Altered Substrate Specificity Research Articles

The D816V Mutation of C-Kit Circumvents a Requirement for Src Family Kinases in C-Kit Mediated Signal Tranduction and Transformation

The receptor tyrosine kinase c-Kit plays a critical role in hematopoiesis and gain-of-function mutations of the receptor are frequently seen in several malignancies, including acute myeloid leukemia (AML), mastocytoma and sinonasal NK/T cell lymphomas. The most common mutation of c-Kit in these disorders is a substitution of the aspartic acid residue in position 816 to a valine (D816V), leading to constitutive activation of the receptor. In this study we aimed to investigate the role of Src family kinases in c-Kit/D816V signaling. Src family kinases are necessary for the phosphorylation of wild-type c-Kit as well as for activation of downstream signaling pathways including receptor ubiquitination and the Ras/MEK/Erk pathway. Tyrosine 568 of c-Kit, that is important for Src activation by wild-type c-Kit, was mutated to phenylalanine in both wild-type c-Kit and c-Kit/D816V and stably transfected into the hematopoietic cell line Ba/F3. Our data demonstrate that, unlike wild-type c-Kit, the phosphorylation of c-Kit/D816V is not dependent on Src family kinases. In addition we found that neither receptor ubiquitination nor Erk activation by c-Kit/D816V required activation of Src family kinases. In vitro kinase assay using synthetic peptides revealed that c-Kit/D816V had an altered substrate specificity resembling Src and Abl tyrosine kinases. The serine/threonine kinases Akt and Erk play important roles in cell survival and proliferation mediated by receptor tyrosine kinases. We could show constitutive activation of both PI3-kinase pathway and Erk in Ba/F3 cells expressing c-Kit/D816V, although ligand stimulation induced even stronger activation. We further present evidence that, in contrast to wild-type c-Kit, Src family kinases are dispensible for c-Kit/D816V cell survival. Taken together, we demonstrate that the signal transduction pathways mediated by c-Kit/D816V are markedly different from those activated by wild-type c-Kit and that altered substrate substrate specificity of c-Kit circumvents a need for Src family kinases in signaling of growth and survival, thereby contributing to the transforming potential of c-Kit/D816V.

Improvement of a Stereoselective Biocatalytic Synthesis by Substrate and Enzyme Engineering: 2-Hydroxy-(4′-oxocyclohexyl)acetonitrile as the Model

Even if biocatalysis is finding increasing application, it still has to gain widespread use in synthetic chemistry. Reasons for this are limitations that enzymes have with regard to substrate range, reaction scope, and insufficient selectivity with unnatural compounds. These shortcomings can be challenged by enzyme and/or substrate engineering, which are employed to alter substrate specificity and enhance the enzyme selectivity toward unnatural substrates. Herein, these two approaches are coupled to improve the hydroxynitrile lyase catalyzed synthesis of 2-hydroxy-(4'-oxocyclohexyl)acetonitrile (4). The ketone functionality is masked as an enol ether, and the oxynitrilase of Hevea brasiliensis is engineered towards this masked substrate to give the product with a high optical purity and to drastically lower the amount of enzyme needed.

Modifications of human carboxylesterase for improved prodrug activation

Background: Carboxylesterases (CEs) are ubiquitous enzymes responsible for the hydrolysis of numerous clinically useful drugs. As ester moieties are frequently included in molecules to improve their water solubility and bioavailability, de facto they become substrates for CEs. Objective: In this review, we describe the properties of human CEs with regard to their ability to activate anticancer prodrugs and demonstrate how structure-based design can be used to modulate substrate specificity and to increase efficiency of hydrolysis. Methods: A specific example using CPT-11 and a human liver CE is discussed. However, these techniques can be applied to other enzymes and their associated prodrugs. Results: Structure-guided mutagenesis of CEs can be employed to alter substrate specificity and generate novel enzymes that are efficacious at anticancer prodrug activation.

