Recombinant human liver betaine-homocysteine S-methyltransferase: identification of three cysteine residues critical for zinc binding.

Betaine-homocysteine S-methyltransferase (BHMT; EC 2.1.1.5) catalyzes the transfer of an N-methyl group from betaine to homocysteine to produce dimethylglycine and methionine, respectively. The enzyme is found in the pathway of choline oxidation and is abundantly expressed in liver and kidney. We have recently shown that human BHMT is a zinc metalloenzyme [Millian, N. S., and Garrow, T. A. (1998) Arch. Biochem. Biophys. 356, 93-98]. To facilitate the rapid purification of human BHMT for further physical and mechanistic studies, including characterizing its metal binding properties, we have overexpressed the enzyme in E. coli as a fusion construct which facilitated its subsequent purification by a self-cleavable affinity tag system (IMPACT T7). Using this expression and purification system in conjunction with site-directed mutagenesis, we have identified Cys217, Cys299, and Cys300 as zinc ligands. Mutating any of these Cys residues to Ala results in the complete loss of activity and a significant reduction in the ability of the protein to bind zinc. Comparing the regions of BHMT amino acid sequence surrounding these Cys residues with similar amino acid sequences retrievable from protein databases, we have identified the following motif: G[ILV]NCX(20,100)[ALV]X(2)[ILV]GGCCX(3)PX(2)I, which we propose to be a signature for a family of zinc-dependent methyltransferases that utilize thiols or selenols as methyl acceptors. Some of the members of this family include the vitamin B(12)-dependent methionine synthases, E. coli S-methylmethionine-S-homocysteine methyltransferase, and A. bisulcatus S-methylmethionine-selenocysteine methyltransferase.     

Dissecting the catalytic mechanism of betaine-homocysteine S-methyltransferase by use of intrinsic tryptophan fluorescence and site-directed mutagenesis

Betaine-homocysteine S-methyltransferase (BHMT) is a zinc-dependent enzyme that catalyzes the transfer of a methyl group from glycine betaine (Bet) to homocysteine (Hcy) to form dimethylglycine (DMG) and methionine (Met). Previous studies in other laboratories have indicated that catalysis proceeds through the formation of a ternary complex, with a transition state mimicked by the inhibitor S-(delta-carboxybutyl)-l-homocysteine (CBHcy). Using changes in intrinsic tryptophan fluorescence to determine the affinity of human BHMT for substrates, products, or CBHcy, we now demonstrate that the enzyme-substrate complex reaches its transition state through an ordered bi-bi mechanism in which Hcy is the first substrate to bind and Met is the last product released. Hcy, Met, and CBHcy bind to the enzyme to form binary complexes with K(d) values of 7.9, 6.9, and 0.28 microM, respectively. Binary complexes with Bet and DMG cannot be detected with fluorescence as a probe, but Bet and DMG bind tightly to BHMT-Hcy to form ternary complexes with K(d) values of 1.1 and 0.73 microM, respectively. Mutation of each of the seven tryptophan residues in human BHMT provides evidence that the enzyme undergoes two distinct conformational changes that are reflected in the fluorescence of the enzyme. The first is induced when Hcy binds, and the second, when Bet binds. As predicted by the crystal structure of BHMT, the amino acids Trp44 and Tyr160 are involved in binding Bet, and Glu159 in binding Hcy. Replacing these residues by site-directed mutagenesis significantly reduces the catalytic efficiency (V(max)/K(m)) of the enzyme. Replacing Tyr77 with Phe abolishes enzyme activity.     

Specific potassium ion interactions facilitate homocysteine binding to betaine‐homocysteine S‐methyltransferase

