Hydrophobic Substrate Binding Research Articles

Hydrophobic substrate binding pocket remodeling of echinocandin B deacylase based on multi-dimensional rational design

Actinoplanes utahensis deacylase (AAC)-catalyzed deacylation of echinocandin B (ECB) is a promising method for the synthesis of anidulafungin, the newest of the echinocandin antifungal agents. However, the low activity of AAC significantly limits its practical application. In this work, we have devised a multi-dimensional rational design strategy for AAC, conducting separate analyses on the substrate-binding pocket's volume, curvature, and length. Furthermore, we quantitatively analyzed substrate properties, particularly on hydrophilic and hydrophobic. Accordingly, we tailored the linoleic acid-binding pocket of AAC to accommodate the extended long lipid chain of ECB. By fine-tuning the key residues, the resulting AAC mutants can accommodate the ECB lipid chain with a lower curvature binding pocket. The D53A/I55F/G57M/F154L/Q661L mutant (MT) displayed 331 % higher catalytic efficiency than the wild-type (WT) enzyme. The MT product conversion was 94.6 %, reaching the highest reported level. Utilizing a multi-dimensional rational design for a customized mutation strategy of the substrate-binding pocket is an effective approach to enhance the catalytic efficiency of enzymes in handling complicated substrates.

Phosphorylated Peptide Derived from the Myosin Phosphatase Target Subunit Is a Novel Inhibitor of Protein Phosphatase-1

Identification of specific protein phosphatase-1 (PP1) inhibitors is of special importance regarding the study of its cellular functions and may have therapeutic values in diseases coupled to signaling processes. In this study, we prove that a phosphorylated peptide of the inhibitory region of myosin phosphatase (MP) target subunit (MYPT1), R690QSRRS(pT696)QGVTL701 (P-Thr696-MYPT1690-701), interacts with and inhibits the PP1 catalytic subunit (PP1c, IC50 = 3.84 µM) and the MP holoenzyme (Flag-MYPT1-PP1c, IC50 = 3.84 µM). Saturation transfer difference NMR measurements established binding of hydrophobic and basic regions of P-Thr696-MYPT1690-701 to PP1c, suggesting interactions with the hydrophobic and acidic substrate binding grooves. P-Thr696-MYPT1690-701 was dephosphorylated by PP1c slowly (t1/2 = 81.6-87.9 min), which was further impeded (t1/2 = 103 min) in the presence of the phosphorylated 20 kDa myosin light chain (P-MLC20). In contrast, P-Thr696-MYPT1690-701 (10-500 µM) slowed down the dephosphorylation of P-MLC20 (t1/2 = 1.69 min) significantly (t1/2 = 2.49-10.06 min). These data are compatible with an unfair competition mechanism between the inhibitory phosphopeptide and the phosphosubstrate. Docking simulations of the PP1c-P-MYPT1690-701 complexes with phosphothreonine (PP1c-P-Thr696-MYPT1690-701) or phosphoserine (PP1c-P-Ser696-MYPT1690-701) suggested their distinct poses on the surface of PP1c. In addition, the arrangements and distances of the surrounding coordinating residues of PP1c around the phosphothreonine or phosphoserine at the active site were distinct, which may account for their different hydrolysis rate. It is presumed that P-Thr696-MYPT1690-701 binds tightly at the active center but the phosphoester hydrolysis is less preferable compared to P-Ser696-MYPT1690-701 or phosphoserine substrates. Moreover, the inhibitory phosphopeptide may serve as a template to synthesize cell permeable PP1-specific peptide inhibitors.

The exceptionally efficient quorum quenching enzyme LrsL suppresses Pseudomonas aeruginosa biofilm production.

