Engineering of phenylacetaldehyde reductase for efficient substrate conversion in concentrated 2-propanol

Phenylacetaldehyde reductase (PAR) is suitable for the conversion of various aryl ketones and 2-alkanones to corresponding chiral alcohols. 2-Propanol acts as a substrate solvent and hydrogen donor of coupled cofactor regeneration during the conversion of substrates catalyzed by PAR. To improve the conversion efficiency in high concentrations of substrate and 2-propanol, selection of a PAR mutant library and the subsequent rearrangement of mutations were attempted. With only a single selection round and following the manual combination of advantageous mutations, PAR was successfully adapted for the conversion of high concentrations of substrate with concentrated 2-propanol. This method will be widely applicable for the engineering of enzymes potentially valuable for industry.     

Engineering the phenylacetaldehyde reductase mutant for improved substrate conversion in the presence of concentrated 2-propanol

Phenylacetaldehyde reductase (PAR) from Rhodococcus sp. ST-10 is useful for chiral alcohol production because of its broad substrate specificity and high stereoselectivity. The conversion of ketones into alcohols by PAR requires the coenzyme NADH. PAR can regenerate NADH by oxidizing additional alcohols, especially 2-propanol. However, substrate conversion by wild-type PAR is suppressed in concentrated 2-propanol. Previously, we developed the Sar268 mutant of PAR, which can convert several substrates in the presence of concentrated 2-propanol. In this paper, further mutational engineering of Sar268 was performed to achieve higher process yield. Each of nine amino acid positions that had been examined for generating Sar268 was subjected to saturation mutagenesis. Two novel substitutions at the 42nd amino acid position increased m-chlorophenacyl chloride (m-CPC) conversion. Moreover, several nucleotide substitutions identified from libraries of random mutations around the start codon also improved the PAR activity. E. coli cells harboring plasmid pHAR1, which has the integrated sequence of the top clones from the above selections, provided greater conversion of m-CPC and ethyl 4-chloro-3-oxobutanoate than the Sar268 mutant, with very high optical purity of products. This mutant is a promising novel biocatalyst for efficient chiral alcohol production.     

Development of an improved phenylacetaldehyde reductase mutant by an efficient selection procedure

Chiral alcohols are valuable as diverse chemicals and synthetic intermediate materials. Phenylacetaldehyde reductase (PAR) is an enzyme that converts a wide variety of ketones into chiral alcohols with high optical purity. When an alcohol such as 2-propanol is used as a hydrogen donor, PAR itself will also mediate the regeneration of the coenzyme NADH in situ. Perceiving a capacity for improvement, we sought to develop a PAR that is able to convert higher concentrations of substrates in the presence of high concentrations of 2-propanol. The selection procedure for mutants was re-examined and a procedure able to select an effective amino acid substitution was established. Two advantageous amino acid substitutions were successfully selected using the procedure. When high-concentration substrate conversion reaction was subjected with a mutant that integrated both the two amino acid substitutions, near-complete conversions of m-chlorophenacyl chloride (m-CPC) (2.1 mmol/ml) and ethyl 4-chloro-3-oxobutanoate (ECOB) (1.9 mmol/ml) were achieved.     

Enhancing the biocatalytic potential of carbonyl reductase of Candida viswanathii using aqueous-organic solvent system

In the present work, the toxic effect of various solvents with different Log P values was studied on the whole cells of Candida viswanathii. Experiments showed that the lower concentrations of some solvent increased both the activity retention and enzyme activity as compared to the control while this was not the case with higher concentrations of the same solvents. The model compound taken in the present study was 1-acetophenone. The percentage conversion improved from 76 to 94%. Addition of 2-propanol increased the substrate tolerance, giving the conversion of 90% compared to 9% in control at a substrate concentration of 70 mM in 1h. The operational stability increased at higher temperatures with the addition of 2-propanol in the reaction mixture with good conversion (90%) and enantiomeric excess (>99%) at 45 degrees C and 50 degrees C. The effect was also found to be prominent in other tested substrates. In order to further stabilize the cells for long term use in higher concentration of organic solvents, the cells were further immobilized, and were found to have higher activity retention than that of free cells.     