Betaine‐homocysteine S‐methyltransferase (BHMT) is a zinc‐dependent methyltransferase that uses betaine as the methyl donor for the remethylation of homocysteine to form methionine. This reaction supports S‐adenosylmethionine biosynthesis, which is required for hundreds of methylation reactions in humans. Herein we report that BHMT is activated by potassium ions with an apparent K_M for K⁺ of about 100 µM. The presence of potassium ions lowers the apparent K_M of the enzyme for homocysteine, but it does not affect the apparent K_M for betaine or the apparent k_cat for either substrate. We employed molecular dynamics (MD) simulations to theoretically predict and protein crystallography to experimentally localize the binding site(s) for potassium ion(s). Simulations predicted that K⁺ ion would interact with residues Asp26 and/or Glu159. Our crystal structure of BHMT bound to homocysteine confirms these sites of interaction and reveals further contacts between K⁺ ion and BHMT residues Gly27, Gln72, Gln247, and Gly298. The potassium binding residues in BHMT partially overlap with the previously identified DGG (Asp26‐Gly27‐Gly28) fingerprint in the Pfam 02574 group of methyltransferases. Subsequent biochemical characterization of several site‐specific BHMT mutants confirmed the results obtained by the MD simulations and crystallographic data. Together, the data herein indicate that the role of potassium ions in BHMT is structural and that potassium ion facilitates the specific binding of homocysteine to the active site of the enzyme. Proteins 2014; 82:2552–2564. © 2014 Wiley Periodicals, Inc.     

Liver betaine-homocysteine S-methyltransferase activity undergoes a redox switch at the active site zinc

Using a redox-inert methyl acceptor, we show that betaine-homocysteine S-methyltransferase (BHMT) requires a thiol reducing agent for activity. Short-term exposure of BHMT to reducing agent-free buffer inactivates the enzyme without causing any loss of its catalytic zinc. Activity can be completely restored by the re-addition of a thiol reducing agent. The catalytic zinc of BHMT is bound by three thiolates and one hydroxyl group. Thiol modification experiments indicate that a disulfide bond is formed between two of the three zinc-binding ligands when BHMT is inactive in a reducing agent-free buffer, and that this disulfide can be readily reduced with the concomitant restoration of activity by re-establishing reducing conditions. Long-term exposure of BHMT to reducing agent-free buffer results in the slow, irreversible loss of its catalytic Zn and a corresponding loss of activity. Experiments using the glutamate-cysteine ligase modifier subunit knockout mice Gclm(−/−), which are severely impaired in glutathione synthesis, show that BHMT activity is reduced about 75% in Gclm(−/−) compared to Gclm(+/+) mice.     

Pyruvate carboxylase: isolation of the biotin-containing tryptic peptide and the determination of its primary sequency

The biotin-containing tryptic peptides of pyruvate carboxylase from sheep, chicken, and turkey liver mitochondria have been isolated and their primary structures determined. The amino acid sequences of the 19 residue peptides from chicken and turkey are identical and share a common sequence of 14 residues around biocytin with the 24-residue peptide isolated from sheep. The sequences obtained were: residue 1 → 11 Avian: Gly Ala Pro Leu Val Leu Ser Ala Met Biocytin Met Sheep: Gly Gln Pro Leu Val Leu Ser Ala Met Biocytin Met residues 12 → 19 or 24 Avian: Glu Thr Val Val Thr Ala Pro Arg Sheep: Glu Thr Val Val Thr Ser Pro Val Thr Glu Gly Val Arg A sensitive radiochemical assay for biotin was developed based on the tight binding of biotin by avidin. The ability of zinc sulfate to precipitate, without dissociating, the avidin-biotin complex provided a convenient procedure for separating free and bound biotin, and hence, for back-titrating a standard amount of avidin with [¹⁴C]biotin.     

盐度和甜菜碱对鲈鱼BHMT mRNA表达的影响

甜菜碱高半胱氨酸甲基转移酶(BHMT,EC2.1.1.5)催化甜菜碱的甲基转移给高半胱氨酸(Hcy),而分别生成二甲基甘氨酸和蛋氨酸。利用RT-PCR和SMART RACE的方法从鲈鱼(Lateolabrax japonicus)肝脏中克隆了BHMT全长cDNA。该序列全长1461bp,5'端非翻译区72bp,3'端非翻译区183bp,开放阅读框1206bp,可编码一个由401个氨基酸组成的蛋白质,该蛋白质相对分子质量为44.32kD,等电点为7.21。氨基酸序列分析表明,BHMT具有较高的保守性,鲈鱼BHMT与人、小鼠等9个物种的同源性为77%～93%,其中与黄鲈(Percaflavescens)同源性最高,为93%。用RT-PCR分析BHMT基因在10个组织中的表达结果表明,只有在肝、肠和肾中有较高的表达。RT-PCR和定量PCR表明,鲈鱼从盐度25的海水转入盐度12的海水后,肝、肠和肾BHMT基因表达量有增加,而将鲈鱼从盐度为25的海水转入盐度为29的海水后,肝、肠和肾的BHMT基因表达则减少。腹腔注射甜菜碱可增加鲈鱼BHMT基因在肝、肠和肾三个组织中的相对表达量。这些结果表明,甜菜碱可诱导鲈鱼BHMT...     