Quorum quenching (QQ) is the enzymatic degradation of molecules used by bacteria for synchronizing their behavior within communities. QQ has attracted wide attention due to its potential to inhibit biofilm formation and suppress the production of virulence factors. Through its capacity to limit biofouling and infections, QQ has applications in water treatment, aquaculture, and healthcare. Several different QQ enzymes have been described; however, they often lack the high stability and catalytic efficiency required for industrial applications. Previously, we identified genes from genome sequences of Red Sea sediment bacteria encoding potential QQ enzymes. In this study, we report that one of them, named LrsL, is a metallo-β-lactamase superfamily QQ enzyme with outstanding catalytic features. X-ray crystallography shows that LrsL is a zinc-binding dimer. LrsL has an unusually hydrophobic substrate binding pocket that can accommodate a broad range of acyl-homoserine lactones (AHLs) with exceptionally high affinity. In vitro, LrsL achieves the highest catalytic efficiency reported thus far for any QQ enzyme with a Kcat/KM of 3 × 107. LrsL effectively inhibited Pseudomonas aeruginosa biofilm formation without affecting bacterial growth. Furthermore, LrsL suppressed the production of exopolysaccharides required for biofilm production. These features, and its capacity to regain its function after prolonged heat denaturation, identify LrsL as a robust and unusually efficient QQ enzyme for clinical and industrial applications.

Transport efficiency of AtGTR1 dependents on the hydrophobicity of transported glucosinolates

Glucosinolates (GLSs) are a group of secondary metabolites that are involved in the defense of herbivores. In Arabidopsis thaliana, Glucosinolate Transporter 1 (AtGTR1) transports GLSs with high affinity via a proton gradient-driven process. In addition to transporting GLSs, AtGTR1 also transports phytohormones, jasmonic acid-isoleucine (JA-Ile), and gibberellin (GA). However, little is known about the mechanisms underlying the broad substrate specificity of AtGTR1. Here, we characterized the substrate preference of AtGTR1 by using a yeast uptake assay, and the results revealed that GLS transport rates are negatively correlated with the hydrophobicity of substrates. Interestingly, the AtGTR1 showed a higher substrate affinity for GLSs with higher hydrophobicity, suggesting a hydrophobic substrate binding pocket. In addition, competition assays revealed that JA, salicylic acid (SA), and indole-3-acetic acid (IAA) competed with GLS for transport in yeast, suggesting a potential interaction of AtGTR1 with these phytohormones. To further characterize the functional properties of AtGTR1, mutagenesis experiments confirmed that the conserved EXXEK motif and Arg166 are essential for the GLS transport function. In addition, the purified AtGTR1 adopts a homodimeric conformation, which is possibly regulated by phosphorylation on Thr105. The phosphomimetic mutation, T105D, reduced its protein expression and completely abrogated its GLS transport function, indicating the essential role of phosphorylation on AtGTR1. In summary, this study investigated various factors associated with the GLS transport and increased our knowledge on the substrate preferences of AtGTR1. These findings contribute to understanding how the distribution of defense GLSs is regulated in plants and could be used to improve crop quality in agriculture.

Crystal structure of steroid reductase SRD5A reveals conserved steroid reduction mechanism

Steroid hormones are essential in stress response, immune system regulation, and reproduction in mammals. Steroids with 3-oxo-Δ4 structure, such as testosterone or progesterone, are catalyzed by steroid 5α-reductases (SRD5As) to generate their corresponding 3-oxo-5α steroids, which are essential for multiple physiological and pathological processes. SRD5A2 is already a target of clinically relevant drugs. However, the detailed mechanism of SRD5A-mediated reduction remains elusive. Here we report the crystal structure of PbSRD5A from Proteobacteria bacterium, a homolog of both SRD5A1 and SRD5A2, in complex with the cofactor NADPH at 2.0 Å resolution. PbSRD5A exists as a monomer comprised of seven transmembrane segments (TMs). The TM1-4 enclose a hydrophobic substrate binding cavity, whereas TM5-7 coordinate cofactor NADPH through extensive hydrogen bonds network. Homology-based structural models of HsSRD5A1 and -2, together with biochemical characterization, define the substrate binding pocket of SRD5As, explain the properties of disease-related mutants and provide an important framework for further understanding of the mechanism of NADPH mediated steroids 3-oxo-Δ4 reduction. Based on these analyses, the design of therapeutic molecules targeting SRD5As with improved specificity and therapeutic efficacy would be possible.

