Substrate Recognition Loop Research Articles

Apis mellifera RidA, a novel member of the canonical YigF/YER057c/UK114 imine deiminase superfamily of enzymes pre-empting metabolic damage

The Reactive intermediate deiminase (Rid) protein family is a group of enzymes widely distributed in all Kingdoms of Life. RidA is one of the eight known Rid subfamilies, and its members act by preventing the accumulation of 2-aminoacrylate, a highly reactive enamine generated during the metabolism of some amino acids, by hydrolyzing the 2-iminopyruvate tautomer to pyruvate and ammonia. RidA members are homotrimers exhibiting a remarkable thermal stability. Recently, a novel subclass of RidA was identified in teleosts, which differs for stability and substrate specificity from the canonical RidA. In this study we structurally and functionally characterized RidA from Apis mellifera (AmRidA) as the first example of an invertebrate RidA to assess its belonging to the canonical RidA group, and to further correlate structural and functional features of this novel enzyme class. Circular dichroism revealed a spectrum typical of the RidA proteins and the high thermal stability. AmRidA exhibits the 2-imino acid hydrolase activity typical of RidA family members with a substrate specificity similar to that of the canonical RidA. The crystal structure confirmed the homotrimeric assembly and the presence of the typical structural features of RidA proteins, such as the proposed substrate recognition loop, and the ß-sheets ß1-ß9 and ß1-ß2. In conclusion, our data define AmRidA as a canonical member of the well-conserved RidA family and further clarify the diagnostic structural features of this class of enzymes.

An engineered old yellow enzyme that enables efficient synthesis of (4R,6R)-Actinol in a one-pot reduction system.

(4R,6R)-Actinol can be stereo-selectively synthesized from ketoisophorone by a two-step conversion using a mixture of two enzymes: Candida macedoniensis old yellow enzyme (CmOYE) and Corynebacterium aquaticum (6R)-levodione reductase. However, (4S)-phorenol, an intermediate, accumulates because of the limited substrate range of CmOYE. To address this issue, we solved crystal structures of CmOYE in the presence and absence of a substrate analogue p-HBA, and introduced point mutations into the substrate-recognition loop. The most effective mutant (P295G) showed two- and 12-fold higher catalytic activities toward ketoisophorone and (4S)-phorenol, respectively, than the wild-type, and improved the yield of the two-step conversion from 67.2 to 90.1%. Our results demonstrate that the substrate range of an enzyme can be changed by introducing mutation(s) into a substrate-recognition loop. This method can be applied to the development of other favorable OYEs with different substrate preferences.

Directed Evolution of Sortase A Mutants with Altered Substrate Selectivity Profiles

The ligation of two polypeptides in a chemoselective manner by the bacterial transpeptidase sortase A has become a versatile tool for protein engineering approaches. When sortase-mediated ligation is used for protein semisynthesis, up to four mutations resulting from the strict requirement of the LPxTG sorting motif are introduced into the target protein. Here we report the directed evolution of a mutant sortase A that possesses broad substrate selectivity. A phage-display screen of a mutant sortase library that was randomized in the substrate recognition loop was used to isolate this mutant. The altered substrate selectivity represents a gain-of-function that was exploited for the traceless semisynthesis of histone H3. Our report is a decisive step toward a platform of engineered sortases with distinct ligation properties that will conceivably allow for more versatile assemblies of modified proteins in biotechnological approaches.

Regioselective Control of β- d-Glucose Oxidation by Pyranose 2-Oxidase Is Intimately Coupled to Conformational Degeneracy

Trametes multicolor pyranose 2-oxidase (P2O) is a flavoprotein oxidase that oxidizes d-glucose at C2 to 2-keto- d-glucose by a highly regioselective mechanism. In this work, fluorinated sugar substrates were used as mechanistic probes to investigate the basis of regioselectivity in P2O. Although frequently used to study the mechanisms of glycoside hydrolases, our work provides the first example of applying these probes to sugar oxidoreductases. Our previous structure of the P2O mutant H167A in complex with the slow substrate 2-deoxy-2-fluoro- d-glucose showed a substrate-binding mode compatible with oxidation at C3. To accommodate the sugar, a gating segment, 454FSY 456, in the substrate recognition loop partly unfolded to create a spacious and more polar active site that is distinct from the closed state of P2O. The crystal structure presented here shows that the preferred C2 oxidation where an ordered complex of P2O H167A with 3-deoxy-3-fluoro- d-glucose at 1.35 Å resolution was successfully trapped. In this semi-open C2-oxidation complex, the substrate recognition loop tightens to form an optimized substrate complex stabilized by interactions between Asp452 and glucose O4, as well as Tyr456 and the glucose O6 group, interactions that are not possible when glucose is positioned for oxidation at C3. The different conformations of the 454FSY 456 gating segment in the semi-open and closed states induce backbone and side-chain movements of Thr169 and Asp452 that add further differential stabilization to the individual states. We expect the semi-open state (C2-oxidation state) and closed state to be good approximations of the active-site structure during the reductive half-reaction (sugar oxidation) and oxidative half-reaction (O 2 reduction).