Molecular dynamics study of the proposed proton transport pathways in [FeFe]-hydrogenase

Possible proton transport pathways in Clostridium pasteurianum (CpI) [FeFe]-hydrogenase were investigated with molecular dynamics simulations. This study was undertaken to evaluate the functional pathway and provide insight into the hydrogen bonding features defining an active proton transport pathway. Three pathways were evaluated, two of which consist of water wires and one of predominantly amino acid residues. Our simulations suggest that protons are not transported through water wires. Instead, the five-residue motif (Glu282, Ser319, Glu279, H₂O, Cys299) was found to be the likely pathway, consistent with previously made experimental observations. The pathway was found to have a persistent hydrogen bonded core (residues Cys299 to Ser319), with less persistent hydrogen bonds at the ends of the pathway for both H₂ release and H₂ uptake. Single site mutations of the four residues have been shown experimentally to deactivate the enzyme. The theoretical evaluation of these mutations demonstrates redistribution of the hydrogen bonds in the pathway, resulting in enzyme deactivation. Finally, coupling between the protein dynamics near the proton transport pathway and the redox partner binding regions was also found as a function of H₂ uptake and H₂ release states, which may be indicative of a correlation between proton and electron movement within the enzyme.     

Functional importance of the conserved N-terminal domain of the mitochondrial replicative DNA helicase

The mitochondrial replicative DNA helicase is an essential cellular protein that shows high similarity with the bifunctional primase-helicase of bacteriophage T7, the gene 4 protein (T7 gp4). The N-terminal primase domain of T7 gp4 comprises seven conserved sequence motifs, I, II, III, IV, V, VI, and an RNA polymerase basic domain. The putative primase domain of metazoan mitochondrial DNA helicases has diverged from T7 gp4 and in particular, the primase domain of vertebrates lacks motif I, which comprises a zinc binding domain. Interestingly, motif I is conserved in insect mtDNA helicases. Here, we evaluate the effects of overexpression in Drosophila cell culture of variants carrying mutations in conserved amino acids in the N-terminal region, including the zinc binding domain. Overexpression of alanine substitution mutants of conserved amino acids in motifs I, IV, V and VI and the RNA polymerase basic domain results in increased mtDNA copy number as is observed with overexpression of the wild type enzyme. In contrast, overexpression of three N-terminal mutants W282L, R301Q and P302L that are analogous to human autosomal dominant progressive external ophthalmoplegia mutations results in mitochondrial DNA depletion, and in the case of R301Q, a dominant negative cellular phenotype. Thus whereas our data suggest lack of a DNA primase activity in Drosophila mitochondrial DNA helicase, they show that specific N-terminal amino acid residues that map close to the central linker region likely play a physiological role in the C-terminal helicase function of the protein.     

Amino acid residue penultimate to the amino-terminal gly residue strongly affects two cotranslational protein modifications, N-myristoylation and N-acetylation

To examine the amino-terminal sequence requirements for cotranslational protein N-myristoylation, a series of site-directed mutagenesis of N-terminal region were performed using tumor necrosis factor as a nonmyristoylated model protein. Subsequently, the susceptibility of these mutants to protein N-myristoylation was evaluated by metabolic labeling in an in vitro translation system or in transfected cells. It was found that the amino acid residue at position 3 in an N-myristoylation consensus motif, Met-Gly-X-X-X-Ser-X-X-X, strongly affected the susceptibility of the protein to two different cotranslational protein modifications, N-myristoylation and N-acetylation; 10 amino acids (Ala, Ser, Cys, Thr, Val, Asn, Leu, Ile, Gln, and His) with a radius of gyration smaller than 1.80 A directed N-myristoylation, two negatively charged residues (Asp and Glu) directed N-acetylation, and two amino acids (Gly and Met) directed heterogeneous modification with both N-myristoylation and N-acetylation. The amino acid requirements at this position for the two modifications were dramatically changed when Ser at position 6 in the consensus motif was replaced with Ala. Thus, the amino acid residue penultimate to the N-terminal Gly residue strongly affected two cotranslational protein modifications, N-myristoylation and N-acetylation, and the amino acid requirements at this position for these two modifications were significantly affected by downstream residues.     

