进化分析表明,该酶与白纹黄单胞菌Xanthomonas albilineans str. GPEPC73的肽酶亲缘性最高。基于预测的三维模型,对高度柔性的位点进行饱和突变,从282株突变体中筛选得到3株T50较野生型高5°C以上的突变体。【结论】对红纹黄单胞菌AEH的氨基酸序列分析有助于探索同源蛋白的进化过程。对高度柔性位点进行饱和突变的策略可以用于提高热稳定性。     

GH42家族保守氨基酸位点累积突变对Geobacillus stearothermophilus来源β-半乳糖苷酶BgaB催化活性的影响

【背景】β-半乳糖苷酶转糖苷活性弱,产物低聚半乳糖(galactooligosaccharides, GOS)易被水解,致其催化得率普遍较低。【目的】以GH42家族Geobacillus stearothermophilus来源β-半乳糖苷酶BgaB为对象,探讨家族保守氨基酸位点突变对β-半乳糖苷酶BgaB催化活性的影响。【方法】在单点突变体功能研究基础上,采用定点突变与化学修饰相结合的方法,对保守氨基酸位点E303与F341进行累积突变。【结果】与野生型酶相比,所构建双点突变体Ox-E303C/F341S水解活性降低为30%;GOS最大得率由0.75%提高到19.50%。【结论】家族保守氨基酸位点累积突变能够使单点突变体功能得到共同进化,降低β-半乳糖苷酶水解活性和底物抑制作用,能够提高其转糖苷催化活性。     

糖基转移酶基因rbfCxoo缺失突变导致水稻白叶枯病菌毒性表达增强

摘 要：【目的】阐明水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,简称Xoo)基因组中推导的脂多糖O抗原合成蛋白基因rbfCxoo (XOO2599) 的结构和生物学功能。【方法】通过基因克隆、序列分析、缺失突变和表型测定,对rbfCxoo的分子特征及其功能进行了鉴定。【结果】 用特异性引物进行PCR扩增,从野生型菌株PXO99A基因组DNA中成功地获得了与己测序菌株KACC10331序列完全一致的全长基因序列; RbfCxoo序列N端和C端分别具有一个糖基转移酶的保守结构域(Glycos_transf_2)。用标记置换法获得了基因缺失突变体△rbfCxoo。与PXO99A相比,△rbfCxoo 脂多糖O抗原合成能力并未发生变化,但鞭毛素糖基化能力有所降低。此外,△rbfCxoo鞭毛运动性、生物膜形成和胞外纤维素酶和木聚糖酶活性都无明显改变,但对水稻的致病性显著增强,毒性相关基因的表达也有所增加。【结论】RbfCxoo可能与细菌鞭毛素糖基化修饰以及毒性表达有关。     

AUR1, a novel gene conferring aureobasidin resistance onSaccharomyces cerevisiae: a study of defective morphologies in Aur1p-depleted cells

Aureobasidin A (AbA), a cyclic depsipeptide produced byAureobasidium pullulans R106, is highly toxic to fungi includingSaccharomyces cerevisiae. We isolated several dominant mutants ofS. cerevisiae which are resistant to more than 25 µg/ml of AbA. From a genomic library of one suchAUR1 mutant, theAUR1 ^R (foraureobasidinresistant) mutant gene was isolated as a gene that confers resistance to AbA on wild-type cells. Its nucleotide sequence showed that the predicted polypeptide is a hydrophobic protein composed of 401 amino acids, which contains several possible transmembrane domains and at least one predicted N-linked glycosylation site. Comparison of the mutant gene with the wild-typeaur1 ⁺ gene revealed that the substitution of Phe at position 158 by Tyr is responsible for acquisition of AbA resistance. We suggest that the gene product of the wild-typeaur1 ⁺ is a target for AbA on the basis of following results. Firstly, cells that overexpress the wild-typeaur1 ⁺ gene become resistant to AbA, just as cells with anAUR1 ^R mutation do. Secondly, disruption of theaur1 ⁺ gene demonstrated that it is essential for growth. Thirdly, in the cells with a disruptedaur1 locus, pleiotropic morphological changes including disappearance of microtubules, degradation of tubulin and abnormal deposition of chitin were observed. Some of these abnormalities are also observed when wild-type cells are treated with AbA. The abnormality in microtubules suggests that the Aur1 protein is involved in microtubule organization and stabilization.     