Local Sequence Targeting in the AID/APOBEC Family Differentially Impacts Retroviral Restriction and Antibody Diversification

Nucleic acid cytidine deaminases of the activation-induced deaminase (AID)/APOBEC family are critical players in active and innate immune responses, playing roles as target-directed, purposeful mutators. AID specifically deaminates the host immunoglobulin (Ig) locus to evolve antibody specificity, whereas its close relative, APOBEC3G (A3G), lethally mutates the genomes of retroviral pathogens such as HIV. Understanding the basis for the target-specific action of these enzymes is essential, as mistargeting poses significant risks, potentially promoting oncogenesis (AID) or fostering drug resistance (A3G). AID prefers to deaminate cytosine in WRC (W = A/T, R = A/G) motifs, whereas A3G favors deamination of CCC motifs. This specificity is largely dictated by a single, divergent protein loop in the enzyme family that recognizes the DNA sequence. Through grafting of this substrate-recognition loop, we have created enzyme variants of A3G and AID with altered local targeting to directly evaluate the role of sequence specificity on immune function. We find that grafted loops placed in the A3G scaffold all produced efficient restriction of HIV but that foreign loops in the AID scaffold compromised hypermutation and class switch recombination. Local targeting, therefore, appears alterable for innate defense against retroviruses by A3G but important for adaptive antibody maturation catalyzed by AID. Notably, AID targeting within the Ig locus is proportionally correlated to its in vitro ability to target WRC sequences rather than non-WRC sequences. Although other mechanisms may also contribute, our results suggest that local sequence targeting by AID/APOBEC3 enzymes represents an elegant example of co-evolution of enzyme specificity with its target DNA sequence.

H-Bonding and Positive Charge at the N(5)/O(4) Locus Are Critical for Covalent Flavin Attachment in Trametes Pyranose 2-Oxidase

Flavoenzymes perform a wide range of redox reactions in nature, and a subclass of flavoenzymes carry covalently bound cofactor. The enzyme–flavin bond helps to increase the flavin's redox potential to facilitate substrate oxidation in several oxidases. The formation of the enzyme–flavin covalent bond—the flavinylation reaction—has been studied for the past 40 years. For the most advocated mechanism of autocatalytic flavinylation, the quinone methide mechanism, appropriate stabilization of developing negative charges at the flavin N(1) and N(5) loci is crucial. Whereas the structural basis for stabilization at N(1) is relatively well studied, the structural requisites for charge stabilization at N(5) remain less clear. Here, we show that flavinylation of histidine 167 of pyranose 2-oxidase from Trametes multicolor requires hydrogen bonding at the flavin N(5)/O(4) locus, which is offered by the side chain of Thr169 when the enzyme is in its closed, but not open, state. Moreover, our data show that additional stabilization at N(5) by histidine 548 is required to ensure high occupancy of the histidyl–flavin bond. The combination of structural and spectral data on pyranose 2-oxidase mutants supports the quinone methide mechanism. Our results demonstrate an elaborate structural fine-tuning of the active site to complete its own formation that couples efficient holoenzyme synthesis to conformational substates of the substrate-recognition loop and concerted movements of side chains near the flavinylation ligand.

Structural and Biochemical Characterization of the Cytochrome P450 CypX (CYP134A1) from Bacillus subtilis: A Cyclo-l-leucyl-l-leucyl Dipeptide Oxidase

Cytochrome P450 CypX (CYP134A1), isolated from Bacillus subtilis, has previously been implicated in the three-step oxidative transformation of the diketopiperazine cyclo-l-leucyl-l-leucyl into pulcherriminic acid, a precursor of the extracellular iron chelate pulcherrimin. In this study, we present the first experimental data relating to CYP134A1, where we show that CYP134A1 binds cyclo-l-leucyl-l-leucyl with an affinity of 24.5 +/- 0.5 muM. Structurally related diketopiperazines sharing similar alkyl side chains to cyclo-l-leucyl-l-leucyl also bind to CYP134A1 with comparable affinity. CYP134A1 is capable of catalyzing the in vitro oxidation of diketopiperazine substrates when supported with several alternate electron transfer partner systems. Products containing one additional oxygen atom and which are intermediate products of the expected pulcherriminic acid were identified by GCMS. The oxidation of related diketopiperazines reveals that different oxidative pathways exist for CYP134A1-catalyzed diketopiperazine oxidation. The crystal structure of CYP134A1 has been determined to 2.7 A resolution in the absence of substrate and in the presence of bound phenylimidazole ligands to 3.1 and 3.2 A resolution. The active site is dominated by hydrophobic residues and contains an unusual proline residue in place of the normally conserved alcohol residue that typically plays an important role in oxygen activation. The B-B(2) substrate recognition loop, which forms part of the active site, shows considerable flexibility and was found in both open and closed conformations in different structures. These results represent the first insights into the structural and biochemical basis underlying the multistep oxidation catalyzed by CYP134A1.