Identification and Analysis of Conserved Sequence Motifs in Cytochrome P450 Family 2: FUNCTIONAL AND STRUCTURAL ROLE OF A MOTIF 187RFDYKD192 IN CYP2B ENZYMES

Using a multiple alignment of 175 cytochrome P450 (CYP) family 2 sequences, 20 conserved sequence motifs (CSMs) were identified with the program PCPMer. Functional importance of the CSM in CYP2B enzymes was assessed from available data on site-directed mutants and genetic variants. These analyses suggested an important role of the CSM 8, which corresponds to(187)RFDYKD(192) in CYP2B4. Further analysis showed that residues 187, 188, 190, and 192 have a very high rank order of conservation compared with 189 and 191. Therefore, eight mutants (R187A, R187K, F188A, D189A, Y190A, K191A, D192A, and a negative control K186A) were made in an N-terminal truncated and modified form of CYP2B4 with an internal mutation, which is termed 2B4dH/H226Y. Function was examined with the substrates 7-methoxy-4-(trifluoromethyl)coumarin (7-MFC), 7-ethoxy-4-(trifluoromethyl)coumarin (7-EFC), 7-benzyloxy-4-(trifluoromethyl)coumarin (7-BFC), and testosterone and with the inhibitors 4-(4-chlorophenyl)imidazole (4-CPI) and bifonazole (BIF). Compared with the template and K186A, the mutants R187A, R187K, F188A, Y190A, and D192A showed > or =2-fold altered substrate specificity, k(cat), K(m), and/or k(cat)/K(m) for 7-MFC and 7-EFC and 3- to 6-fold decreases in differential inhibition (IC(50,BIF)/IC(50,4-CPI)). Subsequently, these mutants displayed 5-12 degrees C decreases in thermal stability (T(m)) and 2-8 degrees C decreases in catalytic tolerance to temperature (T(50)) compared with the template and K186A. Furthermore, when R187A and D192A were introduced in CYP2B1dH, the P450 expression and thermal stability were decreased. In addition, R187A showed increased activity with 7-EFC and decreased IC(50,BIF)/IC(50,4-CPI) compared with 2B1dH. Analysis of long range residue-residue interactions in the CYP2B4 crystal structures indicated strong hydrogen bonds involving Glu(149)-Asn(177)-Arg(187)-Tyr(190) and Asp(192)-Val(194), which were significantly-reduced/abolished by the Arg(187)-->Ala and Asp(192)-->Alasubstitutions, respectively.

Ribozyme mediated trans insertion-splicing of modified oligonucleotides into RNA

The trans insertion-splicing reaction, catalyzed by a group I intron-derived from Pneumocystis carinii, was recently developed for the site-specific insertion of a segment of RNA into a separate RNA substrate. The molecular determinants of this reaction for binding and catalysis are reasonably well understood, making them easily and highly modifiable for altering substrate specificity. To demonstrate proof-of-concept, we now report that the P. carinii ribozyme can except modified oligonucleotides as substrates for catalyzing the trans insertion-splicing reaction. Oligonucleotides that contain one or more sugar modifications (deoxy or methoxy substitution), a backbone modification (phosphorothioate substitution), or a base modification (2-aminopurine or 4-thiouridine) are effective substrates in this reaction. Apparently, trans insertion-splicing is a unique and viable reaction for the site-specific incorporation of modified oligonucleotides into RNAs. This is the first report of a group I intron-derived ribozyme being capable of catalyzing the insertion of a modified oligonucleotide into RNA.

Engineering Substrate Preference in Subtilisin: Structural and Kinetic Analysis of a Specificity Mutant

