A flexible loop in tyrosine hydroxylase controls coupling of amino acid hydroxylation to tetrahydropterin oxidation

The role of a polypeptide loop in tyrosine hydroxylase (TyrH) whose homolog in phenylalanine hydroxylase (PheH) takes on a different conformation when substrates are bound has been studied using site-directed mutagenesis. The loop spans positions 177 to 191; alanine was introduced into those positions, introducing one alanine substitution per TyrH variant. Mutagenesis of residues in the center of the loop resulted in alterations in the KM values for substrates, the Vmax value for dihydroxyphenylalanine (DOPA) synthesis, and the coupling of tetrahydropterin oxidation to tyrosine hydroxylation. The variant with the most altered KM value for 6-methyltetrahydropterin was TyrH F184A. The variants with the most affected K(tyr) values were those with substitutions in the center of the loop, TyrH K183A, F184A, D185A, P186A and D187A. These five variants also had the most reduced Vmax values for DOPA synthesis. Alanine substitution in positions 182-186 resulted in lowered ratios of tyrosine hydroxylation to tetrahydropterin oxidation. TyrH F184Y and PheH Y138F, variants with the residue at the center of the loop substituted with the residue present at the homologous position in the other hydroxylase, were also studied. The V/K(tyr) to V/K(phe) ratios for these variants were altered significantly, but the results did not suggest that F184 of TyrH or Y138 of PheH plays a dominant role in determining amino acid substrate specificity.     

Influence of growth temperature on the acid phosphatase activity in the yeast Yarrowia lipolytica

Abstract The adaptation of the yeast Yarrowia lipolytica 76-18 to growth temperature was studied by measuring the levels of secreted and intracellular acid phosphatase activities during growth at five temperatures from 8 to 36 °C. The intracellular acid phosphatase activity is maximal at a growth temperature of 20 °C. The level of the secreted phosphatase activity decreases as growth temperature increases from 15 to 36 °C. It is the growth temperature itself and not the growth rate that regulates these activities. The observed dependence of the acid phosphatase activity on the growth temperature indicates a possible participation of acid phosphatases in the temperature adaptation of yeast cells.     

Biotechnological production of the European corn borer sex pheromone in the yeast Yarrowia lipolytica

The European corn borer (ECB) Ostrinia nubilalis is a widespread pest of cereals, particularly maize. Mating disruption with the sex pheromone is a potentially attractive method for managing this pest; however, chemical synthesis of pheromones requires expensive starting materials and catalysts and generates hazardous waste. The goal of this study was to develop a biotechnological method for the production of ECB sex pheromone. Our approach was to engineer the oleaginous yeast Yarrowia lipolytica to produce (Z)-11-tetradecenol (Z11-14:OH), which can then be chemically acetylated to (Z)-11-tetradecenyl acetate (Z11-14:OAc), the main pheromone component of the Z-race of O. nubilalis. First, a C14 platform strain with increased biosynthesis of myristoyl-CoA was obtained by introducing a point mutation into the α-subunit of fatty acid synthase, replacing isoleucine 1220 with phenylalanine (Fas2p^I1220F). The intracellular accumulation of myristic acid increased 8.4-fold. Next, fatty acyl-CoA desaturases (FAD) and fatty acyl-CoA reductases (FAR) from nine different species of Lepidoptera were screened in the C14 platform strain, individually and in combinations. A titer of 29.2 ± 1.6 mg L^-1 Z11-14:OH was reached in small-scale cultivation with an optimal combination of a FAD (Lbo_PPTQ) from Lobesia botrana and FAR (HarFAR) from Helicoverpa armigera. When the second copies of FAD and FAR genes were introduced, the titer improved 2.1-fold. The native FAS1 gene's overexpression led to a further 1.5-fold titer increase, reaching 93.9 ± 11.7 mg L^-1 in small-scale cultivation. When the same engineered strain was cultivated in controlled 1 L bioreactors in fed-batch mode, 188.1 ± 13.4 mg L^-1 of Z11-14:OH was obtained. Fatty alcohols were extracted from the biomass and chemically acetylated to obtain Z11-14:OAc. Electroantennogram experiments showed that males of the Z-race of O. nubilalis were responsive to biologically-derived pheromone blend. Behavioral bioassays in a wind tunnel revealed attraction of male O. nubilalis, although full precopulatory behavior was observed less often than for the chemically synthesized pheromone blend. The study paves the way for the production of ECB pheromone by fermentation.     

