Rational Site-directed Mutagenesis Research Articles

Diversity-Oriented Biosynthesis Yields l-Kynurenine Derivative-Based Neurological Drug Candidate Collection

Natural product libraries with a remarkable range of biological activities play pivotal roles in drug discoveries due to their extraordinary structural complexity and immense diversity. l-Kynurenine (l-Kyn)-based derivatives are privileged pharmacophores that exhibit diverse therapeutic implications in neurological disorders. However, the difficulty in obtaining l-Kyn analogues with different skeletal structures has recently led to a decline in its medicinal research. Herein, we report a two-step, one-pot protocol for diversity-oriented biosynthesis of a collection of previously intractable l-Kyn-like compounds. The success of these challenging transformations mainly depends on unlocking the new catalytic scope of tryptophan 2,3-dioxygenases, followed by rational site-directed mutagenesis to modify the substrate domains further. As a result, 18 kynurenine analogues with diverse molecular scaffolds can be rapidly assembled in a predictable manner with 20-83% isolated yields, which not only fill the voids of the catalytic profile of tryptophan 2,3-dioxygenases with an array of substituent groups (e.g., F, Cl, Br, I, CH3, OCH3, and NO2) but also update the current understanding of its substrate spectrum. Our work highlights the great potential of existing enzymes in addressing long-standing synthetic challenges for facilitating the development or discovery of new drug candidates. Furthermore, our approach enables translating the reaction parameters from Eppendorf tubes to 1 L scale, affording l-4-Cl-Kyn and l-5-Cl-Kyn both on a gram scale with more than 80% isolated yields, and provides a promising alternative to further industrial applications.

Rational engineering of AA5_2 copper radical oxidases to probe the molecular determinants governing their substrate selectivity.

Fungal copper radical oxidases (CROs) from the Auxiliary Activity family 5 (AA5) constitute a group of metalloenzymes that oxidize a wide panel of natural compounds, such as galactose-containing saccharides or primary alcohols, into product derivatives exhibiting promising biotechnological interests. Despite a well-conserved first copper-coordination sphere and overall fold, some members of the AA5_2 subfamily are incapable of oxidizing galactose and galactosides but conversely efficiently catalyse the oxidation of diverse aliphatic alcohols. The objective of this study was to understand which residues dictate the substrate preferences between alcohol oxidases and galactose oxidases within the AA5_2 subfamily. Based on structural differences and molecular modelling predictions between the alcohol oxidase from Colletotrichum graminicola (CgrAlcOx) and the archetypal galactose oxidase from Fusarium graminearum (FgrGalOx), a rational mutagenesis approach was developed to target regions or residues potentially driving the substrate specificity of these enzymes. A set of 21 single and multiple CgrAlcOx variants was produced and characterized leading to the identification of six residues (W39, F138, M173, F174, T246, L302), in the vicinity of the active site, crucial for substrate recognition. Two multiple CgrAlcOx variants, i.e. M4F (W39F, F138W, M173R and T246Q) and M6 (W39F, F138W, M173R, F174Y, T246Q and L302P), exhibited a similar affinity for carbohydrate substrates when compared to FgrGalOx. In conclusion, using a rational site-directed mutagenesis approach, we identified key residues involved in the substrate selectivity of AA5_2 enzymes towards galactose-containing saccharides.

Enhancing the catalytic activity of cyanobacterial chlorophyllase from Oscillatoria acuminata PCC 6304 through rational site-directed mutagenesis