Crystal structure of rat liver betaine homocysteine s-methyltransferase reveals new oligomerization features and conformational changes upon substrate binding

Betaine homocysteine S-methyltransferase (BHMT) is one of the two enzymes known to methylate homocysteine to generate methionine in the liver. It presents a Zn(2+) atom linked to three essential Cys residues. The crystal structure of rat liver BHMT has been solved at 2.5A resolution, using crystals with P2(1) symmetry and 45% solvent content in the cell. The asymmetric unit contains the whole functional tetramer showing point symmetry 222. The overall fold of the subunit consists mostly of a (alpha/beta)(8) barrel, as for human BHMT. From the end of the barrel, the polypeptide chain extends away and makes many interactions with a different subunit, forming tight dimers. The most remarkable structural feature of rat liver BHMT is the presence of a helix including residues 381-407, at the C terminus of the chain, which bind together the dimers AB to CD. A strong ion-pair and more than 60 hydrophobic interactions keep this helix stacked to the segment 316-349 from the opposite subunit. Moreover, the crystal structure of free rat liver BHMT clearly shows that Tyr160 is the fourth ligand coordinated to Zn, which is replaced by Hcy upon binding. Two residues essential for substrate recognition, Phe76 and Tyr77, are provided by a conformational change in a partially disordered loop (L2). The crucial role of these residues is highlighted by site-directed mutagenesis.     

Molecular cloning and expression profiling of a chalcone synthase gene from hairy root cultures of Scutellaria viscidula Bunge

A cDNA encoding chalcone synthase (CHS), the key enzyme in flavonoid biosynthesis, was isolated from hairy root cultures of Scutellaria viscidula Bunge by rapid amplification of cDNA ends (RACE). The full-length cDNA of S. viscidula CHS, designated as Svchs (GenBank accession no. EU386767), was 1649 bp with a 1170 bp open reading frame (ORF) that corresponded to a deduced protein of 390 amino acid residues, a calculated molecular mass of 42.56 kDa and a theoretical isoelectric point (pI) of 5.79. Multiple sequence alignments showed that SvCHS shared high homology with CHS from other plants. Functional analysis in silico indicated that SvCHS was a hydrophilic protein most likely associated with intermediate metabolism. The active sites of the malonyl-CoA binding motif, coumaroyl pocket and cyclization pocket in CHS of Medicago sativa were also found in SvCHS. Molecular modeling indicated that the secondary structure of SvCHS contained mainly α-helixes and random coils. Phylogenetic analysis showed that SvCHS was most closely related to CHS from Scutellaria baicalensis. In agreement with its function as an elicitor-responsive gene, the expression of Svchs was induced and coordinated by methyl jasmonate. To our knowledge, this is the first report to describe the isolation and expression of a gene from S. viscidula.     

Enhancing oxidative resistance of Agrobacterium radiobacter N-carbamoyl D-amino acid amidohydrolase by engineering solvent-accessible methionine residues

N-Carbamoyl D-amino acid amidohydrolase (D-NCAase) that catalyzes the stereospecific hydrolysis of N-carbamoyl D-amino acids to their corresponding D-amino acids is valuable in pharmaceutical industry. Agrobacterium radiobacter D-NCAase is sensitive to oxidative damage by hydrogen peroxide. To investigate the role of methionine residues in oxidative inactivation, each of the nine methionine residues in A. radiobacter D-NCAase was substituted with leucine, respectively, by site-directed mutagenesis. Except for two mutants (Met5Leu and Met31Leu) with similar activities, seven mutants (Met73Leu, Met167Leu/Met169Leu, Met184Leu, Met220Leu, Met239Leu, Met244Leu, and Met239Leu/Met244Leu) were found to have reduced activities. In the presence of H(2)O(2), three mutants (Met239Leu, Met244Leu, and Met239Leu/Met244Leu) with substitution of highly solvent-accessible methionines by leucines retained their activities. The other mutants were also considerably resistant to chemical oxidation than was the wild-type enzyme. Thus, substitution of solvent-accessible methionine residues with leucine to enhance oxidative stability of D-NCAase is practical but might be with compromised activity.     