Bacillus subtilisin has been a popular model protein for engineering altered substrate specificity. Although some studies have succeeded in increasing the specificity of subtilisin, they also demonstrate that high specificity is difficult to achieve solely by engineering selective substrate binding. In this paper, we analyze the structure and transient state kinetic behavior of Sbt160, a subtilisin engineered to strongly prefer substrates with phenylalanine or tyrosine at the P4 position. As in previous studies, we measure improvements in substrate affinity and overall specificity. Structural analysis of an inactive version of Sbt160 in complex with its cognate substrate reveals improved interactions at the S4 subsite with a P4 tyrosine. Comparison of transient state kinetic behavior against an optimal sequence (DFKAM) and a similar, but suboptimal, sequence (DVRAF) reveals the kinetic and thermodynamic basis for increased specificity, as well as the limitations of this approach. While highly selective substrate binding is achieved in Sbt160, several factors cause sequence specificity to fall short of that observed with natural processing subtilisins. First, for substrate sequences which are nearly optimal, the acylation reaction becomes faster than substrate dissociation. As a result, the level of discrimination among these substrates diminishes due to the coupling between substrate binding and the first chemical step (acylation). Second, although Sbt160 has 24-fold higher substrate affinity for the optimal substrate DFKAM than for DVRAF, the increased substrate binding energy is not translated into improved transition state stabilization of the acylation reaction. Finally, as interactions at subsites become stronger, the rate-determining step in peptide hydrolysis changes from acylation to product release. Thus, the release of the product becomes sluggish and leads to a low k(cat) for the reaction. This also leads to strong product inhibition of substrate turnover as the reaction progresses. The structural and kinetic analysis reveals that differences in the binding modes at subsites for substrates, transition states, and products are subtle and difficult to manipulate via straightforward protein engineering. These findings suggest several new strategies for engineering highly sequence selective enzymes.

Changes in γ-secretase activity and specificity caused by the introduction of consensus aspartyl protease active motif in Presenilin 1

Presenilin (PS1 or PS2) is an essential component of the active γ-secretase complex that liberates the Aβ peptides from amyloid precursor protein (APP). PS1 is regarded as an atypical aspartyl protease harboring two essential aspartic acids in the context of the sequence D257LV and D385FI, respectively, rather than the typical DTG...DTG catalytic motif of classical aspartyl proteases. In the present studies, we introduced the sequence DTG in PS1 at and around the catalytic D257 and D385 residues to generate three PS1 mutants: D257TG, D385TG, and the double-mutant D257TG/D385TG. The effects of these changes on the γ-secretase activity in the presence or absence of γ-secretase inhibitors and modulators were investigated. The results showed that PS1 mutants having D385TG robustly enhanced Aβ42 production compared to the wild type (wt), and were more sensitive than wt to inhibition by a classical aspartyl protease transition state mimic, and fenchylamine, a sulfonamide derivative. Unlike wt PS1 and some of its clinical mutants, all three PS1 artificial mutants decreased cleavage of Notch S3-site, suggesting that these artificial mutations may trigger conformational changes at the substrate docking and catalytic site that cause alteration of substrate specificity and inhibition pattern. Consistent with this notion, we have found that NSAID enzymatic inhibitors of COX, known modulators of the γ-secretase activity, cause PS1 mutants containing D385TG to produce higher levels of both Aβ38 and Aβ42, but to reduce levels of Aβ39, showing a pattern of Aβ formation different from that observed with wild type PS1 and its clinical mutants. This study provides an important structural clue for the rational design of drugs to inhibit processing of APP at the γ-site without interfering with Notch processing.

A Novel Genetic Selection System for Improved Enantioselectivity of Bacillus subtilis Lipase A

In directed evolution experiments, success often depends on the efficacy of screening or selection methods. Genetic selections have proven to be extremely valuable for evolving enzymes with improved catalytic activity, improved stability, or with altered substrate specificity. In contrast, enantioselectivity is a difficult parameter to select for. In this study, we present a successful strategy that not only selects for catalytic activity, but for the first time also for enantioselectivity, as demonstrated by the selection of Bacillus subtilis lipase A variants with inverted and improved enantioselectivity. A lipase mutant library in an aspartate auxotroph Escherichia coli was plated on minimal medium that was supplemented with the aspartate ester of the desired enantiomer (S)-(+)-1,2-O-isopropylidene-sn-glycerol. To inhibit growth of less enantioselective variants, a covalently binding phosphonate ester of the opposite (R)-(-)-1,2-O-isopropylidene-sn-glycerol enantiomer was added as well. After three selection rounds in which the selection pressure was increased by raising the phosphonate ester concentration, a mutant was selected with an improved enantioselectivity increased from an ee of -29.6 % (conversion 23.4 %) to an ee of +73.1 % (conversion 28.9 %) towards the (S)-(+)-enantiomer. Interestingly, its amino acid sequence showed that the acid of the catalytic triad had migrated to a position further along the loop that connects beta7 and alphaE; this shows that the position of the catalytic acid is not necessarily conserved in this lipase.