In our previous study, a novel cyanobacterial chlorophyllase from Oscillatoria acuminata (OaCLH) was successfully overexpressed and biochemically characterized in Escherichia coli BL21(DE3). Recombinant OaCLH has higher heterologous expression in E. coli than plant chlorophyllases. However, catalytic efficiency (kcat/Km) of the recombinant OaCLH is significantly lower than those of several reported algae and plant chlorophyllases from Chlamydomonas reinhardtii (CrCLH), Triticum aestivum (TaCLH), and Chenopodium album (CaCLH). In this study, three single-site mutant enzymes (W160R, V228C, and D224 N) were constructed by site-directed mutagenesis to improve the catalytic efficiency of OaCLH using rational design based on multiple sequence alignment and homology modeling. Trp160 was found to be important in substrate access and binding, while Val228 was found to be involved in substrate recognition. Mutants W160R and V228C significantly increased the enzyme affinity and catalytic activity against all substrates. Asp224 played an important role in substrate specificity. Mutant D224 N changed its preferred substrate from chlorophyll b to chlorophyll a. According to the circular dichroism spectra, the introduced mutations had no significant effect on the overall secondary structures of three mutant enzymes. When compared to mutant W160R, double mutant W160RV228C showed a slight improvement in enzyme affinity and catalytic activity. These mutation sites are reported for the first time in OaCLH, and provided information about the structural basis of catalytic activity and substrate specificity of OaCLH. Mutant enzymes (W160R, V228C, D224 N, and W160RV228C) with improved catalytic efficiency may be good candidates for further engineering of OaCLH for potential industrial applications.

Rational Site-Directed Mutagenesis of a Diels–Alderase Permits Reversal of Diastereoselectivity

Rational Site-Directed Mutagenesis of a Diels–Alderase Permits Reversal of Diastereoselectivity

Switching the substrate specificity of lysoplasmalogen-specific phospholipase D.

Lysoplasmalogen‐specific phospholipase D (LyPls‐PLD) catalyzes reactions in a manner similar to those catalyzed by glycerophosphodiester phosphodiesterase (GDPD) and other well‐known PLDs. Although these enzymes hydrolyze the glycerophosphodiester bond, their substrate specificities are completely different. Previously, we reported that LyPls‐PLD from Thermocrispum sp. RD004668 shows only 53% activity with 1‐hexadecyl‐2‐hydroxy‐sn‐glycero‐3‐phosphocholine (LysoPAF) relative to the 100% activity it shows with choline lysoplasmalogen (LyPlsCho). Lipoprotein‐associated phospholipase A2 (Lp‐PLA2) activity can be used to evaluate for cardiovascular disease. Hence, development of a point‐of‐care testing kit requires a LysoPAF‐specific PLD (LysoPAF‐PLD) to measure Lp‐PLA2 activity. Rational site‐directed mutagenesis and kinetic analysis were applied to generate LysoPAF‐PLD from LyPls‐PLD and to clarify the mechanisms underlying the substrate‐recognition ability of LyPls‐PLD. Our results suggest that LyPls‐PLD variants A47, M71, N173, F211, and W282 are possibly involved in substrate recognition and that F211L may substantially alter substrate preference. Moreover, the specific activity ratio LysoPAF/LyPlsCho corresponding to F211L was up to 25‐fold higher than that corresponding to the wild‐type enzyme. Thus, we succeeded in switching from LyPlsCho‐ to LysoPAF‐PLD. These results suggest that the F211L variant may be utilized to measure Lp‐PLA2 activity. Kinetic analyses demonstrated that product release was the rate‐limiting step of the reaction, with flexibility of the sn‐1 ether‐linked vinyl/alkyl chain of the substrate being essential for substrate binding and product release. Our findings may lead to a better understanding of the differences between homologous enzymes (such as PLD, sphingomyelinase D, and GDPD of the phosphatidylinositol‐phosphodiesterase superfamily) in relation to substrate recognition.Enzyme EC 3.1.4.2 (currently assigned).

Engineering better catalytic activity and acidic adaptation into Kluyveromyces marxianus exoinulinase using site-directed mutagenesis.

