Structures of an engineered phospholipase D with specificity for secondary alcohol transphosphatidylation: insights into plasticity of substrate binding and activation.

Phospholipase D (PLD) is an enzyme useful for the enzymatic modification of phospholipids. In the presence of primary alcohols, the enzyme catalyses transphosphatidylation of the head group of phospholipid substrates to synthesise a modified phospholipid product. However, the enzyme is specific for primary alcohols and thus the limitation of the molecular size of the acceptor compounds has restricted the type of phospholipid species that can be synthesised. An engineered variant of PLD from Streptomyces antibioticus termed TNYR SaPLD was developed capable of synthesising 1-phosphatidylinositol with positional specificity of up to 98%. To gain a better understanding of the substrate binding features of the TNYR SaPLD, crystal structures have been determined for the free enzyme and its complexes with phosphate, phosphatidic acid and 1-inositol phosphate. Comparisons of these structures with the wild-type SaPLD show a larger binding site able to accommodate a bulkier secondary alcohol substrate as well as changes to the position of a flexible surface loop proposed to be involved in substrate recognition. The complex of the active TNYR SaPLD with 1-inositol phosphate reveals a covalent intermediate adduct with the ligand bound to H442 rather than to H168, the proposed nucleophile in the wild-type enzyme. This structural feature suggests that the enzyme exhibits plasticity of the catalytic mechanism different from what has been reported to date for PLDs. These structural studies provide insights into the underlying mechanism that governs the recognition of myo-inositol by TNYR SaPLD, and an important foundation for further studies of the catalytic mechanism.

Highly selective bile acid hydroxylation by the multifunctional bacterial P450 monooxygenase CYP107D1 (OleP).

OBJECTIVE: Regio- and stereoselective hydroxylation of lithocholic acid (LCA) using CYP107D1 (OleP), a cytochrome P450 monooxygenase from the oleandomycin synthesis pathway of Streptomyces antibioticus. RESULTS: Co-expression of CYP107D1 from S. antibioticus and the reductase/ferredoxin system PdR/PdX from Pseudomonas putida was performed in Escherichia coli whole cells. In vivo hydroxylation of LCA exclusively yielded the 6ß-OH product murideoxycholic acid (MDCA). In resting cells, 19.5% of LCA was converted to MDCA within 24 h, resulting in a space time yield of 0.04 mmol L-1 h-1. NMR spectroscopy confirmed the identity of MDCA as the sole product. CONCLUSIONS: The multifunctional P450 monooxygenase CYP107D1 (OleP) can hydroxylate LCA, forming MDCA as the only product.

Microbial synthesis of the type I polyketide 6-methylsalicylate with Corynebacterium glutamicum.

Type I polyketide synthases (PKSs) are large multi-domain proteins converting simple acyl-CoA thioesters such as acetyl-CoA and malonyl-CoA to a large diversity of biotechnologically interesting molecules. Such multi-step reaction cascades are of particular interest for applications in engineered microbial cell factories, as the introduction of a single protein with many enzymatic activities does not require balancing of several individual enzymatic activities. However, functional introduction of type I PKSs into heterologous hosts is very challenging as the large polypeptide chains often do not fold properly. In addition, PKS usually require post-translational activation by dedicated 4'-phosphopantetheinyl transferases (PPTases). Here, we introduce an engineered Corynebacterium glutamicum strain as a novel microbial cell factory for type I PKS-derived products. Suitability of C. glutamicum for polyketide synthesis could be demonstrated by the functional introduction of the 6-methylsalicylic acid synthase ChlB1 from Streptomyces antibioticus. Challenges related to protein folding could be overcome by translation fusion of ChlB1Sa to the C-terminus of the maltose-binding protein MalE from Escherichia coli. Surprisingly, ChlB1Sa was also active in the absence of a heterologous PPTase, which finally led to the discovery that the endogenous PPTase PptACg of C. glutamicum can also activate ChlB1Sa. The best strain, engineered to provide increased levels of acetyl-CoA and malonyl-CoA, accumulated up to 41 mg/L (0.27 mM) 6-methylsalicylic acid within 48 h of cultivation. Further experiments showed that PptACg of C. glutamicum can also activate nonribosomal peptide synthetases (NRPSs), rendering C. glutamicum a promising microbial cell factory for the production of several fine chemicals and medicinal drugs.

Acyl chain that matters: introducing sn-2 acyl chain preference to a phospholipase D by protein engineering.