Novel aminopeptidase specific for glycine from Actinomucor elegans

Glycyl aminopeptidase was purified 600-fold from a cell extract of Actinomucor elegans by ammonium sulfate fractionation and sequential chromatography on DEAE-Toyopearl, Toyopearl HW65C, and FPLC-Superdex 200 HR, with recovery of 3.3% of the activity. The enzyme highly specifically hydrolyzed Gly-X (amino acid, peptide, or arylamide) bonds. The enzyme hydrolyzed other amino acid residues but at a rate of less than one fifth that with Gly. The order was Gly > Ala > Met > Arg > Ser > Leu. The Km value for glycyl-2-naphthylamide was 0.24 mM. The enzyme was most active at pH 8.0 with glycyl-2-naphthylamide as the substrate and its optimal temperature was 40 degrees C. The enzyme was inhibited by iodoacetic acid, and p-chloromercuribenzoate but not done by diisopropylfluorophosphate, o-phenanthroline, or EDTA. Magnesium and calcium had no effect on enzymic activity, but the activity was suppressed by cadmium, zinc, and copper ions. The molecular mass was estimated to be 320 kDa by gel filtration on FPLC-Superdex 200 HR and 56.5 kDa by SDS-PAGE, so the enzyme probably was a hexamer.     

Betaine-homocysteine methyltransferase: zinc in a distorted barrel

Betaine-homocysteine methyl transferase (BHMT) catalyzes the synthesis of methionine from betaine and homocysteine (Hcy), utilizing a zinc ion to activate Hcy. BHMT is a key liver enzyme that is important for homocysteine homeostasis. X-ray structures of human BHMT in its oxidized (Zn-free) and reduced (Zn-replete) forms, the latter in complex with the bisubstrate analog, S(delta-carboxybutyl)-L-homocysteine, were determined at resolutions of 2.15 A and 2.05 A. BHMT is a (beta/alpha)(8) barrel that is distorted to construct the substrate and metal binding sites. The zinc binding sequences G-V/L-N-C and G-G-C-C are at the C termini of strands beta6 and beta8. Oxidation to the Cys217-Cys299 disulfide and expulsion of Zn are accompanied by local rearrangements. The structures identify Hcy binding fingerprints and provide a prototype for the homocysteine S-methyltransferase family.     

In Silico Analog Design for Terbinafine Against Trichophyton rubrum: A Preliminary Study

The diseases caused by dermatophytes are common among several other infections which cause serious threat to human health. It is evident that enzyme squalene epoxidase is responsible for prolonged dermatophyte infection and it is appealing to note that this enzyme is also responsible for fatty acid synthesis in these groups of fungi. In the present study, terbinafine drug which targets enzyme squalene epoxidase has been explored to design its various novel analogues. The present study suggests that many more prominent drug analogues could be constituted which may be crucial towards designing new drug candidates. In the present study, we have designed a series of such analogues viz. [(2E)-6,6-dimethylhept-2-en-4-yn-1-yl](methyl)(naphthalen-1-ylmethyl)amine, N-[8-({[(2E)-6,6-dimethylhept-2-en-4-yn-1-yl](methyl)amino}methyl)naphthalen-1-yl]-2-(sulfoamino) acetamide, {[4-(dihydroxyamino)-8-({[(2E)-6,6-dimethylhept-2-en-4-yn-1-yl](methyl)amino}methyl)naphthalen-1-yl]sulfanyl}methanol and (R)-{[4-({[(2E,6R)-6,7-dimethyloct-2-en-4-yn-1-yl](methyl)amino}methyl)-5-[(hydroxysulfamoyl)amino]naphthalen-1-yl]amino}sulfinic acid. Moreover, further by molecular docking approach the binding between enzyme and designed analogues was further analysed. The present preliminary report suggested a considerably good docking interaction score of −338.75 kcal/mol between terbinafine and squalene epoxidase from Trichophyton rubrum. This preliminary study implies that few designed candidate ligands can be effectual towards the activity of this enzyme and can play crucial role in pathogenesis control of T. rubrum.     