Association with lipids or detergents is essential for preservation of the active structure of lipoprotein-associated phospholipase A2

Recombinant lipoprotein-associated phospholipase A2 (rLp-PLA2) expressed in HEK293 cells has a propensity to form oligomers in the absence of detergents. Dilution of rLp-PLA2 in the absence of detergents results in irreversible inactivation of the enzyme. The monomeric rLp-PLA2 may expose its hydrophobic interfacial binding region or substrate binding compartment to water and that may cause structural collapsing of the enzyme. Formation of self-aggregate, complex with binding partners or association with detergent micelles is to block the access of aqueous solvent to the hydrophobic substrate binding site and therefore prevents the structural collapsing. Dilution inactivation of the enzyme can be prevented in the presence of LDL or HDL suggesting that Lp-PLA2 association with lipoprotein particles (LDL and HDL) is necessary for Lp-PLA2 to maintain its enzymatic activity in human plasma. Formation of higher affinity complex gave better protection of rLp-PLA2 structure and activity. The method can be harnessed to detect the interaction between rLp-PLA2 and components of lipoprotein particles. Apo(a), ApoB 100 and ApoA1 were found to protect the enzyme from inactivation at roughly the similar level (˜80 ± 5%) comparing to human serum albumin control (˜40%). One mg/ml pig brain phospholipid showed 100% protection under the same conditions.

Mapping the Hydrophobic Substrate Binding Site of Phenylalanine Ammonia-Lyase from Petroselinum crispum

Modification of the hydrophobic binding pocket of phenylalanine ammonia-lyase from Petroselinum crispum (PcPAL) enables increased activity and selectivity toward phenylalanines and cinnamic acids m...

Enhancing butanol tolerance of Escherichia coli reveals hydrophobic interaction of multi-tasking chaperone SecB

BackgroundEscherichia coli has been proved to be one promising platform chassis for the production of various natural products, such as biofuels. Product toxicity is one of the main bottlenecks for achieving maximum production of biofuels. Host strain engineering is an effective approach to alleviate solvent toxicity issue in fermentation.ResultsThirty chaperones were overexpressed in E. coli JM109, and SecB recombinant strain was identified with the highest n-butanol tolerance. The tolerance (T) of E. coli overexpressing SecB, calculated by growth difference in the presence and absence of solvents, was determined to be 9.13% at 1.2% (v/v) butanol, which was 3.2-fold of the control strain. Random mutagenesis of SecB was implemented and homologously overexpressed in E. coli, and mutant SecBT10A was identified from 2800 variants rendering E. coli the highest butanol tolerance. Saturation mutagenesis on T10 site revealed that hydrophobic residues were required for high butanol tolerance of E. coli. Compared with wild-type (WT) SecB, the T of SecBT10A strain was further increased from 9.14 to 14.4% at 1.2% butanol, which was 5.3-fold of control strain. Remarkably, E. coli engineered with SecBT10A could tolerate as high as 1.8% butanol (~ 14.58 g/L). The binding affinity of SecBT10A toward model substrate unfolded maltose binding protein (preMBP) was 11.9-fold of that of WT SecB as determined by isothermal titration calorimetry. Residue T10 locates at the entrance of hydrophobic substrate binding groove of SecB, and might play an important role in recognition and binding of cargo proteins.ConclusionsSecB chaperone was identified by chaperone mining to be effective in enhancing butanol tolerance of E. coli. Maximum butanol tolerance of E. coli could reach 1.6% and 1.8% butanol by engineering single gene of SecB or SecBT10A. Hydrophobic interaction is vital for enhanced binding affinity between SecB and cargo proteins, and therefore improved butanol tolerance.

Molecular architectures of benzoic acid-specific type III polyketide synthases.