Phospholipase D (PLD) is an enzyme widely used for enzymatic synthesis of structured phospholipids (PLs) with modified head groups. These PLs are mainly used as food supplements and liposome ingredients. Still, there is a need for an enzyme that discriminates between PLs and lysoPLs, for specific detection of lysoPLs in various specimens and enzymatic synthesis of certain PLs from a mixed substrate. To meet this demand, we aimed at altering sn-2 acyl chain recognition of a PLD, leading to a variant enzyme preferably reacting on lysoPLs, by protein engineering. Based on the crystal structure of Streptomyces antibioticus PLD, W166 was targeted for saturation mutagenesis due to its strong interaction with the sn-2 acyl chain of the PL. Screening result pointed at W166R and W166K PLDs to selectively react on lysophosphatidylcholine (lysoPC), while not on PC. These variants showed a negative correlation between activity and sn-2 chain length of PL substrates. This behavior was not observed in the wild-type (WT)-PLD. Kinetic analysis revealed that the W166R and W166K variants have 7-10 times higher preference to lysoPC compared to the WT-PLD. Additionally, W166R PLD showed detectable activity toward glycero-3-phosphocholine, unlike the WT-PLD. Applicability of the lysoPC-preferring PLD was demonstrated by detection of lysoPC in the mixed PC/lysoPC sample and by the synthesis of cyclic phosphatidic acid. Structure model analyses supported the experimental findings and provided a basis for the structure model-based hypothesis on the observed behavior of the enzymes.

Ketonization of Proline Residues in the Peptide Chains of Actinomycins by a 4-Oxoproline Synthase.

X-type actinomycins (Acms) contain 4-hydroxyproline (Acm X0 ) or 4-oxoproline (Acm X2 ) in their ß-pentapeptide lactone rings, whereas their α ring contains proline. We demonstrate that these Acms are formed through asymmetric condensation of Acm half molecules (Acm halves) containing proline with 4-hydroxyproline- or 4-oxoproline-containing Acm halves. In turn, we show-using an artificial Acm half analogue (PPL 1) with proline in its peptide chain-their conversion into the 4-hydroxyproline- and 4-oxoproline-containing Acm halves, PPL 0 and PPL 2, in mycelial suspensions of Streptomyces antibioticus. Two responsible genes of the Acm X biosynthetic gene cluster of S. antibioticus, saacmM and saacmN, encoding a cytochrome P450 monooxygenase (Cyp) and a ferredoxin were identified. After coexpression in Escherichia coli, their gene products converted PPL 1 into PPL 0 and PPL 2 in vivo as well as in situ in permeabilized cell of the transformed E. coli strain in conjunction with the host-encoded ferredoxin reductase in a NADH (NADPH)-dependent manner. saAcmM has high sequence similarity to the Cyp107Z (Ema) family of Cyps, which can convert avermectin B1 into its keto derivative, 4''-oxoavermectin B1. Determination of the structure of saAcmM reveals high similarity to the Ema structure but with significant differences in residues decorating their active sites, which defines saAcmM and its orthologues as a distinct new family of peptidylprolineketonizing Cyp.

Inhibition of homologous phosphorolytic ribonucleases by citrate may represent an evolutionarily conserved communicative link between RNA degradation and central metabolism.

Ribonucleases play essential roles in all aspects of RNA metabolism, including the coordination of post-transcriptional gene regulation that allows organisms to respond to internal changes and environmental stimuli. However, as inherently destructive enzymes, their activity must be carefully controlled. Recent research exemplifies the repertoire of regulatory strategies employed by ribonucleases. The activity of the phosphorolytic exoribonuclease, polynucleotide phosphorylase (PNPase), has previously been shown to be modulated by the Krebs cycle metabolite citrate in Escherichia coli. Here, we provide evidence for the existence of citrate-mediated inhibition of ribonucleases in all three domains of life. In silico molecular docking studies predict that citrate will bind not only to bacterial PNPases from E. coli and Streptomyces antibioticus, but also PNPase from human mitochondria and the structurally and functionally related archaeal exosome complex from Sulfolobus solfataricus. Critically, we show experimentally that citrate also inhibits the exoribonuclease activity of bacterial, eukaryotic and archaeal PNPase homologues in vitro. Furthermore, bioinformatics data, showing key citrate-binding motifs conserved across a broad range of PNPase homologues, suggests that this regulatory mechanism may be widespread. Overall, our data highlight a communicative link between ribonuclease activity and central metabolism that may have been conserved through the course of evolution.