Engineering of protease variants exhibiting altered substrate specificity

By using an improved genetic screening system, variants of the HAV 3CP protease which exhibit altered P2 specificity were obtained. We randomly mutated the His145, Lys146, Lys147, and Leu155 residues that constitute the S2 pocket of 3CP and then isolated variants that preferred substrates with Gln over the original Thr at the P2 position using a yeast-based screening method. One of the isolated variants cleaved the Gln-containing peptide substrate more efficiently in vitro, proving the efficiency of our method in isolating engineered proteases with desired substrate selectivity.

Streptomyces Phospholipase D Mutants with Altered Substrate Specificity Capable of Phosphatidylinositol Synthesis

The substrate specificity of a phospholipase D (PLD) from Streptomyces antibioticus was altered by site-directed saturation mutagenesis, so that it was able to synthesize phosphatidylinositol (PI). Mutations were introduced in the pld gene at the positions corresponding to three amino acid residues that might be involved in substrate recognition, and the mutated genes were expressed in Escherichia coli BL21 (DE3). High-throughput screening of approximately 10,000 colonies for PI-synthesizing activity identified 25 PI-synthesizing mutant PLDs. One of these mutant enzymes was chosen for further analysis. The structure of the PI synthesized with the mutant enzyme was analyzed by HPLC-MS and NMR. It was found that the mutant enzyme generated a mixture of structural isomers of PIs with the phosphatidyl groups connected at different positions of the inositol ring. The phosphatidylcholine-hydrolyzing activity of the mutant PLD was much lower than that of the wild-type enzyme. The mutant enzyme was able to transphosphatidylate various cyclohexanols with a preference for bulkier compounds. This is the first example of alteration of the substrate specificity of PLD and of PI synthesis by Streptomyces PLD.

Characterization of thermostable aminoacylase from hyperthermophilic archaeon Pyrococcus horikoshii

The gene encoding putative aminoacylase (ORF: PH0722) in the genome sequence of a hyperthermophilic archaeon, Pyrococcus horikoshii, was cloned and overexpressed in Escherichia coli. The recombinant enzyme was determined to be thermostable aminoacylase (PhoACY), forming a homotetramer. Purified PhoACY showed the ability to release amino acid molecules from the substrates N-acetyl-L-Met, N-acetyl-L-Gln and N-acetyl-L-Leu, but had a lower hydrolytic activity towards N-acetyl-L-Phe. The kinetic parameters K(m) and k(cat) were determined to be 24.6 mm and 370 s(-1), respectively, for N-acetyl-l-Met at 90 degrees C. Purified PhoACY contained one zinc atom per subunit. EDTA treatment resulted in the loss of PhoACY activity. Enzyme activity was fully recovered by the addition of divalent metal ions (Zn(2+), Mn(2+) and Ni(2+)), and Mn(2+) addition caused an alteration in substrate specificity. Site-directed mutagenesis analysis and structural modeling of PhoACY, based on Arabidopsis thaliana indole-3-acetic acid amino acid hydrolase as a template, revealed that, amongst the amino acid residues conserved in PhoACY, His106, Glu139, Glu140 and His164 were related to the metal-binding sites critical for the expression of enzyme activity. Other residues, His198 and Arg260, were also found to be involved in the catalytic reaction, suggesting that PhoACY obeys a similar reaction mechanism to that proposed for mammalian aminoacylases.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of variants of monoamine oxidase from Aspergillus niger.

Monoamine oxidase from Aspergillus niger (MAO-N) is an FAD-dependent enzyme that catalyses the conversion of terminal amines to their corresponding aldehydes. Variants of MAO-N produced by directed evolution have been shown to possess altered substrate specificity. Crystals of two of these variants (MAO-N-3 and MAO-N-5) have been obtained; the former displays P2(1) symmetry with eight molecules per asymmetric unit and the latter has P4(1)2(1)2 or P4(3)2(1)2 symmetry and two molecules per asymmetric unit. Solution of these structures will help shed light on the molecular determinants of improved activity and high enantioselectivity towards a broad range of substrates.