Enantioselectivity of Candida rugosa lipases (Lip1, Lip3, and Lip4) towards 2-bromo phenylacetic acid octyl esters controlled by a single amino acid

Enantiomer discrimination by enzymes is a very accurate mechanism, which often involves few amino acids located at the active site. Lipase isoforms from Candida rugosa are very good enzymatic models to study this phenomenon as they display high sequence homology (>80%) and their enantioselectivity is often pointed out. In the present work, we investigated three lipases from C. rugosa (Lip1, Lip3, and Lip4, respectively) towards the resolution of 2-bromo-arylacetic acid esters, an important class of chemical intermediates in the pharmaceutical industry. All exhibited a high enantioselectivity, with Lip4 preferring the R-enantiomer (E-value = 15), while Lip1 and Lip3 showed an S-enantioselectivity >200. A combination of sequence and structure analysis of the three C. rugosa lipases suggested that position 296 could play a role in S- or R-enantiomer preference of C. rugosa lipases. This led to the construction by site-directed mutagenesis of Lip1 and Lip4 variants in which position 296 was, respectively, exchanged by a Gly, Ala, Leu, or Phe amino acid. Screening of these variants for their enantioselectivity toward 2-bromo phenyl acetic acid octyl esters revealed that steric hindrance of the amino acid residue introduced at position 296 controls both the enantiopreference and the enantioselectivity value of the lipase: bulkier is the amino acid at position 296, larger is the selectivity towards the S-enantiomer. To investigate further these observations at an atomic level, we carried out a preliminary modeling study of the tetrahedral intermediates formed by Lip1 and Lip4 with the (R, S)-2-bromo-phenylacetic acid octyl ester enantiomers that provides some insight regarding the determinants responsible for lipase enantiodiscrimination.     

Identification of amino acid residues involved in the binding of Huperzine A to cholinesterases.

Huperzine A, a potential agent for therapy in Alzheimer's disease and for prophylaxis of organophosphate toxicity, has recently been characterized as a reversible inhibitor of cholinesterases. To examine the specificity of this novel compound in more detail, we have examined the interaction of the 2 stereoisomers of Huperzine A with cholinesterases and site-specific mutants that detail the involvement of specific amino acid residues. Inhibition of fetal bovine serum acetylcholinesterase by (-)-Huperzine A was 35-fold more potent than (+)-Huperzine A, with KI values of 6.2 nM and 210 nM, respectively. In addition, (-)-Huperzine A was 88-fold more potent in inhibiting Torpedo acetylcholinesterase than (+)-Huperzine A, with KI values of 0.25 microM and 22 microM, respectively. Far larger KI values that did not differ between the 2 stereoisomers were observed with horse and human serum butyrylcholinesterases. Mammalian acetylcholinesterase, Torpedo acetylcholinesterase, and mammalian butyrylcholinesterase can be distinguished by the amino acid Tyr, Phe, or Ala in the 330 position, respectively. Studies with mouse acetylcholinesterase mutants, Tyr 337 (330) Phe and Tyr 337 (330) Ala yielded a difference in reactivity that closely mimicked the native enzymes. In contrast, mutation of the conserved Glu 199 residue to Gln in Torpedo acetylcholinesterase produced only a 3-fold increase in KI value for the binding of Huperzine A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alteration of a single amino acid changes the substrate specificity of dihydroflavonol 4-reductase

Many plant species exhibit a reduced range of flower colors due to the lack of an essential gene or to the substrate specificity of a biosynthetic enzyme. Petunia does not produce orange flowers because dihydroflavonol 4-reductase (DFR) from this species, an enzyme involved in anthocyanin biosynthesis, inefficiently reduces dihydrokaempferol, the precursor to orange pelargonidin-type anthocyanins. The substrate specificity of DFR, however, has not been investigated at the molecular level. By analyzing chimeric DFRs of Petunia and Gerbera, we identified a region that determines the substrate specificity of DFR. Furthermore, by changing a single amino acid in this presumed substrate-binding region, we developed a DFR enzyme that preferentially reduces dihydrokaempferol. Our results imply that the substrate specificity of DFR can be altered by minor changes in DFR.     