Exoinulinase catalyzes the successive removal of individual fructose moiety from the non-reducing end of the inulin molecule, which is useful for biotechnological applications like producing fructan-based non-grain biomass energy and high-fructose syrup. In this study, an exoinulinase (KmINU) from Kluyveromyces marxianus DSM 5418 was tailored for increased catalytic activity and acidic adaptation for inulin hydrolysis processes by rational site-directed mutagenesis. Three mutations, S124Y, N158S and Q215V distal to the catalytic residues of KmINU were designed and heterologously expressed in Pichia pastoris GS115. Compared to the wild-type, S124Y shifted the pH-activity profile towards acidic pH values and increased the catalytic activity and catalytic efficiency by 59% and 99% to 688.4 ± 17.03 s-1 and 568.93 L mmol-1 s-1 , respectively. N158S improved the catalytic activity under acidic pH conditions, giving a maximum value of 464.06 ± 14.06 s-1 on inulin at pH 4.5. Q215V markedly improved the substrate preference for inulin over sucrose by 5.56-fold, and showed catalytic efficiencies of 208.82 and 6.88 L mmol-1 s-1 towards inulin and sucrose, respectively. Molecular modeling and computational docking indicated that structural reorientation may underlie the increased catalytic activity, acidic adaptation and substrate preference. The KmINU mutants may serve as industrially promising candidates for inulin hydrolysis. Protein engineering of exoinulinase here provides a successful example of the extent to which mutating non-conserved substrate recognition and binding residues distal to the active site can be used for industrial enzyme improvements. © 2020 Society of Chemical Industry.

Amino acid residues important for D-galactose recognition by the F-type lectin, Ranaspumin-4

F-type lectins are typically L-fucose binding proteins with characteristic L-fucose-binding and calcium-binding sequence motifs, and an F-type lectin fold. An exception is Ranaspumin-4, an F-type lectin of the Tungra frog, Engystomops pustulosus. Ranaspumin-4 is D-galactose specific and does not bind to L-fucose although it has the conserved L-fucose binding sequence motif and shares overall sequence similarity with other F-type lectins. Here, we report the detailed glycan-binding profile of wild-type Ranaspumin-4 using hemagglutination inhibition assays, flow cytometry assays and enzyme-linked lectin assays, and identify residues important for D-galactose recognition using rational site-directed mutagenesis. We demonstrate that Ranaspumin-4 binds to terminal D-galactose in α or β linkage with preference for α1-3, α1-4, β1-3, and β1-4 linkages. Further, we find that a methionine residue (M31) in Ranaspumin-4 that occurs in place of a conserved Gln residue (in other F-type lectins), supports D-galactose recognition. Resides Q42 and F156 also likely aid in D-galactose recognition.

Structure-based design approach to rational site-directed mutagenesis of β-lactoglobulin

Recombinant proteins play an important role in medicine and have diverse applications in industrial biotechnology. Lactoglobulin has shown great potential for use in targeted drug delivery and body fluid detoxification because of its ability to bind a variety of molecules. In order to modify the biophysical properties of β-lactoglobulin, a series of single-site mutations were designed using a structure-based approach. A 3-dimensional structure alignment of homologous molecules led to the design of nine β-lactoglobulin variants with mutations introduced in the binding pocket region. Seven stable and correctly folded variants (L39Y, I56F, L58F, V92F, V92Y, F105L, M107L) were thoroughly characterized by fluorescence, circular dichroism, isothermal titration calorimetry, size-exclusion chromatography, and X-ray structural investigations. The effects of the amino acid substitutions were observed as slight rearrangements of the binding pocket geometry, but they also significantly influenced the global properties of the protein. Most of the mutations increased the thermal/chemical stability without altering the dimerization constant or pH-dependent conformational behavior. The crystal structures reveal that the I56F and F105L mutations reduced the depth of the binding pocket, which is advantageous since it can reduce the affinity to endogenous fatty acids. The F105L mutant created a unique binding mode for a fatty acid, supporting the idea that lactoglobulin can be altered to bind unique molecules. Selected variants possessing a unique combination of their individual properties can be used for further, more advanced mutagenesis, and the presented results support further research using β-lactoglobulin as a therapeutic delivery agent or a blood detoxifying molecule.

Tuning the pH profile of β-glucuronidase by rational site-directed mutagenesis for efficient transformation of glycyrrhizin.