Isolation of a distinct class of gain-of-function SHP-2 mutants with oncogenic RAS-like transforming activity from solid tumors

SHP-2 protein tyrosine phosphatase plays an important role in activation of the RAS-dependent signaling. Gain-of-function mutations in the PTPN11 gene, which encodes SHP-2, have been found in the leukemia-prone developmental disorder Noonan syndrome as well as sporadic childhood leukemias, indicating that SHP-2 is a bona fide human oncoprotein. However, the role of SHP-2 mutations in non-hematological malignancies remains obscure. Here, we screened for PTPN11 mutations in primary solid tumors and identified a 1520C>A mutation that causes threonine-507 to lysine (T507K) substitution in the phosphatase domain of SHP-2 in a case of hepatocellular carcinoma. T507K SHP-2 exhibited altered substrate specificity with slightly elevated basal phosphatase activity. Upon expression in NIH3T3 cells, T507K SHP-2 induced transformed foci, which was not observed with wild type, Noonan-specific or leukemia-specific SHP-2. Furthermore, NIH3T3 cells transformed by T507K SHP-2 showed anchorage-independent growth and developed tumors in nude mice. These results indicate that quantitative and/or qualitative alteration in phosphatase activity determines the transforming potential as well as target cell/tissue spectrum of individual SHP-2 mutants as oncoproteins. Although rare in solid tumors, the identified T507K SHP-2 represents a distinct class of SHP-2 mutants with oncogenic RAS-like transforming activity, which could contribute to the development of solid tumors.

Laboratory Evolution of Escherichia coli Thioredoxin for Enhanced Catalysis of Protein Oxidation in the Periplasm Reveals a Phylogenetically Conserved Substrate Specificity Determinant

Thioredoxin exported into the Escherichia coli periplasm catalyzes the oxidation of protein thiols in a DsbB-dependent function. However, the oxidative activity of periplasmic thioredoxin is insufficient to render dsbA(-) cells susceptible to infection by M13, a phenotype that is critically dependent on disulfide bond formation in the cell envelope. We sought to examine the molecular determinants that are required in order to convert thioredoxin from a reductant into an efficient periplasmic oxidant. A genetic screen for mutations in thioredoxin that render dsbA(-) cells sensitive to infection by M13 led to the isolation of a single amino acid substitution, G74S. In vivo the TrxA(G74S) mutant exhibited enhanced catalytic activity in the oxidation of alkaline phosphatase but was unable to oxidize FlgI and restore cell motility. In vitro studies revealed that the G74S substitution does not affect the redox potential of the thioredoxin-active site or its kinetics of oxidation by DsbB. Thus, the gain of function afforded by G74S stems in part from its altered substrate specificity, which also rendered the protein more resistant to reduction by DsbD/DsbC in the periplasm.

Engineering the Properties of D-Amino Acid Oxidases by a Rational and a Directed Evolution Approach

D-amino acid oxidase (DAAO) is a FAD-containing flavoprotein that dehydrogenates the D-isomer of amino acids to the corresponding imino acids, coupled with the reduction of FAD. The cofactor then reoxidizes on molecular oxygen and the imino acid hydrolyzes spontaneously to the alpha-keto acid and ammonia. In vitro DAAO displays broad substrate specificity, acting on several neutral and basic D-amino acids: the most efficient substrates are amino acids with hydrophobic side chains. D-aspartic acid and D-glutamic acid are not substrates for DAAO. Through the years, it has been the subject of a number of structural, functional and kinetic investigations. The most recent advances are represented by site-directed mutagenesis studies and resolution of the 3D-structure of the enzymes from pig, human and yeast. The two approaches have given us a deeper understanding of the structure-function relationships and promoted a number of investigations aimed at the modulating the protein properties. By a rational and/or a directed evolution approach, DAAO variants with altered substrate specificity (e.g., active on acidic or on all D-amino acids), increased stability (e.g., stable up to 60 degrees C), modified interaction with the flavin cofactor, and altered oligomeric state were produced. The aim of this paper is to provide an overview of the most recent research on the engineering of DAAOs to illustrate their new intriguing properties, which also have enabled us to pursue new biotechnological applications.

Mice Engineered To Express a Form of Thrombin Favoring Protein C Are Resistant to S. aureus-Induced Sepsis.