解脂耶氏酵母中囊泡蛋白YlSec15的鉴定及功能研究

解脂耶氏酵母(Yarrowia lipolytica)进行出芽繁殖时,决定未来分裂平面的出芽位点不是随机选取的,而是选择在前一次细胞分裂位置的对侧出芽,即进行双极出芽。目前对解脂耶氏酵母双极出芽的分子调控机制并不清楚。通过观察蛋白定位及过量表达的方法研究了解脂耶氏酵母中囊泡蛋白YlSec15的功能。结果表明:YlSec15在细胞中有明显的极性定位,在细胞的小芽内以及大中芽的芽颈处富集,过量表达YlSec15抑制了菌丝的形成并使得部分细胞的出芽位点选择方式由双极出芽转变为随机出芽,而引起这一变化的原因可能是由于过量的YlSec15在细胞中不能进行正常的极性定位。此外,YlSec15可能是通过YlRas2介导的信号通路参与调控细胞的菌丝形成及双极出芽。这一发现丰富了解脂耶氏酵母中双极出芽的分子调控机制,也证明了极性生长与囊泡运输之间是相互影响的。     

解脂耶氏酵母基于木质纤维素原料生产化学品研究进展*

解脂耶氏酵母具有遗传背景清晰、分子操作体系较为成熟、抗逆性强、底物谱广、有机酸和蛋白质分泌能力强等优点,在微生物发酵生产化学品领域极具应用潜力。木质纤维素是丰富的可再生生物质资源,以木质纤维素原料替代化石原料生产化学品对于缓解全球能源危机、保障粮食安全等意义重大。解脂耶氏酵母可以天然代谢木质纤维素水解产生的葡萄糖,但对其他水解产物(如木糖)的利用效率极低。综述解脂耶氏酵母利用木质纤维素原料的代谢途径及改造策略,以木质纤维素原料生产化学品为例,重点讨论该过程中的主要瓶颈问题及解决办法,为后续研究提供参考。     

Properties of recombinant Rhizomucor miehei lipase with amino acid substitutions of Phe94 in the substrate binding domain

The chain length specificity of Rhizomucor miehei lipase was altered by substituting Phe94 in the protein groove which is responsible for accommodating the acyl chain of the substrate. Three recombinant enzymes, Phe94Arg, Phe94Glu and Phe94Gln, were expressed in Pichia pastoris, purified and their ability to hydrolyse p-nitrophenyl esters and triacylglycerols of different chain length was studied.     

Substitution of the amino acid at position 102 with polar and aromatic residues influences substrate specificity of lactate dehydrogenase

The Gin residue at amino acid position 102 ofBacillus stearothermophilus lactate dehydrogenase was replaced with Ser, Thr, Tyr, or Phe to investigate the effect on substrate recognition. The Q102S and Q102T mutant enzymes were found to have a broader range of substrate specificity (measured byk _cat/K _m) than the wild-type enzyme. However, it is evident that either Ser or Thr at position 102 are of a size able to accommodate a wide variety of substrates in the active site and substrate specificity appears to rely largely on size discrimination in these mutants. The Q102F and Q102Y mutant enzymes have low catalytic efficiency and do not show this relaxed substrate specificity. However, their activities are restored by the presence of an aromatic substrate. All of the enzymes have a very low catalytic efficiency with branched chain aliphatic substrates.Abbreviations used BSLDH Bacillus stearothermophilus lactate dehydrogenase - FBP fructose-1,6-bisphosphate - HP hydroxypyruvate - KB ketobutyrate - KC ketocaproate - KV ketovalerate - MDH malate dehydrogenase - PP phenylpyruvate - PYR pyruvate - RBE relative binding energy     

Identification of the amino acid residues in trichosanthin crucial for IgE response

Trichosanthin (TCS) is the major effective component from Chinese herb Trichosanthes kirilowii. TCS has been approved to be effective in clinical treatment of HIV infection and leukemia, but its allergenicity has limited its clinical usage. To identify amino acid residues in TCS with an important role in IgE induction, TCS-specific IgE mAb (TE1) was used to serve as a probe and TE1 epitope was determined by a random phage-peptide library. Based on phage peptide sequences, TE1 epitope was predicted at amino acid residues 169-174 (QQIGKR) of TCS protein. Based on modeling data, two amino acids (Lys173 and Arg174) on TCS were considered to have a crucial role in binding to TE1. After lysine 173 and arginine 174 were mutated to glycine, the mutant TCS protein specifically lost the binding activity to TE1 mAb and exhibited reduced IgE induction in the immunized mice. The data showed that the IgE epitope of TCS was determined and shown to play a critical role in induction of IgE, and the modification of IgE-epitope may be a useful strategy to reduce the allergenicity of an allergen.     