Biphenyl synthase and benzophenone synthase constitute an evolutionarily distinct clade of type III polyketide synthases (PKSs) that use benzoic acid-derived substrates to produce defense metabolites in plants. The use of benzoyl-CoA as an endogenous substrate is unusual for type III PKSs. Moreover, sequence analyses indicate that the residues responsible for the functional diversification of type III PKSs are mutated in benzoic acid-specific type III PKSs. In order to gain a better understanding of structure-function relationships within the type III PKS family, the crystal structures of biphenyl synthase from Malus × domestica and benzophenone synthase from Hypericum androsaemum were compared with the structure of an archetypal type III PKS: chalcone synthase from Malus × domestica. Both biphenyl synthase and benzophenone synthase contain mutations that reshape their active-site cavities to prevent the binding of 4-coumaroyl-CoA and to favor the binding of small hydrophobic substrates. The active-site cavities of biphenyl synthase and benzophenone synthase also contain a novel pocket associated with their chain-elongation and cyclization reactions. Collectively, these results illuminate structural determinants of benzoic acid-specific type III PKSs and expand the understanding of the evolution of specialized metabolic pathways in plants.

Substrate Recognition by a Dual-Function P450 Monooxygenase GfsF Involved in FD-891 Biosynthesis.

GfsF is a multifunctional P450 monooxygenase that catalyzes epoxidation and subsequent hydroxylation in the biosynthesis of macrolide polyketide FD-891. Here, we describe the biochemical and structural analysis of GfsF. To obtain the structural basis of a dual-function reaction, we determined the crystal structure of ligand-free GfsF, which revealed GfsF to have a predominantly hydrophobic substrate binding pocket. The docking models, in conjunction with the results of the enzymatic assay with substrate analogues and site-directed mutagenesis suggested two distinct substrate binding modes for epoxidation and hydroxylation reactions, which explained how GfsF regulates the order of two oxidative reactions. These findings provide new insights into the reaction mechanism of multifunctional P450 monooxygenases.

Enzymatic Activity and Thermodynamic Stability of Biliverdin IXβ Reductase Are Maintained by an Active Site Serine.

Biliverdin reductase IXβ (BLVRB) is a crucial enzyme in heme metabolism. Recent studies in humans have identified a loss-of-function mutation (Ser111Leu) that unmasks a fundamentally important role in hematopoiesis. We have undertaken experimental and thermodynamic modeling studies to provide further insight into the role of the cofactor in substrate accessibility and protein folding properties regulating BLVRB catalytic mechanisms. Site-directed mutagenesis with molecular dynamic (MD) simulations establish the critical role of NAD(P)H-dependent conformational changes on substrate accessibility by forming the "hydrophobic pocket", along with identification of a single key residue (Arg35) modulating NADPH/NADH selectivity. Loop80 and Loop120 block the hydrophobic substrate binding pocket in apo BLVRB (open), whereas movement of these structures after cofactor binding results in the "closed" (catalytically active) conformation. Both enzymatic activity and thermodynamic stability are affected by mutation(s) involving Ser111, which is located in the core of the BLVRB active site. This work 1) elucidates the crucial role of Ser111 in enzymatic catalysis and thermodynamic stability by active site hydrogen bond network; 2) defines a dynamic model for apo BLVRB extending beyond the crystal structure of the binary BLVRB/NADP+ complex; 3) provides a structural basis for the "encounter" and "equilibrium" states of the binary complex, which are regulated by NAD(P)H.

Structure and Function of a C-C Bond Cleaving Oxygenase in Atypical Angucycline Biosynthesis.

C-C bond ring cleaving oxygenases represent a unique family of enzymes involved in the B ring cleavage reaction only observed in atypical angucycline biosynthesis. B ring cleavage is the key reaction leading to dramatic divergence in the final structures of atypical angucyclines. Here, we present the crystal structure of AlpJ, the first structure of this family of enzymes. AlpJ has been verified as the enzyme catalyzing C-C bond cleavage in kinamycin biosynthesis. The crystal structure of the AlpJ monomer resembles the dimeric structure of ferredoxin-like proteins. The N- and C-terminal halves of AlpJ are homologous, and both contain a putative hydrophobic substrate binding pocket in the "closed" and "open" conformations, respectively. Structural comparison of AlpJ with ActVA-Orf6 and protein-ligand docking analysis suggest that the residues including Asn60, Trp64, and Trp181 are possibly involved in substrate recognition. Site-directed mutagenesis results supported our hypothesis, as mutation of these residues led to nearly a complete loss of the activity of AlpJ. Structural analysis also revealed that AlpJ possesses an intramolecular domain-domain interface, where the residues His50 and Tyr178 form a hydrogen bond that probably stabilizes the three-dimensional structure of AlpJ. Site-directed mutagenesis showed that the two residues, His50 and Tyr178, were vital for the activity of AlpJ. Our findings shed light on the structure and catalytic mechanism of the AlpJ family of oxygenases, which presumably involves two active sites that might function in a cooperative manner.