Thrombin is central in thrombus formation as both a positive mediator of thrombus formation through the proteolytic activation of PARs, fibrinogen, fXI and other prothrombotic substrates, and a negative modulator of the coagulation cascade through the activation of protein C. Detailed structure-function studies have revealed that thrombin can be redesigned to favor either procoagulant or anticoagulant substrates. The introduction of W215A/E217A substitutions in the murine thrombin active site (fIIWE) results in a pronounced “specificity switch” that reduces catalytic efficiency with fibrinogen by at least 3-orders-magnitude while only modestly reducing activity for protein C activation. To evaluate the effects of fIIWE activity in vivo, we have used a gene-targeting strategy to generate mice carrying the W215A/E217A mutations in the endogenous murine prothrombin gene. The mutant allele was transmitted through the germline and was found to support the expression of normal levels of hepatic fII mRNA and plasma fII in both heterozygous and homozygous neonates. Unlike fII knockout mice, homozygous fIIWE mice were observed at term with the expected Mendelian frequency. Nevertheless, homozygous fIIWE offspring uniformly succumbed to spontaneous bleeding events within days of birth. Heterozygous fIIWE/WT animals generally survived to adulthood, were capable of carrying multiple liters to term, and unchallenged mice displayed a hematological profile similar to wildtype mice. However, consistent with a predicted anticoagulant phenotype, adult fIIWE/WT heterozygotes exhibited significantly delayed thrombus formation following ferric chloride injury of mesenteric arterioles and extended bleeding times following tail tip excision relative to control mice expressing wildtype fII. Given that activated protein C has been shown to be efficacious in the treatment of sepsis, we explored whether the shift in thrombin specificity in heterozygous fIIWE/WT mice would confer the benefit of rendering animals tolerant to acute septic challenges. Kaplan-Meier analyses following intravenous administration of S. aureus revealed that fIIWE/WT mice exhibited a significant survival advantage over littermate wildtype animals challenged in parallel and tracked over a 7-day observation period. Notably, extended thrombus formation and bleeding times as well as resistance to sepsis was not simply a function of half normal wildtype fII expression. When these analyses were performed in animals carrying one wildtype allele and one null mutation allele, results were similar to wiltype. These studies further underscore the interplay between the hemostatic and inflammatory systems in vivo and highlight the possible therapeutic utility of recombinant (pro)thrombin derivatives with selected alterations in substrate specificity.

Polymorphisms in the MGMT Gene as Predictors of Leukemia Risk and Response to Therapy in Children with AML.

Approximately 40% of patients treated with chemotherapy for pediatric AML will relapse after initial treatment and 8–10% will die of toxic side effects of chemotherapy. This highly variable response to standardized treatment protocols is due in part to genetic variation among patients in the metabolism of chemotherapeutic agents and in the response to the DNA damage they induce. We have analyzed polymorphisms in the coding region of the DNA alkylation repair gene O6-Methylguanine-DNA Methyltransferase (MGMT) in 356 patients with de novo pediatric AML to determine whether naturally occurring MGMT variant alleles correlate with leukemia susceptibility, treatment efficacy, and the incidence of adverse effects of chemotherapy. The polymorphism rs2308321 (Ile143Val) has been shown to modify biochemical properties of the MGMT protein, with altered substrate specificity of the rare valine allele (G genotype) compared with the isoleucine allele (A genotype) (Margison et al, Carcinogenesis , 26;1473, 2005). All patients were treated on Children's Cancer Group protocols 2941 and 2961. Outcomes were examined stratified by genotype. All patients received intensively timed induction therapy with IDA-DCTER (idarubicin, dexamethasone, Ara-C, thioguanine, etoposide and daunomycin) given on days 0 to 3 followed by DCTER given on days 10 to 13. On recovery of ANC and platelet counts, patients were randomized to consolidation therapy with a further course of IDA-DCTER/DCTER or IDA-FLAG (idarubicin, fludarabine, Ara-C and G-CSF). Patients received intensification with a single course of high dose Ara-C and L-asparaginase unless a family donor was available when allogeneic transplant was performed after busulfan and cyclophosphamide. Black children were significantly more likely to have a homozygous wild-type AA genotype than white children (p=0.002). When compared to a healthy control population, there was no correlation between MGMT polymorphisms and the risk of AML overall. However, in a case-case comparison of genetic sub-types of AML, children with a variant allele (AG or G/G genotype) were significantly more likely to have monosomy 7 than AA cases (9% vs 1%; p=0.01); in contrast, children with trisomy 8 were significantly more likely to have an AA genotype (8% vs 0%; p=0.028). The presence of at least one G allele was associated with a significant reduction in the incidence of relapse. There was no effect on treatment-related mortality (TRM). In a multivariate Cox model adjusted for race, the increased risk of relapse (RR) associated with the variant G allele remained significant (hazard ratio 1.72 95% CI 1.03–2.86; p=0.037). These results suggest that genotyping at the rs2308321 polymorphism in the MGMT gene could be useful in the risk stratification of patients with AML prior to therapy.A/AA/G+G/GN% ± 2 SEN% ± 2 SEp-value5yr OS from end of course 128054 ± 67671 ± 110.0315yr RR from end of course 128043 ± 67627 ± 110.0145yr TRM from end of course 128014 ± 47614 ± 80.11