In this study, we aimed to shift the optimal pH of acidic β-glucuronidase from Aspergillus oryzae Li-3 (PGUS) to the neutral region by site-directed mutagenesis, thus allowing high efficient biotransformation of glycyrrhizin (GL) into glycyrrhetinic acid (GA) under higher pH where the solubility of GL could be greatly enhanced. Based on PGUS structure analysis, five critical aspartic acid and glutamic acid residues were replaced with arginine on the surface to generate a variant 5Rs with optimal pH shifting from 4.5 to 6.5. The catalytic efficiency (kcat /Km) value of 5Rs at pH6.5 was 10.7-fold higher than that of PGUS wild-type at pH6.5, even 1.4-fold higher than that of wild-type at pH4.5. Molecular dynamics simulation was performed to explore the molecular mechanism for the shifted pH profile and enhanced pH stability of 5Rs.

Modification of quaternary structure of Candida albicans GlcN-6-P synthase and its desensitization to inhibition by UDP-GlcNAc by site-directed mutagenesis

Site-directed mutagenesis of the CaGFA1 gene encoding glucosamine-6-phosphate synthase from Candida albicans was performed. Desensitization of the enzyme to inhibition by UDPGlcNAc was achieved upon T487I and H492F substitutions at the UDP-GlcNAc binding site, exchange of D524, S525 and S527 for Ala at the dimer:dimer interface and construction of the tail-lock array (L434R and L460A) at the C-tail region. The first two sets if mutageneses but not the last one resulted in conversion of the tetrameric enzyme into its dimeric form. Evidence for links and communication between the UDP-GlcNAc binding site and the dimer-dimer contact areas are presented. The CaGfa1-T487IH492F and CaGfa1-KHSH-D524AS525AS527A muteins are the first examples of the successful conversion of eukaryotic GlcN-6-P synthase into its prokaryotic-like version upon rational site-directed mutagenesis.

Catalytic efficiency and thermostability improvement of Suc2 invertase through rational site-directed mutagenesis.

Engineering of invertases has come to attention because of increasing demand for possible applications of invertases in various industrial processes. Due to the known physicochemical properties, invertases from micro-organisms such as Saccharomyces cerevisiae carrying SUC2 gene are considered as primary models. To improve thermostability and catalytic efficiency of SUC2 invertase (SInv), six influential residues with Relative Solvent Accessibility<5% were selected through multiple-sequence alignments, molecular modelling, structural and computational analyses. Consequently, SInv and 5 mutants including three mutants with single point substitution [Mut1=P152V, Mut2=S85V and Mut3=K153F)], one mutant with two points [Mut4=S305V-N463V] and one mutant with three points [Mut5=S85V-K153F-T271V] were developed via site-directed mutagenesis and produced using Pichia pastoris as the host. Physicochemical studies on these enzymes indicated that the selected amino acids which were located in the active site region mainly influenced catalytic efficiency. The best improvement belonged to Mut1 (54% increase in Kcat/Km) and Mut3 exhibited the worst effect (90% increase in Km). These results suggest that Pro152 and Lys153 play key role in preparation of the right substrate lodging in the active site of SInv. The best thermostability improvement (16%) was observed for Mut4 in which two hydrophilic residues located on the loops, far from the active site, were replaced by Valines. These results suggest that tactful simultaneous substitution of influential hydrophilic residues in both active site region and peripheral loops with hydrophobic amino acids could result in more thermostable invertases with enhanced catalytic efficiency.

P212A Mutant of Dihydrodaidzein Reductase Enhances (S)-Equol Production and Enantioselectivity in a Recombinant Escherichia coli Whole-Cell Reaction System.