Functional analysis and crystallographic structure of clotrimazole bound OleP, a cytochrome P450 epoxidase from Streptomyces antibioticus involved in oleandomycin biosynthesis.

BACKGROUND: OleP is a cyt P450 from Streptomyces antibioticus carrying out epoxigenation of the antibiotic oleandomycin during its biosynthesis. The timing of its reaction has not been fully clarified, doubts remain regarding its substrate and catalytic mechanism. METHODS: The crystal structure of OleP in complex with clotrimazole, an inhibitor of P450s used in therapy, was solved and the complex formation dynamics was characterized by equilibrium and kinetic binding studies and compared to ketoconazole, another azole differing for the N1-substituent. RESULTS: Clotrimazole coordinates the heme and occupies the active site. Most of the residues interacting with clotrimazole are conserved and involved in substrate binding in MycG, the P450 epoxigenase with the highest homology with OleP. Kinetic characterization of inhibitor binding revealed OleP to follow a simple bimolecular reaction, without detectable intermediates. CONCLUSIONS: Clotrimazole-bound OleP adopts an open form, held by a π-π stacking chain that fastens helices F and G and the FG loop. Affinity is affected by the interactions of the N1 substituent within the active site, given the one order of magnitude difference of the off-rate constants between clotrimazole and ketoconazole. Based on structural similarities with MycG, we propose a binding mode for both oleandomycin intermediates, that are the candidate substrates of OleP. GENERAL SIGNIFICANCE: Among P450 epoxigenases OleP is the only one that introduces an epoxide on a non-activated C­C bond. The data here presented are necessary to understand the rare chemistry carried out by OleP, to engineer it and to design more selective and potent P450-targeted drugs.

Directing positional specificity in enzymatic synthesis of bioactive 1-phosphatidylinositol by protein engineering of a phospholipase D.

Phosphatidylinositol (PI) holds a potential of becoming an important dietary supplement due to its effects on lipid metabolism in animals and humans manifested as a decrease of the blood cholesterol and lipids, and relief of the metabolic syndrome. To establish an efficient, enzymatic system for PI production from phosphatidylcholine and myo-inositol as an alcohol acceptor, our previous study started with the wild-type Streptomyces antibioticus phospholipase D (SaPLD) as a template for generation of PI-synthesizing variants by saturation mutagenesis targeting positions involved in acceptor accommodation, W187, Y191, and Y385. The isolated variants generated PI as a mixture of positional isomers, among which only 1-PI exists in nature. Thus, the current study has focused to improve positional specificity of W187N/Y191Y/Y385R SaPLD (NYR) which generates PI as a mixture of 1-PI and 3-PI in the ratio of 76/24, by subjecting four residues of its acceptor-binding site to saturation mutagenesis. Subsequent screening pointed at NYR-186T and NYR-186L as the most improved variants producing PI with a ratio of 1-/3-PI = 93/7 and 87/13, respectively, at 37°C. Lowering the reaction temperature further improved the specificity of both variants to 1-/3-PI > 97/3 at 20°C with no change in total PI yield. Structure model analyses imply that G186T and G186L mutations increased rigidity of the acceptor-binding site, thus limiting the possible orientations of myo-inositol. The two newly isolated PLDs are promising for future application in large-scale 1-PI production.

SimC7 Is a Novel NAD(P)H-Dependent Ketoreductase Essential for the Antibiotic Activity of the DNA Gyrase Inhibitor Simocyclinone.

Simocyclinone D8 (SD8) is a potent DNA gyrase inhibitor produced by Streptomyces antibioticus Tü6040. The simocyclinone (sim) biosynthetic gene cluster has been sequenced and a hypothetical biosynthetic pathway has been proposed. The tetraene linker in SD8 was suggested to be the product of a modular type I polyketide synthase working in trans with two monofunctional enzymes. One of these monofunctional enzymes, SimC7, was proposed to supply a dehydratase activity missing from two modules of the polyketide synthase. In this study, we report the function of SimC7. We isolated the entire ~72-kb sim cluster on a single phage artificial chromosome clone and produced simocyclinone heterologously in a Streptomyces coelicolor strain engineered for improved antibiotic production. Deletion of simC7 resulted in the production of a novel simocyclinone, 7-oxo-SD8, which unexpectedly carried a normal tetraene linker but was altered in the angucyclinone moiety. We demonstrate that SimC7 is an NAD(P)H-dependent ketoreductase that catalyzes the conversion of 7-oxo-SD8 into SD8. 7-oxo-SD8 was essentially inactive as a DNA gyrase inhibitor, and the reduction of the keto group by SimC7 was shown to be crucial for high-affinity binding to the enzyme. Thus, SimC7 is an angucyclinone ketoreductase that is essential for the biological activity of simocyclinone.