On the molecular mechanism of lactoperoxidase-catalyzed H2O2 metabolism and irreversible enzyme inactivation

Lactoperoxidase-catalyzed H2O2 metabolism proceeds through one of three different pathways, depending on the nature and the concentration of the second substrate as an e- donor and/or on pH conditions. In the lactoperoxidase (LPO)-H2O2 system, at low H2O2 concentrations and/or alkaline conditions the peroxidatic cycle involves ferric LPO----compound I----compound II----ferric LPO conversion, whereas high H2O2 concentrations and/or acidic conditions favor the ferric LPO----compound I----compound II----compound III----ferrous LPO----ferric LPO pathway. The compound III/ferroperoxidase states are associated with irreversible enzyme inactivation by cleavage of the heme moiety and liberation of iron. It is likely that either singlet oxygen or superoxide and hydroxyl radicals are involved in the attack on heme iron, because inactivation correlates with oxygen production and can be decreased to a certain degree by scavengers such as ethanol, 1-propanol, 2-propanol, or mannitol. In the LPO-H2O2-I- system, the enzyme may also be inactivated by I2 generated in the course of enzymatic I- oxidation (i.e. during ferric LPO----compound I----ferric LPO cycles).     

Efficient synthesis of optically pure alcohols by asymmetric hydrogen-transfer biocatalysis: application of engineered enzymes in a 2-propanol–water medium

We describe an efficient method for producing both enantiomers of chiral alcohols by asymmetric hydrogen-transfer bioreduction of ketones in a 2-propanol (IPA)–water medium with E. coli biocatalysts expressing phenylacetaldehyde reductase (PAR: wild-type and mutant enzymes) from Rhodococcus sp. ST-10 and alcohol dehydrogenase from Leifsonia sp. S749 (LSADH). We also describe the detailed properties of mutant PARs, Sar268, and HAR1, which were engineered to have high activity and productivity in media composed of polar organic solvent and water, and the construction of three-dimensional structure of PAR by homology modeling. The K _m and V _max values for some substrates and the substrate specificity of mutant PARs were quite different from those of wild-type PAR. The results well explained the increased productivity of engineered PARs in IPA–water medium.     

PAR对氨基酰化酶不可逆抑制动力学研究

本文将邹氏的在酶的活性修饰剂存在下的底物反应动力学理论应用于氨基酰化酶被金属螯合剂PAR脱锌而失活的动力学研究。通过对不同浓度的PAR存在下底物反应过程和含有PAR的不同浓度的底物中酶促反应的分析,讨论了PAR对氨基酰化酶的脱锌机制。这一过程很可能按如下机制进行:首先,PAR与酶分子活性部位的锌结合,形成一复合物,这一步是较快的反应,然后发生一个可逆的构象变化,最后是不可逆的去锌步骤。锌的存在显然稳定了酶活性部位的构象,而这正是酶活性所必需的。     

Chiral alcohol production by NADH-dependent phenylacetaldehyde reductase coupled with in situ regeneration of NADH.

Phenylacetaldehyde reductase (PAR) produced by styrene-assimilating Corynebacterium strain ST-10 was used to synthesize chiral alcohols. This enzyme with a broad substrate range reduced various prochiral aromatic ketones and beta-ketoesters to yield optically active secondary alcohols with an enantiomeric purity of more than 98% enantiomeric excess (e.e.). The Escherichia coli recombinant cells which expressed the par gene could efficiently produce important pharmaceutical intermediates; (R)-2-chloro-1-(3-chlorophenyl)ethanol (28 mg.mL-1) from m-chlorophenacyl chloride, ethyl (R)-4-chloro-3-hydroxy butanoate) (28 mg.mL-1) from ethyl 4-chloro-3-oxobutanoate and (S)-N-tert-butoxycarbonyl(Boc)-3-pyrrolidinol from N-Boc-3-pyrrolidinone (51 mg.mL-1), with more than 86% yields. The high yields were due to the fact that PAR could concomitantly reproduce NADH in the presence of 3-7% (v/v) 2-propanol in the reaction mixture. This biocatalytic process provided one of the best asymmetric reductions ever reported.     

PAR1 cleavage and signaling in response to activated protein C and thrombin

Activated protein C (APC), a natural anticoagulant protease, can trigger cellular responses via protease-activated receptor-1 (PAR1), a G protein-coupled receptor for thrombin. Whether this phenomenon contributes to the physiological effects of APC is unknown. Toward answering this question, we compared the kinetics of PAR1 cleavage on endothelial cells by APC versus thrombin. APC did cleave PAR1 on the endothelial surface, and antibodies to the endothelial protein C receptor inhibited such cleavage. Importantly, however, APC was approximately 10(4)-fold less potent than thrombin in this setting. APC and thrombin both triggered PAR1-mediated responses in endothelial cells including expression of antiapoptotic (tumor necrosis factor-alpha-induced a20 and iap-1) and chemokine (interleukin-8 (il-8) and cxcl3) genes, but again, APC was approximately 10(4)-fold less potent than thrombin. The addition of zymogen protein C to endothelial cultures did not alter the rate of PAR1 cleavage at low or high concentrations of thrombin, and PAR1 cleavage was substantial at thrombin concentrations too low to trigger detectable conversion of protein C to APC. Thus, locally generated APC did not contribute to PAR1 cleavage beyond that effected by thrombin in this system. Although consistent with reports that sufficiently high concentrations of APC can cleave and activate PAR1 in culture, our data suggest that a significant physiological role for PAR1 activation by APC is unlikely.     