The N-Terminal α-Helix Domain of Pseudomonas aeruginosa Lipoxygenase Is Required for Its Soluble Expression in Escherichia coli but Not for Catalysis.

Lipoxygenase (LOX) is an industrial enzyme with wide applications in food and pharmaceutical industries. The available structure information indicates that eukaryotic LOXs consist of N terminus β-barrel and C terminus catalytic domains. However, the latest crystal structure of Pseudomonas aeruginosa LOX shows it is significantly different from those of eukaryotic LOXs, including the N-terminal helix domain. In this paper, the functions of this N-terminal helix domain in the soluble expression and catalysis of P. aeruginosa LOX were analyzed. Genetic truncation of this helix domain resulted in an insoluble P. aeruginosa LOX mutant. The active C-terminal domain was obtained by dispase digestion of the P. aeruginosa LOX derivative containing the genetically introduced dispase recognition sites. This functional C-terminal domain showed raised substrate affinity but reduced catalytic activity and thermostability. Crystal structure analyses demonstrate that the broken polar contacts connecting the two domains and the exposed hydrophobic substrate binding pocket may contribute to the insoluble expression of the C terminus domain and the changes in the enzyme properties. Our data suggest that the N terminus domain of P. aeruginosa LOX is required for its soluble expression in E. coli, which is different from that of the eukaryotic LOXs. Besides this, this N-terminal domain is not necessary for catalysis but shows positive effects on the enzyme properties. The results presented here provide new and valuable information on the functions of the N terminus helix domain of P. aeruginosa LOX and further improvement of its enzyme properties by molecular modification.

Mia40 is a trans-site receptor that drives protein import into the mitochondrial intermembrane space by hydrophobic substrate binding.

Many proteins of the mitochondrial IMS contain conserved cysteines that are oxidized to disulfide bonds during their import. The conserved IMS protein Mia40 is essential for the oxidation and import of these proteins. Mia40 consists of two functional elements: an N-terminal cysteine-proline-cysteine motif conferring substrate oxidation, and a C-terminal hydrophobic pocket for substrate binding. In this study, we generated yeast mutants to dissect both Mia40 activities genetically and biochemically. Thereby we show that the substrate-binding domain of Mia40 is both necessary and sufficient to promote protein import, indicating that trapping by Mia40 drives protein translocation. An oxidase-deficient Mia40 mutant is inviable, but can be partially rescued by the addition of the chemical oxidant diamide. Our results indicate that Mia40 predominantly serves as a trans-site receptor of mitochondria that binds incoming proteins via hydrophobic interactions thereby mediating protein translocation across the outer membrane by a 'holding trap' rather than a 'folding trap' mechanism.

Structural–functional organization of cytochrome P450 containing monooxygenase and some aspects of modeling