Investigation of binding-site homology between mushroom and bacterial tyrosinases by using aurones as effectors.

Tyrosinase is a copper-containing enzyme found in plants and bacteria, as well as in humans, where it is involved in the biosynthesis of melanin-type pigments. Tyrosinase inhibitors have attracted remarkable research interest as whitening agents in cosmetology, antibrowning agents in food chemistry, and as therapeutics. In this context, commercially available tyrosinase from mushroom (TyM) is frequently used for the identification of inhibitors. This and bacterial tyrosinase (TyB) have been the subjects of intense biochemical and structural studies, including X-ray diffraction analysis, and this has led to the identification of structural homology and divergence among enzymes from different sources. To better understand the behavior of potential inhibitors of TyM and TyB, we selected the aurone family-previously identified as potential inhibitors of melanin biosynthesis in human melanocytes. In this study, a series of 24 aurones with different hydroxylation patterns at the A- and B-rings were evaluated on TyM and TyB. The results show that, depending on the hydroxylation pattern of A- and B-rings, aurones can behave as inhibitors, substrates, and activators of both enzymes. Computational analysis was performed to identify residues surrounding the aurones in the active sites of both enzymes and to rationalize the interactions. Our results highlight similarities and divergence in the behavior of TyM and TyB toward the same set of molecules.

A simple strategy for glycosyltransferase-catalyzed aminosugar nucleotide synthesis.

A set of 2-chloro-4-nitrophenyl glucosamino-/xylosaminosides were synthesized and assessed as potential substrates in the context of glycosyltransferase-catalyzed formation of the corresponding UDP/TDP-α-D-glucosamino-/xylosaminosugars and in single-vessel model transglycosylation reactions. This study highlights a robust platform for aminosugar nucleotide synthesis and reveals OleD Loki to be a proficient catalyst for U/TDP-aminosugar synthesis and utilization

Inulavosin and its benzo-derivatives, melanogenesis inhibitors, target the copper loading mechanism to the active site of tyrosinase.

Tyrosinase, a melanosomal membrane protein containing copper, is a key enzyme for melanin synthesis in melanocytes. Inulavosin inhibits melanogenesis by enhancing a degradation of tyrosinase in lysosomes. However, the mechanism by which inulavosin redirects tyrosinase to lysosomes is yet unknown. The analyses of structure-activity relationship of inulavosin and its benzo-derivatives reveal that the hydroxyl and the methyl groups play a critical role in their inhibitory activity. Intriguingly, the docking studies to tyrosinase suggest that the compounds showing inhibitory activity bind through hydrophobic interactions to the cavity of tyrosinase below which the copper-binding sites are located. This cavity is proposed to be required for the association with a chaperon that assists in copper loading to tyrosinase in Streptomyces antibioticus. Inulavosin and its benzo-derivatives may compete with the copper chaperon and result in a lysosomal mistargeting of apo-tyrosinase that has a conformational defect.

Diverse levels of sequence selectivity and catalytic efficiency of protein-tyrosine phosphatases.

The sequence selectivity of 14 classical protein-tyrosine phosphatases (PTPs) (PTPRA, PTPRB, PTPRC, PTPRD, PTPRO, PTP1B, SHP-1, SHP-2, HePTP, PTP-PEST, TCPTP, PTPH1, PTPD1, and PTPD2) was systematically profiled by screening their catalytic domains against combinatorial peptide libraries. All of the PTPs exhibit similar preference for pY peptides rich in acidic amino acids and disfavor positively charged sequences but differ vastly in their degrees of preference/disfavor. Some PTPs (PTP-PEST, SHP-1, and SHP-2) are highly selective for acidic over basic (or neutral) peptides (by >10(5)-fold), whereas others (PTPRA and PTPRD) show no to little sequence selectivity. PTPs also have diverse intrinsic catalytic efficiencies (kcat/KM values against optimal substrates), which differ by >10(5)-fold due to different kcat and/or KM values. Moreover, PTPs show little positional preference for the acidic residues relative to the pY residue. Mutation of Arg47 of PTP1B, which is located near the pY-1 and pY-2 residues of a bound substrate, decreased the enzymatic activity by 3-18-fold toward all pY substrates containing acidic residues anywhere within the pY-6 to pY+5 region. Similarly, mutation of Arg24, which is situated near the C-terminus of a bound substrate, adversely affected the kinetic activity of all acidic substrates. A cocrystal structure of PTP1B bound with a nephrin pY(1193) peptide suggests that Arg24 engages in electrostatic interactions with acidic residues at the pY+1, pY+2, and likely other positions. These results suggest that long-range electrostatic interactions between positively charged residues near the PTP active site and acidic residues on pY substrates allow a PTP to bind acidic substrates with similar affinities, and the varying levels of preference for acidic sequences by different PTPs are likely caused by the different electrostatic potentials near their active sites. The implications of the varying sequence selectivity and intrinsic catalytic activities with respect to PTP in vivo substrate specificity and biological functions are discussed.