Activity variation in alcohol dehydrogenase paralogs is associated with adaptation to cactus host use in cactophilic Drosophila

Drosophila mojavensis and Drosophila arizonae are species of cactophilic flies that share a recent duplication of the alcohol dehydrogenase (Adh) locus. One paralog (Adh-2) is expressed in adult tissues and the other (Adh-1) in larvae and ovaries. Enzyme activity measurements of the ADH-2 amino acid polymorphism in D. mojavensis suggest that the Fast allozyme allele has a higher activity on 2-propanol than 1-propanol. The Fast allele was found at highest frequency in populations that utilize hosts with high proportions of 2-propanol, while the Slow allele is most frequent in populations that utilize hosts with high proportions of 1-propanol. This suggests that selection for ADH-2 allozyme alleles with higher activity on the most abundant alcohols is occurring in each D. mojavensis population. In the other paralog, ADH-1, significant differences between D. mojavensis and D. arizonae are associated with a previously shown pattern of adaptive protein evolution in D. mojavensis. Examination of protein sequences showed that a large number of amino acid fixations between the paralogs have occurred in catalytic residues. These changes are potentially responsible for the significant difference in substrate specificity between the paralogs. Both functional and sequence variation within and between paralogs suggests that Adh has played an important role in the adaptation of D. mojavensis and D. arizonae to their cactophilic life.     

水稻冠层光截获、光能利用与产量的关系

以两优培九和武香粳14号水稻品种为材料,在不同栽插密度和施氮水平下进行2年田间试验,研究水稻冠层光合有效辐射(PAR)截获率、光能利用率与水稻产量的关系.结果表明:分蘖期至成熟期,各处理水稻冠层平均PAR反射率为3.45％,其中,分蘖期至抽穗期的冠层反射PAR占冠层总PAR损失的10.90％,显著小于抽穗期至成熟期的22.06％.分蘖期至成熟期的冠层PAR转化率随栽插密度的增加而减少,随施氮量的增加而增大;分蘖期至抽穗期的冠层PAR转化率高于抽穗期至成熟期.在分蘖期至成熟期,冠层PAR利用率随栽插密度和施氮量的增加而增大,各处理中两优培九的平均PAR利用率(1.83 g· MJ-1)显著高于武香粳14(1.42 g·MJ-1);武香粳14因生育期较长,分蘖期至成熟期的入射PAR及中、高栽插密度处理的PAR截获量均高于两优培九.水稻不同生长阶段冠层PAR截获率和利用率与产量呈显著正相关,PAR转化率与产量也呈正相关,但相关性不显著.因此,在保持较高PAR截获率的基础上提高冠层PAR转化率,进而提高冠层PAR利用率,有利于水稻高产.     

水稻冠层光截获、光能利用与产量的关系

以两优培九和武香粳14号水稻品种为材料,在不同栽插密度和施氮水平下进行2年田间试验,研究水稻冠层光合有效辐射(PAR)截获率、光能利用率与水稻产量的关系.结果表明： 分蘖期至成熟期,各处理水稻冠层平均PAR反射率为3.45%,其中,分蘖期至抽穗期的冠层反射PAR占冠层总PAR损失的10.90%,显著小于抽穗期至成熟期的22.06%.分蘖期至成熟期的冠层PAR转化率随栽插密度的增加而减少,随施氮量的增加而增大;分蘖期至抽穗期的冠层PAR转化率高于抽穗期至成熟期.在分蘖期至成熟期,冠层PAR利用率随栽插密度和施氮量的增加而增大,各处理中两优培九的平均PAR利用率(1.83 g·MJ^-1)显著高于武香粳14(1.42 g·MJ^-1);武香粳14因生育期较长,分蘖期至成熟期的入射PAR及中、高栽插密度处理的PAR截获量均高于两优培九.水稻不同生长阶段冠层PAR截获率和利用率与产量呈显著正相关,PAR转化率与产量也呈正相关,但相关性不显著.因此,在保持较高PAR截获率的基础上提高冠层PAR转化率,进而提高冠层PAR利用率,有利于水稻高产.     