AUR1, a novel gene conferring aureobasidin resistance onSaccharomyces cerevisiae: a study of defective morphologies in Aur1p-depleted cells

Aureobasidin A (AbA), a cyclic depsipeptide produced byAureobasidium pullulans R106, is highly toxic to fungi includingSaccharomyces cerevisiae. We isolated several dominant mutants ofS. cerevisiae which are resistant to more than 25 µg/ml of AbA. From a genomic library of one suchAUR1 mutant, theAUR1 ^R (foraureobasidinresistant) mutant gene was isolated as a gene that confers resistance to AbA on wild-type cells. Its nucleotide sequence showed that the predicted polypeptide is a hydrophobic protein composed of 401 amino acids, which contains several possible transmembrane domains and at least one predicted N-linked glycosylation site. Comparison of the mutant gene with the wild-typeaur1 ⁺ gene revealed that the substitution of Phe at position 158 by Tyr is responsible for acquisition of AbA resistance. We suggest that the gene product of the wild-typeaur1 ⁺ is a target for AbA on the basis of following results. Firstly, cells that overexpress the wild-typeaur1 ⁺ gene become resistant to AbA, just as cells with anAUR1 ^R mutation do. Secondly, disruption of theaur1 ⁺ gene demonstrated that it is essential for growth. Thirdly, in the cells with a disruptedaur1 locus, pleiotropic morphological changes including disappearance of microtubules, degradation of tubulin and abnormal deposition of chitin were observed. Some of these abnormalities are also observed when wild-type cells are treated with AbA. The abnormality in microtubules suggests that the Aur1 protein is involved in microtubule organization and stabilization.     

Aureobasidin A arrests growth of yeast cells through both ceramide intoxication and deprivation of essential inositolphosphorylceramides

All mature Saccharomyces cerevisiae sphingolipids comprise inositolphosphorylceramides containing C26:0 or C24:0 fatty acids and either phytosphingosine or dihydrosphingosine. Here we analysed the lipid profile of lag1 Δ lac1 Δ mutants lacking acyl-CoA-dependent ceramide synthesis, which require the reverse ceramidase activity of overexpressed Ydc1p for sphingolipid biosynthesis and viability. These cells, termed 2Δ.YDC1, make sphingolipids containing exclusively dihydrosphingosine and an abnormally wide spectrum of fatty acids with between 18 and 26 carbon atoms. Like wild-type cells, 2Δ.YDC1 cells stop growing when exposed to Aureobasidin A (AbA), an inhibitor of the inositolphosphorylceramide synthase AUR1 , yet their ceramide levels remain very low. This finding argues against a current hypothesis saying that yeast cells do not require inositolphosphorylceramides and die in the presence of AbA only because ceramides build up to toxic concentrations. Moreover, W303 lag1 Δ lac1 Δ ypc1 Δ ydc1 Δ cells, reported to be AbA resistant, stop growing on AbA after a certain number of cell divisions, most likely because AbA blocks the biosynthesis of anomalous inositolphosphorylsphingosides. Thus, data argue that inositolphosphorylceramides of yeast, the equivalent of mammalian sphingomyelins, are essential for growth. Data also clearly confirm that wild-type strains, when exposed to AbA, immediately stop growing because of ceramide intoxication, long before inositolphosphorylceramide levels become subcritical.     

The protozoan inositol phosphorylceramide synthase: a novel drug target that defines a new class of sphingolipid synthase

Sphingolipids are ubiquitous and essential components of eukaryotic membranes, particularly the plasma membrane. The biosynthetic pathway for the formation of these lipid species is conserved up to the formation of sphinganine. However, a divergence is apparent in the synthesis of complex sphingolipids. In animal cells, ceramide is a substrate for sphingomyelin (SM) production via the enzyme SM synthase. In contrast, fungi utilize phytoceramide in the synthesis of inositol phosphorylceramide (IPC) catalyzed by IPC synthase. Because of the absence of a mammalian equivalent, this essential enzyme represents an attractive target for anti-fungal compounds. In common with the fungi, the kinetoplastid protozoa (and higher plants) synthesize IPC rather than SM. However, orthologues of the gene believed to encode the fungal IPC synthase (AUR1) are not readily identified in the complete genome data bases of these species. By utilizing bioinformatic and functional genetic approaches, we have isolated a functional orthologue of AUR1 in the kinetoplastids, causative agents of a range of important human diseases. Expression of this gene in a mammalian cell line led to the synthesis of an IPC-like species, strongly indicating that IPC synthase activity is reconstituted. Furthermore, the gene product can be specifically inhibited by an anti-fungal-targeting IPC synthase. We propose that the kinetoplastid AUR1 functional orthologue encodes an enzyme that defines a new class of protozoan sphingolipid synthase. The identification and characterization of the protozoan IPC synthase, an enzyme with no mammalian equivalent, will raise the possibility of developing anti-protozoal drugs with minimal toxic side affects.     

Kei1: A Novel Subunit of Inositolphosphorylceramide Synthase,Essential for Its Enzyme Activity and Golgi Localization

Fungal sphingolipids have inositol-phosphate head groups, which are essential for the viability of cells. These head groups are added by inositol phosphorylceramide (IPC) synthase, and AUR1 has been thought to encode this enzyme. Here, we show that an essential protein encoded by KEI1 is a novel subunit of IPC synthase of Saccharomyces cerevisiae. We find that Kei1 is localized in the medial-Golgi and that Kei1 is cleaved by Kex2, a late Golgi processing endopeptidase; therefore, it recycles between the medial- and late Golgi compartments. The growth defect of kei1-1, a temperature-sensitive mutant, is effectively suppressed by the overexpression of AUR1, and Aur1 and Kei1 proteins form a complex in vivo. The kei1-1 mutant is hypersensitive to aureobasidin A, a specific inhibitor of IPC synthesis, and the IPC synthase activity in the mutant membranes is thermolabile. A part of Aur1 is missorted to the vacuole in kei1-1 cells. We show that the amino acid substitution in kei1-1 causes release of Kei1 during immunoprecipitation of Aur1 and that Aur1 without Kei1 has hardly detectable IPC synthase activity. From these results, we conclude that Kei1 is essential for both the activity and the Golgi localization of IPC synthase.     