Optimization of phenoxazinone synthase production by response surface methodology and its application in Congo red decolourization

Background: Enzymatic decolourization has been recently proposed as a promising and eco-friendly method for treatment of synthetic dye-contaminated wastewaters. However, the processes require large quantities of enzymes, attracting significant attention in developing efficient methods for mass production of multifunctional enzymes. Several methods such as response surface methodology (RSM) and orthogonal experiment have been applied to optimize the parameters in bioprocesses for enzyme production. Results: In the present study, a laccase-like enzyme, phenoxazinone synthase (PHS) originated from Streptomyces antibioticus was recombinantly expressed in Escherichia coli BL21 (DE3). The production of PHS in E. coli BL21 was optimized by response surface methodology based on Box-Behnken design. A full third-order polynomial model was generated by data analysis with Statistica 8.0 in which the optimal conditions for PHS production were calculated to be 1.525 mM CuSO4 and 16.096 hrs induction at temperature of 29.88ºC. The highest PHS production under optimal conditions was calculated to be 4098.51 U/l using the established model. Average PHS production obtained from actual production processes carried out under the calculated optimal conditions was 4052.00 U/l, very close to the value predicted by the model. Crude PHS was subsequently tested in Congo red decolourization which exhibited a low decolourization rate of 27% without mediator. Several mediators were found to improve PHS-catalyzed Congo red decolourization, with the highest rate of 73.89% obtained with 2,2’-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) as mediator under optimized conditions of 4000 U/l PHS activity, 10 μM ABTS, 100 μM Congo red, and 8 hrs reaction time. Conclusion: Our results indicated that PHS recombinantly produced in E. coli BL21 was a prospective enzyme for decolorizing reactive dye Congo red.

RNase III is required for actinomycin production in Streptomyces antibioticus.

Using insertional mutagenesis, we have disrupted the RNase III gene, rnc, of the actinomycin-producing streptomycete, Streptomyces antibioticus. Disruption was verified by Southern blotting. The resulting strain grows more vigorously than its parent on actinomycin production medium but produces significantly lower levels of actinomycin. Complementation of the rnc disruption with the wild-type rnc gene from S. antibioticus restored actinomycin production to nearly wild-type levels. Western blotting experiments demonstrated that the disruptant did not produce full-length or truncated forms of RNase III. Thus, as is the case in Streptomyces coelicolor, RNase III is required for antibiotic production in S. antibioticus. No differences in the chemical half-lives of bulk mRNA were observed in a comparison of the S. antibioticus rnc mutant and its parental strain.

Assessing the regioselectivity of OleD-catalyzed glycosylation with a diverse set of acceptors.

To explore the acceptor regioselectivity of OleD-catalyzed glucosylation, the products of OleD-catalyzed reactions with six structurally diverse acceptors flavones- (daidzein), isoflavones (flavopiridol), stilbenes (resveratrol), indole alkaloids (10-hydroxycamptothecin), and steroids (2-methoxyestradiol)-were determined. This study highlights the first synthesis of flavopiridol and 2-methoxyestradiol glucosides and confirms the ability of OleD to glucosylate both aromatic and aliphatic nucleophiles. In all cases, molecular dynamics simulations were consistent with the determined product distribution and suggest the potential to develop a virtual screening model to identify additional OleD substrates.

Crystallization, optimization and preliminary X-ray characterization of a metal-dependent PI-PLC from Streptomyces antibioticus.