PAR4, but not PAR1, signals human platelet aggregation via Ca2+ mobilization and synergistic P2Y12 receptor activation

Regulation of platelet activation plays a central role in hemostasis and pathophysiological processes such as coronary artery disease. Thrombin is the most potent activator of platelets. Human platelets express two thrombin receptors, PAR1 and PAR4, both of which signal platelet activation. Evidence is lacking on the mechanism by which PAR1 and PAR4 may differentially signal platelet aggregation. Here we show that at the relatively high concentration of agonist most likely found at the site of a local thrombus, dual inhibition of the P2Y12 receptor and calcium mobilization result in a complete inhibition of PAR4-induced aggregation, while having no effect on either thrombin or PAR1-mediated platelet aggregation. Both PAR1- and PAR4mediated aggregation are independent of calcium mobilization. Furthermore, we show that P2Y12 receptor activation is not required for protease-activated receptor-mediated aggregation at higher agonist concentrations and is only partially required for Rap1 as well as GPIIbIIIa activation. P2Y12 receptor inhibitors clinically in use such as clopidogrel are postulated to decrease platelet aggregation through partial inhibition of PAR1 signaling. Our data, however, indicate that at high local concentrations of thrombin, it is the signaling through PAR4 rather than PAR1 that may be regulated through purinergic feedback. Thus, our data identify an intra-platelet mechanism that may function as a future site for therapeutic intervention.     

THE EVOLUTIONARY DYNAMICS OF SEXUALLY ANTAGONISTIC MUTATIONS IN PSEUDOAUTOSOMAL REGIONS OF SEX CHROMOSOMES

Sex chromosomes can evolve gene contents that differ from the rest of the genome, as well as larger sex differences in gene expression compared with autosomes. This probably occurs because fully sex‐linked beneficial mutations substitute at different rates from autosomal ones, especially when fitness effects are sexually antagonistic (SA). The evolutionary properties of genes located in the recombining pseudoautosomal region (PAR) of a sex chromosome have not previously been modeled in detail. Such PAR genes differ from classical sex‐linked genes by having two alleles at a locus in both sexes; in contrast to autosomal genes, however, variants can become associated with gender. The evolutionary fates of PAR genes may therefore differ from those of either autosomal or fully sex‐linked genes. Here, we model their evolutionary dynamics by deriving expressions for the selective advantages of PAR gene mutations under different conditions. We show that, unless selection is very strong, the probability of invasion of a population by an SA mutation is usually similar to that of an autosomal mutation, unless there is close linkage to the sex‐determining region. Most PAR genes should thus evolve similarly to autosomal rather than sex‐linked genes, unless recombination is very rare in the PAR.     

Isolation and preliminary characterization of amino acid substitution mutations that increase the activity of the osmoregulated ProP protein of Salmonella enterica serovar Typhimurium

In Enterobacteriaceae, the ProP protein, which takes up proline and glycine betaine, is subject to a post-translational control mechanism that increases its activity at high osmolarity. In order to investigate the osmoregulatory mechanism of the Salmonella enterica ProP, we devised a positive selection for mutations that conferred increased activity on this protein at low osmolarity. The selection involved the isolation of mutations in a proline auxotroph that resulted in increased accumulation of proline via the ProP system in the presence of glycine betaine, which is a competitive inhibitor of proline uptake by this permease. This selection was performed by first-year undergraduates in two semesters of a research-based laboratory course. The students generated sixteen mutations resulting in six different single amino acids substitutions. They determined the effects of the mutations on the growth rates of the cells in media of high and low osmolarity in the presence of low concentrations of proline or glycine betaine. Furthermore, they identified the mutations by DNA sequencing and displayed the mutated amino acids on a putative three-dimensional structure of the protein. This analysis suggested that all six amino acid substitutions are residues in trans-membrane helices that have been proposed to contribute to the formation of the transport pore, and, thus, may affect the substrate binding site of the protein.     

Calcium Mobilization And Protein Kinase C Activation Downstream Of Protease Activated Receptor 4 (PAR4) Is Negatively Regulated By PAR3 In Mouse Platelets