(S)-Equol, a gut bacterial isoflavone derivative, has drawn great attention because of its potent use for relieving female postmenopausal symptoms and preventing prostate cancer. Previous studies have reported on the dietary isoflavone metabolism of several human gut bacteria and the involved enzymes for conversion of daidzein to (S)-equol. However, the anaerobic growth conditions required by the gut bacteria and the low productivity and yield of (S)-equol limit its efficient production using only natural gut bacteria. In this study, the low (S)-equol biosynthesis of gut microorganisms was overcome by cloning the four enzymes involved in the biosynthesis from Slackia isoflavoniconvertens into Escherichia coli BL21(DE3). The reaction conditions were optimized for (S)-equol production from the recombinant strain, and this recombinant system enabled the efficient conversion of 200 μM and 1 mM daidzein to (S)-equol under aerobic conditions, achieving yields of 95% and 85%, respectively. Since the biosynthesis of trans-tetrahydrodaidzein was found to be a rate-determining step for (S)-equol production, dihydrodaidzein reductase (DHDR) was subjected to rational site-directed mutagenesis. The introduction of the DHDR P212A mutation increased the (S)-equol productivity from 59.0 mg/liter/h to 69.8 mg/liter/h in the whole-cell reaction. The P212A mutation caused an increase in the (S)-dihydrodaidzein enantioselectivity by decreasing the overall activity of DHDR, resulting in undetectable activity for (R)-dihydrodaidzein, such that a combination of the DHDR P212A mutant with dihydrodaidzein racemase enabled the production of (3S,4R)-tetrahydrodaidzein with an enantioselectivity of >99%.

Identification of residues involved in nucleotidyltransferase activity of JHP933 from helicobacter pyloriby site-directed mutagenesis

Helicobacter pylori is a well-known bacterial pathogen involved in the development of peptic ulcer, gastric adenocarcinoma and other forms of gastric cancer. Evidence has suggested that certain strain-specific genes in the plasticity region may play key roles in the pathogenesis of H. pylori-associated gastroduodenal diseases. Therefore there is considerable interest in the strain-specific genes located in the plasticity regions of H. pylori. JHP933 is encoded by the gene in the plasticity region of H. pylori strain J99. Recently, the crystal structure of JHP933 has confirmed it as a nucleotidyltransferase (NTase) superfamily protein and a putative active site has been proposed. However, no evidence from direct functional assay has been presented to confirm the active site and little is known about the functional mechanism of JHP933. Here, through superimposition with Cid1/NTP complex structures, we modelled the complex structures of JHP933 with different NTPs. Based on the models and using rational site-directed mutagenesis combined with enzymatic activity assays, we confirm the active site and identify several residues important for the nucleotidyl transferring function of JHP933. Furthermore, mutations of these active site residues result in the abolishment of the nucleotidyltransferase activity of JHP933. This work provides preliminary insight into the molecular mechanism underlying the pathophysiological role in H. pylori infection of JHP933 as a novel NTase superfamily protein.

The aspartate aminotransferase-like domain of Firmicutes MocR transcriptional regulators

Bacterial MocR transcriptional regulators possess an N-terminal DNA-binding domain containing a conserved helix-turn-helix module and an effector-binding and/or oligomerization domain at the C-terminus, homologous to fold type-I pyridoxal 5′-phosphate (PLP) enzymes. Since a comprehensive structural analysis of the MocR regulators is still missing, a comparisons of Firmicutes MocR sequences was undertook to contribute to the understanding of the structural characteristics of the C-terminal domain of these proteins, and to shed light on the structural and functional relationship with fold type-I PLP enzymes. Results of this work suggest the presence of at least three subgroups within the MocR sequences and provide a guide for rational site-directed mutagenesis studies aimed at deciphering the structure-function relationships in this new protein family.

A saxitoxin-binding aptamer with higher affinity and inhibitory activity optimized by rational site-directed mutagenesis and truncation.

Saxitoxin (STX), a member of the family of paralytic shellfish poisoning toxins, poses toxicological and ecotoxicological risks. To develop an analytical recognition element for STX, a DNA aptamer (APT(STX1)) was previously discovered via an iterative process known as Systematic Evolution of Ligands by Exponential Enrichment (SELEX) by Handy etal. Our study focused on generating an improved aptamer based on APT(STX1) through rational site-directed mutation and truncation. In this study, we generated the aptamer, M-30f, with a 30-fold higher affinity for STX compared with APT(STX1). The Kd value for M-30f was 133nM, which was calculated by Bio-Layer Interferometry. After optimization, we detected and compared the interaction of STX with aptamers (APT(STX1) or M-30f) through several techniques (ELISA, cell bioassay, and mouse bioassay). Both aptamers' STX-binding ability was demonstrated in all three methods. Moreover, M-30f performs better than its parent sequence with higher suppressive activity against STX. As a molecular recognition element, M-30f has good prospects for practical application.