A mutation in the secretion pathway of the yeast Yarrowia lipolytica that displays synthetic lethality in combination with a mutation affecting the signal recognition particle.

In an attempt to identify proteins involved in the translocation step of protein secretion, a genetic screen was carried out in the yeast Yarrowia lipolytica. A conditional lethal mutant which has a defect in the 7S RNA of the signal recognition particle was mutagenized and screened for second-site mutations that specifically exacerbate its temperature sensitivity. This approach had previously allowed the characterization of an endoplasmic reticulum component, Sls1p, involved in protein translocation. A second mutation, sls2-1, was isolated that causes synthetic lethality when combined with the 7S RNA mutation. On its own, the sls2-1 mutation confers a temperature-sensitive growth phenotype. The secretory phenotype of the sls2 mutant consists in abnormal secretion of several polypeptides, and thus differs from the defect in secretory protein synthesis associated with the 7S RNA and sls1-1 mutations. Two new Y. lipolytica genes were identified which can relieve the growth defect of sls2-1 cells: SLS2 itself and SSL2, a multicopy suppressor of the temperature sensitivity of the sls2 mutant. The SLS2 gene encodes a polypeptide that can potentially be farnesylated and phosphorylated, and shares some homology with an S. cerevisiae protein of unknown function. Ssl2p resembles calmodulin-dependent serine/threonine protein kinases. These two proteins may interact to regulate protein sorting. Received: 9 June 1998 / Accepted: 10 February 1999     

Production of succinic acid at low pH by a recombinant strain of the aerobic yeast Yarrowia lipolytica

Biotechnological production of weak organic acids such as succinic acid is most economically advantageous when carried out at low pH. Among naturally occurring microorganisms, several bacterial strains are known to produce considerable amounts of succinic acid under anaerobic conditions but they are inefficient in performing the low‐pH fermentation due to their physiological properties. We have proposed therefore a new strategy for construction of an aerobic eukaryotic producer on the basis of the yeast Yarrowia lipolytica with a deletion in the gene coding one of succinate dehydrogenase subunits. Firstly, an original in vitro mutagenesis‐based approach was proposed to construct strains with Ts mutations in the Y. lipolytica SDH1 gene. These mutants were used to optimize the composition of the media for selection of transformants with the deletion in the Y. lipolytica SDH2 gene. Surprisingly, the defects of each succinate dehydrogenase subunit prevented the growth on glucose but the mutant strains grew on glycerol and produced succinate in the presence of the buffering agent CaCO₃. Subsequent selection of the strain with deleted SDH2 gene for increased viability allowed us to obtain a strain capable of accumulating succinate at the level of more than 45 g L^?1 in shaking flasks with buffering and more than 17 g L^?1 without buffering. The possible effect of the mutations on the utilization of different substrates and perspectives of constructing an industrial producer is discussed. Biotechnol. Bioeng. 2010;107:673–682. © 2010 Wiley Periodicals, Inc.     

Investigation of the effect of biologically active threo-Ds-isocitric acid on oxidative stress in Paramecium caudatum

The effect of biologically active form (threo-Ds-) of isocitric acid (ICA) on oxidative stress was studied using the infusorian Paramecium caudatum stressed by hydrogen peroxide and salts of some heavy metals (Cu, Pb, Zn, and Cd). ICA at concentrations between 0.5 and 10?mM favorably influenced the infusorian cells with oxidative stress induced by the toxicants studied. The maximal antioxidant effect of ICA was observed at its concentration 10?mM irrespective of the toxicant used (either H₂O₂ or heavy metal ions). ICA was found to be a more active antioxidant than ascorbic acid. Biologically active pharmaceutically pure threo-Ds-ICA was produced through cultivation of the yeast Yarrowia lipolytica and isolated from the culture liquid in the form of crystalline monopotassium salt with a purity of 99.9%.     