A recombinant metal-dependent phosphatidylinositol-specific phospholipase C (PI-PLC) from Streptomyces antibioticus has been crystallized by the hanging-drop method with and without heavy metals. The native crystals belonged to the orthorhombic space group P222, with unit-cell parameters a=41.26, b=51.86, c=154.78 Å. The X-ray diffraction results showed significant differences in the crystal quality of samples soaked with heavy atoms. Additionally, drop pinning, which increases the surface area of the drops, was also used to improve crystal growth and quality. The combination of heavy-metal soaks and drop pinning was found to be critical for producing high-quality crystals that diffracted to 1.23 Šresolution.

High-throughput colorimetric assays for nucleotide sugar formation and glycosyl transfer.

Glycosyltransferases are ubiquitous in nature, catalyzing glycosidic bond formation in the context of an enormous range of substrates, which include all major classes of biological molecules. Because this wide range of substrates lacks a shared, distinguishable feature that can be altered by glycosyl transfer, general assays for detection of glycosyltransferase activity have long been largely limited to low-throughput methods. Of those high-throughput assays reported in the literature, many are confined to specific glycosyl transfer reactions with modified aglycon acceptors selected for their unique analytical properties. Herein are described a series of protocols centered on the use of 2-chloro-4-nitrophenyl glycoside donors and the reversibility of glycosyltransferase-catalyzed reactions to enable a colorimetric assay for the formation of sugar nucleotides, coupled reaction systems for the glycodiversification of small molecules, and a general colorimetric assay for glycosyltransfer, applicable to drug discovery, protein engineering, and other fundamental sugar nucleotide-dependent investigations.

Insights into a divergent phenazine biosynthetic pathway governed by a plasmid-born esmeraldin gene cluster.

Phenazine-type metabolites arise from either phenazine-1-carboxylic acid (PCA) or phenazine-1,6-dicarboxylic acid (PDC). Although the biosynthesis of PCA has been studied extensively, PDC assembly remains unclear. Esmeraldins and saphenamycin, the PDC originated products, are antimicrobial and antitumor metabolites isolated from Streptomyces antibioticus Tü 2706. Herein, the esmeraldin biosynthetic gene cluster was identified on a dispensable giant plasmid. Twenty-four putative esm genes were characterized by bioinformatics, mutagenesis, genetic complementation, and functional protein expressions. Unlike enzymes involved in PCA biosynthesis, EsmA1 and EsmA2 together decisively promoted the PDC yield. The resulting PDC underwent a series of conversions to give 6-acetylphenazine-1-carboxylic acid, saphenic acid, and saphenamycin through a unique one-carbon extension by EsmB1-B5, a keto reduction by EsmC, and an esterification by EsmD1-D3, the atypical polyketide sythases, respectively. Two transcriptional regulators, EsmT1 and EsmT2, are required for esmeraldin production.

Improving thermostability of phosphatidylinositol-synthesizing Streptomyces phospholipase D.

Aimed to produce thermostable phosphatidylinositol (PI)-synthesizing phospholipase D (PLD), we initiated site-directed combinatorial mutagenesis followed by high-throughput screening. Previous site-directed combinatorial mutagenesis of wild-type Streptomyces PLD produced a mutant, DYR (W187D/Y191Y/Y385R) with PI-synthesizing ability. Deriving PI as a product of transphosphatidylation between phosphatidylcholine and myo-inositol, with myo-inositol in excess at high-temperature reaction conditions can increase yield due to enhanced solubility of this substrate. Thus, we improved DYR's thermostability by introduction of random mutations into selected amino acid positions having high B-factor. Screening of the libraries under restricted conditions yielded single-point mutants, specifically D40H, T291Y and R329G. Combinations of these point mutations yielded double (D40H/T291Y, D40H/R329G and T291Y/R329G) and triple (D40H/T291Y/R329G) mutants. PI synthesis at elevated temperatures pointed at D40H/T291Y as the most efficient enzyme. Circular dichroism analysis revealed D40H/T291Y to have increased melting temperature and postponed onset of thermal unfolding compared with DYR. Thermal tolerance study at 65°C confirmed D40H/T291Y's thermostability as its half-inactivation time was 8.7 min longer compared with DYR. This mutant had significantly less root-mean-square deviation change compared with DYR and showed no change in root-mean-square fluctuation when temperature shifts from 40 to 60°C, as determined by molecular dynamics analysis. Acquired different degrees of thermostability were also observed for several other DYR mutants.