Characterization and modification of enzymes in the 2-ketoisovalerate biosynthesis pathway of Ralstonia eutropha H16.

2-Ketoisovalerate is an important cellular intermediate for the synthesis of branched-chain amino acids as well as other important molecules, such as pantothenate, coenzyme A, and glucosinolate. This ketoacid can also serve as a precursor molecule for the production of biofuels, pharmaceutical agents, and flavor agents in engineered organisms, such as the betaproteobacterium Ralstonia eutropha. The biosynthesis of 2-ketoisovalerate from pyruvate is carried out by three enzymes: acetohydroxyacid synthase (AHAS, encoded by ilvBH), acetohydroxyacid isomeroreductase (AHAIR, encoded by ilvC), and dihydroxyacid dehydratase (DHAD, encoded by ilvD). In this study, enzymatic activities and kinetic parameters were determined for each of the three R. eutropha enzymes as heterologously purified proteins. AHAS, which serves as a gatekeeper for the biosynthesis of all three branched-chain amino acids, demonstrated the tightest regulation through feedback inhibition by L-valine (IC50=1.2 mM), L-isoleucine (IC50=2.3 mM), and L-leucine (IC50=5.4 mM). Intermediates in the valine biosynthesis pathway also exhibit feedback inhibitory control of the AHAS enzyme. In addition, AHAS has a very weak affinity for pyruvate (KM=10.5 μM) and is highly selective towards 2-ketobutyrate (R=140) as a second substrate. AHAIR and DHAD are also inhibited by the branched-chain amino acids, although to a lesser extent when compared to AHAS. Experimental evolution and rational site-directed mutagenesis revealed mutants of the regulatory subunit of AHAS (IlvH) (N11S, T34I, A36V, T104S, N11F, G14E, and N29H), which, when reconstituted with wild-type IlvB, lead to AHAS having reduced valine, leucine, and isoleucine sensitivity. The study of the kinetics and inhibition mechanisms of R. eutropha AHAS, AHAIR, and DHAD has shed light on interactions between these enzymes and the products they produce; it, therefore, can be used to engineer R. eutropha strains with optimal production of 2-ketoisovalerate for value-added materials.

Improving Pseudomonas alcaligenes lipase’s diastereopreference in hydrolysis of diastereomeric mixture of menthyl propionate by site-directed mutagenesis

The resolutions of racemic diastereomeric mixtures of menthyl propionate was performed by Pseudomonas alcaligenes lipase (PaL) to produce (2S, 5R) L-menthol. Because of the inherently low diastereopreference of PaL, covalent docking and molecular dynamic (MD) simulations were used to investigate possible avenues of improvement. Rational site-directed mutagenesis of PaL revealed residues V180 and A272 to be the hotspots for diastereopreference. The double V180L/A272F mutant exhibited the highest degree of diastereopreference, as the diastereomeric ratio of (2S, 5R) L-menthol increased towards both (2R, 5S) L-neomenthol (dr1) and (2R, 5R) D-isoneomenthol (dr2) (diastereomeric ratios dr1 and dr2 increased to 4.65 and 2.13 times that of wild-type PaL). MD simulation analysis indicated that these mutations decrease the flexibility of the surrounding protein regions. The combination of increased steric exclusion and decreased flexibility results in less favorable binding of the non-target substrates, (2R, 5S) L-neomenthyl propionate and (2R, 5R) D-isoneomenthyl propionate, to the V180L/A272F mutant. These results confirmed and further improved our previously proposed model of the diastereomer recognition mechanism based on the combined effect of steric exclusion and regional flexibility.

Altering the activation mechanism in Thermomyces lanuginosus lipase.