Identification of amino acid sequences in the integrin beta 1 cytoplasmic domain implicated in cytoskeletal association

Wild-type and mutant chicken integrin beta 1 subunit (beta 1c) cDNAs were expressed in NIH 3T3 cells and assayed for localization in focal adhesions of cells plated on fibronectin substrates. Focal adhesion localization in stable transfected cells was assayed by indirect immunofluorescent staining with chicken-specific anti-beta 1c antibodies. Mutant beta 1c integrins containing internal deletions of 13 amino acids adjacent to the membrane, delta 759-771, and 20 centrally located amino acids, delta 771-790, localized in focal adhesions demonstrating that sequences required for direction to focal adhesion structures were not limited to one region of the cytoplasmic domain. Point mutations revealed three clusters of amino acids which contribute to localization in focal adhesions. These three clusters or signals are: cyto-1 (764-774), cyto-2 (785-788), and cyto-3 (797-800). The 11-residue cyto-1 signal is only found on integrin beta subunit sequences, except beta 4. Four residues within this region, D764, F768, F771, and E774, could not be altered without reducing focal adhesion staining intensities, and likely form a signal that occupies one side of an alpha helix. Mutations involving two cyto-1 residues, K770 and F771, also appeared to affect heterodimer affinity and specificity. Cyto-2 (785-788,), NPIY, is an NPXY signal that forms a tight turn motif. Cyto-2 provides a structural conformation, which when perturbed by proline removal or addition, inhibits integrin localization in focal adhesions. Cyto-3 (797-800), NPKY, resembles cyto-2, however, the nonconserved proline residue can be replaced without alteration of the localization phenotype. Cyto-3, therefore, constitutes a unique integrin signal, NXXY. Both serine and tyrosine residues at positions 790 and 788, respectively, which have been implicated in integrin phosphorylation/regulation, were conservatively replaced without detectable effect on focal adhesion localization. However, acidic replacements for these amino acids reduced focal adhesion staining intensities, suggesting that phosphorylation at these sites may negatively regulate integrin function.     

STE11 disruption reveals the central role of a MAPK pathway in dimorphism and mating in Yarrowia lipolytica

Yarrowia lipolytica is a dimorphic fungus whose morphology is controlled by several factors such as pH and different compounds. To determine if the STE11-mitogen-activated protein kinase (MAPK) pathway plays a role in dimorphism of Y. lipolytica, we isolated the gene encoding a Mapkkk. The isolated gene (STE11) has an ORF of 2832 bp without introns, encoding a protein of 944 amino acids, with a theoretical Mr of 100.9 kDa, that exhibits high homology to fungal Mapkkks. Disruption of the STE11 gene was achieved by the pop-in/pop-out procedure. Growth rate and response to osmotic stress or agents affecting wall integrity were unaffected in the deleted mutants, but they lost the capacity to mate and to grow in the mycelial form. Both alterations were reverted by transformation with the wild-type STE11 gene. The Y. lipolytica STE11 gene driven by two different promoters was unable to complement Saccharomyces cerevisiae ste11Delta mutants, although the gene was transcribed. Also, a wild-type MAPKKK gene from Ustilago maydis failed to complement Y. lipolyticaDeltaste11 mutants. Both negative results were attributed to a failure of the transgenic gene products to interact with the corresponding regulatory and scaffold proteins. This hypothesis was supported by the observation that a truncated version of the U. maydis MAPKKK gene reversed mating and dimorphic defects in the mutants. All these results demonstrate that the MAPK pathway is essential for both morphogenesis and mating in Y. lipolytica.     

Mutations at different sites in members of the Gpr1/Fun34/YaaH protein family cause hypersensitivity to acetic acid in Saccharomyces cerevisiae as well as in Yarrowia lipolytica

The Gpr1 protein of the ascomycetous yeast Yarrowia lipolytica belongs to the poorly characterized Gpr1/Fun34/YaaH protein family, members of which have thus far only been found in prokaryotes and lower eukaryotes. Trans-dominant mutations in the GPR1 gene result in acetic acid sensitivity of cells at low pH. Moreover, Gpr1p is subjected to phosphorylation at serine-37 in a carbon source-dependent manner. Here we show that several mutations within the ORFs of the GPR1 orthologues of Saccharomyces cerevisiae, YCR010c (ATO1) and YNR002c (ATO2), also trans-dominantly induce acetic acid hypersensitivity in this yeast. We demonstrate that the C-termini of mutated Gpr1p, Ycr010cp and Ynr002cp are necessary for the triggering of acetic acid sensitivity. Phosphorylation of Y. lipolytica Gpr1p was also affected by several mutations. Data further suggest that Gpr1p exists in an oligomeric state.