The article deals with a superfamily of monooxygenases, a multienzymeproteinaceous complex containing cytochrome P450 as a terminal electron acceptor. Due to the low substrate specificity, this monooxygenase actively participates in biosynthesis and oxidation of numerous endogenous cell compounds, and also in oxidation and detoxification of wide spectrum of compounds that are foreign to an organism (xenobiotics). The main peculiarity of animal monooxygenase is its interaction with the membrane: all three components of it are true membrane proteins that gain some advantages (binding of hydrophobic substrates to cytochrome P450, creation of optimal conditions by reaction with its own reductase, modulation of electron transport, etc) over other soluble oxidases. In plants, cytochrome P450 is embedded in the membrane but simultaneously, it occurs in a soluble form as well. As a result, a stronger protection barrier against the impact of xenobiotic-toxicants is created in it. In addition, it is shown that in plants, the same isoforms of cytochrome P450 participate in biosyntheses and xenobiotic degradation. Penetration of xenobiotics into a plant cell and especially, its polarity represents a regulatory signal, which ensures proper distribution of hemoprotein pool in these processes. Action mechanism of cytochrome P450, i.e. the functioning of monooxygenase cycle and its completion stage, when oxygen activated by two electrons is inserted into the substrate and hydroxylation product is formed, is described in more detail. Nowadays, functional models of monooxygenases are used effectively to study this stage. It is considered that in oxygenase reaction, oxygen atom formed by its interaction with ferric ion complex and redox-active ligand is transferred to the substrate via so-called oxenoid mechanism. The rate of the whole hydroxylation process is limited by the stage of insertion of activated oxygen into the substrate. It deserves consideration that hydroperoxides, which are reduced by two electrons and perform shunting of the cycle, model incompletely its operation. Action mechanism of monooxygenase essentially differs from this model and the cycle prefers phasic successive proceeding.

Crystal structure of Thermotoga maritima acetyl esterase complex with a substrate analog: Insights into the distinctive substrate specificity in the CE7 carbohydrate esterase family

The carbohydrate esterase family 7 (CE7) members are acetyl esterases that possess unusual substrate specificity for cephalosporin C and 7-amino-cephalosporanic acid. This family containing the α/β hydrolase fold has a distinctive substrate profile that allows it to carry out hydrolysis of esters containing diverse alcohol moieties while maintaining narrow specificity for an acetate ester. Here we investigate the structural basis of this preference for small acyl groups using the crystal structure of the thermostable Thermotoga maritima CE7 acetyl esterase (TmAcE) complexed with a non-cognate substrate analog. The structure determined at 1.86 Å resolution provides direct evidence for the location of the largely hydrophobic and rigid substrate binding pocket in this family. Furthermore, a three-helix insertion domain near the catalytic machinery shapes the substrate binding site. The structure reveals two residues (Pro228 and Ile276) which constitute a hydrophobic rigid binding surface for the acyl group of the ester and thus restricts the size of the acyl group that be accommodated. In combination with previous literature on kinetic properties of the enzyme, our studies suggest that these residues determine the unique specificity of the TmAcE for short straight chain esters. The structure provides a template for focused attempts to engineer the CE7 enzymes for enhanced stability, selectivity or activity for biocatalytic applications.

Identification of imine reductase-specific sequence motifs.

Chiral amines are valuable building blocks for the production of a variety of pharmaceuticals, agrochemicals and other specialty chemicals. Only recently, imine reductases (IREDs) were discovered which catalyze the stereoselective reduction of imines to chiral amines. Although several IREDs were biochemically characterized in the last few years, knowledge of the reaction mechanism and the molecular basis of substrate specificity and stereoselectivity is limited. To gain further insights into the sequence-function relationships, the Imine Reductase Engineering Database (www.IRED.BioCatNet.de) was established and a systematic analysis of 530 putative IREDs was performed. A standard numbering scheme based on R-IRED-Sk was introduced to facilitate the identification and communication of structurally equivalent positions in different proteins. A conservation analysis revealed a highly conserved cofactor binding region and a predominantly hydrophobic substrate binding cleft. Two IRED-specific motifs were identified, the cofactor binding motif GLGxMGx(5 )[ATS]x(4) Gx(4) [VIL]WNR[TS]x(2) [KR] and the active site motif Gx[DE]x[GDA]x[APS]x(3){K}x[ASL]x[LMVIAG]. Our results indicate a preference toward NADPH for all IREDs and explain why, despite their sequence similarity to β-hydroxyacid dehydrogenases (β-HADs), no conversion of β-hydroxyacids has been observed. Superfamily-specific conservations were investigated to explore the molecular basis of their stereopreference. Based on our analysis and previous experimental results on IRED mutants, an exclusive role of standard position 187 for stereoselectivity is excluded. Alternatively, two standard positions 139 and 194 were identified which are superfamily-specifically conserved and differ in R- and S-selective enzymes.