It is shown by rational site-directed mutagenesis of the lid region in Thermomyces lanuginosus lipase that it is possible to generate lipase variants with attractive features, e.g., high lipase activity, fast activation at the lipid interface, ability to act on water-soluble substrates, and enhanced calcium independence. The rational design was based on the lid residue composition in Aspergillus niger ferulic acid esterase (FAEA). Five constructs included lipase variants containing the full FAEA lid, a FAEA-like lid, an intermediate lid of FAEA and TlL character, and the entire lid region from Aspergillus terreus lipase (AtL). To investigate an altered activation mechanism for each variant compared to that of TlL, a combination of activity- and spectroscopic-based measurements were applied. The engineered variant with a lid from AtL displayed interfacial activation comparable to that of TlL, whereas variants with FAEA lid character showed interfacial activation independence with pronounced activity toward pNP-acetate and pNP-butyrate below the critical micelle concentration. For variants with lipase and esterase character, lipase activity measurements further indicated a faster activation at the lipid interface. Relative to their activity toward pNP-ester substrates in calcium-rich buffer, all lid variants retained between 15 and 100% activity in buffer containing 5 mM EDTA whereas TlL activity was reduced to less than 2%, demonstrating the lid's central role in governing calcium dependency. For FAEA-like lid variants, accessible hydrophobic surface area measurements showed an approximate 10-fold increase in the level of binding of extrinsic fluorophores to the protein surface relative to that of TlL accompanied by a blue shift in emission indicative of an open lid in aqueous solution. Together, these studies report on the successful alteration of the activation mechanism in TlL by rational design creating novel lipases with new, intriguing functionalities.

Bpur, the Lyme Disease Spirochete's PUR Domain Protein: IDENTIFICATION AS A TRANSCRIPTIONAL MODULATOR AND CHARACTERIZATION OF NUCLEIC ACID INTERACTIONS

The PUR domain is a nucleic acid-binding motif found in critical regulatory proteins of higher eukaryotes and in certain species of bacteria. During investigations into mechanisms by which the Lyme disease spirochete controls synthesis of its Erp surface proteins, it was discovered that the borrelial PUR domain protein, Bpur, binds with high affinity to double-stranded DNA adjacent to the erp transcriptional promoter. Bpur was found to enhance the effects of the erp repressor protein, BpaB. Bpur also bound single-stranded DNA and RNA, with relative affinities RNA > double-stranded DNA > single-stranded DNA. Rational site-directed mutagenesis of Bpur identified amino acid residues and domains critical for interactions with nucleic acids, and it revealed that the PUR domain has a distinct mechanism of interaction with each type of nucleic acid ligand. These data shed light on both gene regulation in the Lyme spirochete and functional mechanisms of the widely distributed PUR domain.

Redirecting P450 EryK Specificity by Rational Site-Directed Mutagenesis

The C-12 hydroxylase EryK is a bacterial cytochrome P450, active during one of the final tailoring steps of erythromycin A (ErA) biosynthesis. Its tight substrate specificity, restricted to the metabolic intermediate ErD, leads to the accumulation in the culture broth of a shunt metabolite, ErB, that originates from the competitive action of a methyltranferase on the substrate of EryK. Although the methylation of the mycarosyl moiety represents the only difference between the two metabolites, EryK exhibits very low conversion of ErB in ErA via a parallel pathway. Given its limited antimicrobial activity and its moderate toxicity, contamination by such a byproduct decreases the yield and purity of the antibiotic. In this study, EryK has been redesigned to make it suitable for industrial application. Taking advantage of the three-dimensional structure of the enzyme in complex with ErD, three single active-site mutants of EryK (M86A, H88E, and E89L) have been designed to allow hydroxylation of the nonphysiological substrate ErB. The binding and catalytic properties of these three variants on both ErD and ErB have been analyzed. Interestingly, we found the mutation of Met 86 to Ala to yield enzymatic activity on both ErB and ErD. The three-dimensional structure of the complex of mutated EryK with ErB revealed that the mutation allows ErB to accommodate in the active site of the enzyme and to induce its closure, thus assuring the progress of the catalytic reaction. Therefore, by single mutation the fine substrate recognition, active site closure, and locking were recovered.