Thrombin activates platelets through protease activated receptors (PARs). Mouse platelets express PAR3 and PAR4. PAR3 does not signal in platelets. However, PAR4 is a relatively poor thrombin substrate and requires PAR3 as a cofactor at low thrombin concentrations. In this study we show that PAR3 also regulates PAR4 signaling. In response to thrombin (30–100 nM) or PAR4 activating peptide (AYPGKF), platelets from PAR3^−/− mice had increased G_q signaling compared to wild type mice as demonstrated by a 1.6-fold increase in the maximum intracellular calcium (Ca²⁺) mobilization, an increase in phosphorylation level of protein kinase C (PKC) substrates, and a 2-fold increase of Ca²⁺ release from intracellular stores. Moreover, platelets from heterozygous mice (PAR3^+/−) had an intermediate increase in maximum Ca²⁺ mobilization. Treatment of PAR3^−/− mice platelets with P2Y₁₂ antagonist (2MeSAMP) did not affect Ca²⁺ mobilization from PAR4 in response to thrombin or AYPGKF. The activation of RhoA-GTP downstream G_12/13 signaling in response to thrombin was not significantly different between wild type and PAR3^−/− mice. Since PAR3 influenced PAR4 signaling independent of agonist, we examined the direct interaction between PAR3 and PAR4 with bioluminescence resonance energy transfer (BRET). PAR3 and PAR4 form constitutive homodimers and heterodimers. In summary, our results demonstrate that in addition to enhancing PAR4 activation at low thrombin concentrations, PAR3 negatively regulates PAR4-mediated maximum Ca²⁺ mobilization and PKC activation in mouse platelets by physical interaction.     

A kinetic study of lipase-catalyzed reversible kinetic resolution involving verification at miniplant-scale

Lipase-catalyzed kinetic resolution of racemates is a popular method for synthesis of chiral synthons. Most of these resolutions are reversible equilibrium limited reactions. For the first time, an extensive kinetic model is proposed for kinetic resolution reactions, which takes into account the full reversibility of the reaction, substrate inhibition by an acyl donor and an acyl acceptor as well as alternative substrate inhibition by each enantiomer. For this purpose, the reversible enantioselective transesterification of (R/S)-1-methoxy-2-propanol with ethyl acetate catalyzed by Candida antarctica lipase B (CAL-B) is investigated. The detailed model presented here is valid for a wide range of substrate and product concentrations. Following model discrimination and the application of Haldane equations to reduce the degree of freedom in parameter estimation, the 11 free parameters are successfully identified. All parameters are fitted to the complete data set simultaneously. Six types of independent initial rate studies provide a solid data basis for the model. The effect of changes in substrate and product concentration on reaction kinetics is discussed. The developed model is used for simulations to study the behavior of reaction kinetics in a fixed bed reactor. The typical plot of enantiomeric excess versus conversion of substrate and product is evaluated at various initial substrate mixtures. The model is validated by comparison with experimental results obtained with a fixed bed reactor, which is part of a fully automated state-of-the-art miniplant.     

Three different coupled enzymatic systems for in situ regeneration of NADPH

Three different coupled enzymatic systems used in the reduction of sulcatone by alcohol dehydrogenase from Thermoanaerobium brockii (TBADH), were kinetically compared. The first one involved the use of TBADH for both the principal and recycling reactions and 2-propanol 20%, v/v as the recycling substrate. The other two were based on the use of a different enzyme, glucose- or glucose-6-phosphate dehydrogenases, for in situ regeneration of NADPH. The coupled-substrate approach achieved 100% of conversion against 84% of the other two systems.     

The genes from the pseudoautosomal region 1 (PAR1) of the mammalian sex chromosomes: Synteny,phylogeny and selection

Sex chromosomes recombine restrictly in their homologous area, the pseudoautosomal region (PAR), represented by PAR1 and PAR2, which behave like an autosome in both pairing and recombination. The PAR1, common to most of the eutherian mammals, is located at the terminus of the sex chromosomes short arm and exhibit recombination rates ~20 times higher than the autosomes. Here, we assessed the interspecific evolutionary genomic dynamics of 15 genes of the PAR1 across 41 mammalian genera (representing six orders). The strong negative selection detected in most of the assessed groups reinforces the presence of evolutionary constraints, imposed by the important function of the PAR1 genes. Indeed, mutations in these genes are associated with various diseases in humans, including stature problems (Klinefelter Syndrome), leukemia and mental diseases. Yet, a few genes exhibiting positive selection (ω-value >1) were depicted in Rodentia (ASMT and ZBED1) and Primates (CRLF2 and CSF2RA). Rodents have the smallest described PAR1, while that of simian primates/humans underwent a 3 to 5 fold size reduction. The assessment of the PAR1 genes synteny revealed differences among the mammalian species, especially in the Rodentia order where chromosomic translocations from the sex chromosomes to the autosomes were observed. Such syntenic changes may be an evidence of the rapid evolution in rodents, as previous referred in other papers, also depicted by their increased branch lengths in the phylogenetic analyses. Concluding, we suggest that genome migration is an important factor influencing the evolution of mammals and may result in changes of the selective pressures operating on the genome.