Protein interactions between the C-terminus of Aβ-peptide and phospholipase A2--a structure biology based approach to identify novel Alzheimer's therapeutics.

Amyloid β (Aβ) polypeptide plays a key role in determining the state of protein aggregation in Alzheimer's disease. The hydrophobic C-terminal part of the Aβ peptide is critical in triggering the transformation from α-helical to β- sheet structure. We hypothesized that phospholipase A2 (PLA2) may inhibit the aggregation of Aβ peptide by interacting with the peptide and keeping the two peptide chains apart. In order to examine the nature of interactions between PLA2 and Aβ peptide, we prepared and crystallized complex of Naja naja sagittifera PLA2 with the C-terminal hepta-peptide Val-Gly-Gly-Val-Val-Ile-Ala. The X-ray intensity data were collected to 2.04 A resolution and the structure was determined by molecular replacement and refined to the crystallographic R factor of 0.186. The structural analysis revealed that the peptide binds to PLA2 at the hydrophobic substrate binding cavity forming at least eight hydrogen bonds and approximately a two dozen Van der Waals interactions. The number and nature of interactions indicate that the affinity between PLA2 and the hepta-peptide is greater than the affinity between two Aβ peptide chains. Therefore, PLA2 is proposed as a probable ligand to prevent the aggregation of Aβ peptides.

Chiral Hydroxylation at the Mononuclear Nonheme Fe(II) Center of 4-(S) Hydroxymandelate Synthase – A Structure-Activity Relationship Analysis

(S)-Hydroxymandelate synthase (Hms) is a nonheme Fe(II) dependent dioxygenase that catalyzes the oxidation of 4-hydroxyphenylpyruvate to (S)-4-hydroxymandelate by molecular oxygen. In this work, the substrate promiscuity of Hms is characterized in order to assess its potential for the biosynthesis of chiral α-hydroxy acids. Enzyme kinetic analyses, the characterization of product spectra, quantitative structure activity relationship (QSAR) analyses and in silico docking studies are used to characterize the impact of substrate properties on particular steps of catalysis. Hms is found to accept a range of α-oxo acids, whereby the presence of an aromatic substituent is crucial for efficient substrate turnover. A hydrophobic substrate binding pocket is identified as the likely determinant of substrate specificity. Upon introduction of a steric barrier, which is suspected to obstruct the accommodation of the aromatic ring in the hydrophobic pocket during the final hydroxylation step, the racemization of product is obtained. A steady state kinetic analysis reveals that the turnover number of Hms strongly correlates with substrate hydrophobicity. The analysis of product spectra demonstrates high regioselectivity of oxygenation and a strong coupling efficiency of C-C bond cleavage and subsequent hydroxylation for the tested substrates. Based on these findings the structural basis of enantioselectivity and enzymatic activity is discussed.

Use of anion–aromatic interactions to position the general base in the ketosteroid isomerase active site

Although the cation-pi pair, formed between a side chain or substrate cation and the negative electrostatic potential of a pi system on the face of an aromatic ring, has been widely discussed and has been shown to be important in protein structure and protein-ligand interactions, there has been little discussion of the potential structural and functional importance in proteins of the related anion-aromatic pair (i.e., interaction of a negatively charged group with the positive electrostatic potential on the ring edge of an aromatic group). We posited, based on prior structural information, that anion-aromatic interactions between the anionic Asp general base and Phe54 and Phe116 might be used instead of a hydrogen-bond network to position the general base in the active site of ketosteroid isomerase from Comamonas testosteroni as there are no neighboring hydrogen-bonding groups. We have tested the role of the Phe residues using site-directed mutagenesis, double-mutant cycles, and high-resolution X-ray crystallography. These results indicate a catalytic role of these Phe residues. Extensive analysis of the Protein Data Bank provides strong support for a catalytic role of these and other Phe residues in providing anion-aromatic interactions that position anionic general bases within enzyme active sites. Our results further reveal a potential selective advantage of Phe in certain situations, relative to more traditional hydrogen-bonding groups, because it can simultaneously aid in the binding of hydrophobic substrates and positioning of a neighboring general base.