Functional effects of protein sequence polymorphisms in the organic cation/ergothioneine transporter OCTN1 (SLC22A4)

OCTN1 is a multispecific transporter of organic cations and zwitterions, including several clinically important drugs as well as the antioxidant ergothioneine. OCTN1 is highly expressed in the kidney, where it is thought to aid in active secretion of organic cations, and may facilitate the active reabsorption of ergothioneine. Genetic variation in OCTN1 may help to explain interindividual variability in the pharmacokinetics of many cationic or zwitterionic drugs. We screened for human genetic variants in the OCTN1 coding region by direct sequencing in a large sample (n=270) of ethnically diverse healthy volunteers. Six protein sequence-altering variants were identified, including five-amino-acid substitutions and one nonsense mutation. Two of the variants, T306I and L503F, were polymorphic, occurring at frequencies of 37 and 19%, respectively, in the total sample. Allele frequencies are varied by ethnicity. In biochemical assays, two of the variants (D165G and R282X) resulted in complete loss of transport function, and one variant (M205I) caused a reduction in activity to approximately 50% of the reference sequence protein. One variant, L503F, showed altered substrate specificity; this variant occurred at particularly high allele frequency (42%) in the European-American participants in our sample. Subcellular localization and ergothioneine inhibition kinetics were similar among the common amino-acid sequence variants of OCTN1. The common OCTN1-L503F variant may explain a significant amount of population variation in the pharmacokinetics of OCTN1 substrate drugs. The rare loss-of-function variants provide a rational tool for studying the importance of ergothioneine in humans in vivo.

Alteration of substrate specificity of phenylalanine dehydrogenase from Bacillus sphaericus towards an aliphatic unsaturated amino acid by directed evolution

Alteration of substrate specificity of phenylalanine dehydrogenase from Bacillus sphaericus towards an aliphatic unsaturated amino acid by directed evolution

Development of a selection system for the detection of L-ribose isomerase expressing mutants of Escherichia coli

L-Arabinose isomerase (E.C. 5.3.1.14) catalyzes the reversible isomerization between L-arabinose and L-ribulose and is highly selective towards L-arabinose. By using a directed evolution approach, enzyme variants with altered substrate specificity were created and screened in this research. More specifically, the screening was directed towards the identification of isomerase mutants with L-ribose isomerizing activity. Random mutagenesis was performed on the Escherichia coli L-arabinose isomerase gene (araA) by error-prone polymerase chain reaction to construct a mutant library. To enable screening of this library, a selection host was first constructed in which the mutant genes were transformed. In this selection host, the genes encoding for L-ribulokinase and L-ribulose-5-phosphate-4-epimerase were brought to constitutive expression and the gene encoding for the native L-arabinose isomerase was knocked out. L-Ribulokinase and L-ribulose-5-phosphate-4-epimerase are necessary to ensure the channeling of the formed product, L-ribulose, to the pentose phosphate pathway. Hence, the mutant clones could be screened on a minimal medium with L-ribose as the sole carbon source. Through the screening, two first-generation mutants were isolated, which expressed a small amount of L-ribose isomerase activity.