Site-directed mutagenesis of the conserved Ala348 and Gly350 residues at the putative active site of Bacillus kaustophilus leucine aminopeptidase

Leucine aminopeptidase (LAP) is an exopeptidase that catalyzes the hydrolysis of amino acid residues from the amino terminus of proteins and peptides. Sequence alignment shows that the conserved Ala348 and Gly350 residues of Bacillus kaustophilus LAP (BkLAP) are located right next to a coordinated ligand. We further investigated the roles of these two residues by performing computer modeling and site-directed mutagenesis. Based on the modeling, the carbonyl group of Ala348 interacts with Asn345 and Asn435, and that of Gly350 with Ile353 and Leu354, where these interactions might maintain the zinc-coordinated residues at their correct positions. Replacement of Ala348 with Arg resulted in a dramatic reduction in LAP activity. A complete loss of the activity was also observed in A348E, A348V, and the Gly350 variants. Measurement of intrinsic tryptophan fluorescence revealed alteration of the microenvironment of aromatic amino acid residues, while circular dichroism spectra were nearly identical for wild-type and all mutant enzymes. Protein modeling and site-directed mutagenesis suggest that residues Ala348 and Gly350 are essential for BkLAP in maintaining a stable active-site environment for the catalytic reaction.     

Betaine-homocysteine S-methyltransferase-2 is an S-methylmethionine-homocysteine methyltransferase

We demonstrate that purified recombinant human betainehomocysteine methyltransferase-2 (BHMT-2) is a zinc metalloenzyme that uses S-methylmethionine (SMM) as a methyl donor for the methylation of homocysteine. Unlike the highly homologous betaine-homocysteine methyltransferase (BHMT), BHMT-2 cannot use betaine. The K(m) of BHMT-2 for SMM was determined to be 0.94 mm, and it has a turnover number similar to BHMT. Several compounds were tested as inhibitors of recombinant human BHMT and BHMT-2. The SMM-specific methyltransferase activity of BHMT-2 is not inhibited by dimethylglycine and betaine, whereas the former is a potent inhibitor of BHMT. Methionine is a stronger inhibitor of BHMT-2 than BHMT, and S-adenosylmethionine does not inhibit BHMT but is a weak inhibitor of BHMT-2. BHMT can use SMM as a methyl donor with a k(cat)/K(m) that is 5-fold lower than the k(cat)/K(m) for betaine. However, SMM does not inhibit BHMT activity when it is presented to the enzyme at concentrations that are 10-fold greater than the subsaturating amounts of betaine used in the assay. Based on these data, it is our current hypothesis that in vivo most if not all of the SMM-dependent methylation of homocysteine occurs via BHMT-2.     

Cloning and characterization of antioxidant enzyme methionine sulfoxide-S-reductase from Caenorhabditis elegans

Methionine (Met) residues in proteins are susceptible to oxidation. The resulting methionine sulfoxide can be reduced back to methionine by methionine sulfoxide-S-reductase (MsrA). The MsrA gene, isolated from Caenorhabditis elegans, was cloned and expressed in Escherichia coli. The resultant enzyme was able to revert both free Met and Met in proteins in the presence of either NADPH or dithiothreitol (DTT). However, approximately seven times higher enzyme activity was observed in the presence of DTT than of NADPH. The enzyme had an absolute specificity for the reduction of l-methionine-S-sulfoxide but no specificity for the R isomer. K(m) and k(cat) values for the enzyme were approximately 1.18 mM and 3.64 min(-1), respectively. Other kinetics properties of the enzyme were also evaluated.     

C-terminal loop of Streptomyces phospholipase D has multiple functional roles

We have recently shown that two flexible loops of Streptomyces phospholipase D (PLD) affect the catalytic reaction of the enzyme by a comparative study of chimeric PLDs. Gly188 and Asp191 of PLD from Streptomyces septatus TH-2 (TH-2PLD) were identified as the key amino acid residues involved in the recognition of phospholipids. In the present study, we further investigated the relationship between a C-terminal loop of TH-2PLD and PLD activities to elucidate the reaction mechanism and the recognition of the substrate. By analyzing chimeras and mutants in terms of hydrolytic and transphosphatidylation activities, Ala426 and Lys438 of TH-2PLD were identified as the residues associated with the activities. We found that Gly188 and Asp191 recognized substrate forms, whereas residues Ala426 and Lys438 enhanced transphosphatidylation and hydrolysis activities regardless of the substrate form. By substituting Ala426 and Lys438 with Phe and His, respectively, the mutant showed not only higher activities but also higher thermostability and tolerance against organic solvents. Furthermore, the mutant also improved the selectivity of the transphosphatidylation activity. The residues Ala426 and Lys438 were located in the C-terminal flexible loop of Streptomyces PLD separate from the highly conserved catalytic HxKxxxxD motifs. We demonstrated that this C-terminal loop, which formed the entrance of the active well, has multiple functional roles in Streptomyces PLD.