细菌群体感应参与铜绿假单胞菌胞内聚羟基脂肪酸酯合成的调控

群体感应是细菌根据细胞密度变化调控基因表达的一种调节机制。铜绿假单胞菌中QS系统由lasI和rhlI合成的信号分子3OC12-HSL和C4-HSL以及各自的受体蛋白LasR、RhlR组成,它们以级联方式调控多个基因表达。【目的】研究细菌群体感应(QS)对聚羟基脂肪酸酯合成的调控。【方法】利用铜绿假单胞菌PAO1及其QS突变株为材料通过气相色谱、荧光定量PCR在生理和分子水平上研究QS对聚羟基脂肪酸酯合成的调控。【结果】QS信号分子合成抑制剂阿奇霉素处理铜绿假单胞菌PAO1和QS突变株导致胞内PHA积累量显著减少;铜绿假单胞菌PAO1中C4-HSL合成酶基因rhlI缺失突变株PAO210胞内PHA积累量与野生型无差别;而3OC12-HSL合成酶基因lasI缺失突变株PAO55、3OC12-HSL受体合成酶基因lasR缺失突变株PAO56以及lasI/lasR双缺失突变株PAO57胞内PHA含量与野生型相比明显减少;lasI和lasR的突变株体内PHA合成酶基因phaC1的表达量显著降低,信号分子3OC12-HSL回补实验使phaC1的表达量可恢复到野生株水平,但只可部分恢复lasI缺失导致的胞内PHA合成。【结论】由此推测,铜绿假单胞菌群体感应系统中lasI/lasR系统参与胞内聚羟基脂肪酸酯合成的调控。     

Inositol phosphoryl transferases from human pathogenic fungi

The IPC1 gene from Saccharomyces cerevisiae, which encodes inositolphosphorylceramide (IPC) synthase, was first identified as a novel and essential gene encoding resistance to the natural product antifungal aureobasidin A (AUR1). The formation of IPC in fungi is essential for viability, suggesting inhibitors of IPC1p function would make ideal antifungal drug candidates. Homologs of the AUR1/IPC1 gene were identified from a number of human pathogenic fungi, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis and Cryptococcus neoformans. Comparison of these genes with other homologous genes from Candida albicans, Aspergillus fumigatus, Aspergillus nidulans, Saccharomyces cerevisiae and Schizosaccharomyces pombe reveals a conserved structural motif for inositolphosphoryl transferases which is similar to a motif recently described for lipid phosphatases, but with unique characteristics.     

Single point mutations in domain II of the yeast mitochondrial release factor mRF-1 affect ribosome binding.

We have recently described two yeast strains that are mutated in the MRF1 gene encoding the mitochondrial release factor mRF-1. Both mutants provoke gene-specific defects in mitochondrial translational termination. In the present study we report the cloning, sequencing, as well as an analysis of residual activities of both mutant mrf1 alleles. Each allele specifies a different single amino acid substitution located one amino acid apart. The amino acid changes do not affect the level or cellular localization of the mutant proteins, since equal amounts of wild type and mutant mRF-1 were detected in the mitochondrial compartment. Over-expression of the mutant alleles in wild type and mrf1 mutant yeast strains produces a phenotype consistent with a reduced affinity of the mutant release factors for the ribosome, indicating that the mutations map in a release factor domain involved in ribosome binding. We also demonstrate that nonsense suppression caused by a mutation in the mitochondrial homolog of the E. coli small ribosomal protein S4 can be reversed by a slight over-expression of the MRF1 gene.     

Inhibition of yeast inositol phosphorylceramide synthase by aureobasidin A measured by a fluorometric assay

Inositol phosphorylceramide synthase (IPC synthase) is an essential and unique enzyme in fungal sphingolipid biosynthesis and is the target of the cyclic nonadepsipeptide antibiotic aureobasidin A. As a first step towards understanding the mechanism of aureobasidin A inhibition, we developed a fluorometric HPLC assay for IPC synthase using the Saccharomyces cerevisiae enzyme and the fluorescent substrate analog 6-[N-(7-nitro-2,1, 3-benzoxadiazol-4-yl)amino]-hexanoyl ceramide (C(6)-NBD-cer). The kinetic parameters for C(6)-NBD-cer were comparable to those for the synthetic substrate N-acetylsphinganine used previously. Aureobasidin A acted as a tight-binding, non-competitive inhibitor with respect to C(6)-NBD-cer and had a K(i) of 0.55 nM.