Adaptive Evolution of Mus Apobec3 Includes Retroviral Insertion and Positive Selection at Two Clusters of Residues Flanking the Substrate Groove

Mouse APOBEC3 (mA3) is a cytidine deaminase with antiviral activity. mA3 is linked to the Rfv3 virus resistance factor, a gene responsible for recovery from infection by Friend murine leukemia virus, and mA3 allelic variants differ in their ability to restrict mouse mammary tumor virus. We sequenced mA3 genes from 38 inbred strains and wild mouse species, and compared the mouse sequence and predicted structure with human APOBEC3G (hA3G). An inserted sequence was identified in the virus restrictive C57BL strain allele that disrupts a splice donor site. This insertion represents the long terminal repeat of the xenotropic mouse gammaretrovirus, and was acquired in Eurasian mice that harbor xenotropic retrovirus. This viral regulatory sequence does not alter splicing but is associated with elevated mA3 expression levels in spleens of laboratory and wild-derived mice. Analysis of Mus mA3 coding sequences produced evidence of positive selection and identified 10 codons with very high posterior probabilities of having evolved under positive selection. Six of these codons lie in two clusters in the N-terminal catalytically active cytidine deaminase domain (CDA), and 5 of those 6 codons are polymorphic in Rfv3 virus restrictive and nonrestrictive mice and align with hA3G CDA codons that are critical for deaminase activity. Homology models of mA3 indicate that the two selected codon clusters specify residues that are opposite each other along the predicted CDA active site groove, and that one cluster corresponds to an hAPOBEC substrate recognition loop. Substitutions at these clustered mA3 codons alter antiviral activity. This analysis suggests that mA3 has been under positive selection throughout Mus evolution, and identified an inserted retroviral regulatory sequence associated with enhanced expression in virus resistant mice and specific residues that modulate antiviral activity.

Importance of the gating segment in the substrate‐recognition loop of pyranose 2‐oxidase

Pyranose 2-oxidase from Trametes multicolor is a 270 kDa homotetrameric enzyme that participates in lignocellulose degradation by wood-rotting fungi and oxidizes a variety of aldopyranoses present in lignocellulose to 2-ketoaldoses. The active site in pyranose 2-oxidase is gated by a highly conserved, conformationally degenerate loop (residues 450-461), with a conformer ensemble that can accommodate efficient binding of both electron-donor substrate (sugar) and electron-acceptor substrate (oxygen or quinone compounds) relevant to the sequential reductive and oxidative half-reactions, respectively. To investigate the importance of individual residues in this loop, a systematic mutagenesis approach was used, including alanine-scanning, site-saturation and deletion mutagenesis, and selected variants were characterized by biochemical and crystal-structure analyses. We show that the gating segment ((454)FSY(456)) of this loop is particularly important for substrate specificity, discrimination of sugar substrates, turnover half-life and resistance to thermal unfolding, and that three conserved residues (Asp(452), Phe(454) and Tyr(456)) are essentially intolerant to substitution. We furthermore propose that the gating segment is of specific importance for the oxidative half-reaction of pyranose 2-oxidase when oxygen is the electron acceptor. Although the position and orientation of the slow substrate 2-deoxy-2-fluoro-glucose when bound in the active site of pyranose 2-oxidase variants is identical to that observed earlier, the substrate-recognition loop in F454N and Y456W displays a high degree of conformational disorder. The present study also lends support to the hypothesis that 1,4-benzoquinone is a physiologically relevant alternative electron acceptor in the oxidative half-reaction.

Structure of the bifunctional isocitrate dehydrogenase kinase/phosphatase

The Escherichia coli isocitrate dehydrogenase kinase/phosphatase (AceK) is a unique bifunctional enzyme that phosphorylates or dephosphorylates isocitrate dehydrogenase (ICDH) in response to environmental changes, resulting in the inactivation or, respectively, activation of ICDH. ICDH inactivation short-circuits the Krebs cycle by enabling the glyoxlate bypass. It was the discovery of AceK and ICDH that established the existence of protein phosphorylation regulation in prokaryotes. As a 65-kDa protein, AceK is significantly larger than typical eukaryotic protein kinases. Apart from the ATP-binding motif, AceK does not share sequence homology with any eukaryotic protein kinase or phosphatase. Most intriguingly, AceK possesses the two opposing activities of protein kinase and phosphatase within one protein, and specifically recognizes only intact ICDH. Additionally, AceK has strong ATPase activity. It has been shown that AceK kinase, phosphatase and ATPase activities reside at the same site, although the molecular basis of such multifunctionality and its regulation remains completely unknown. Here we report the structures of AceK and its complex with ICDH. The AceK structure reveals a eukaryotic protein-kinase-like domain containing ATP and a regulatory domain with a novel fold. As an AceK phosphatase activator and kinase inhibitor, AMP is found to bind in an allosteric site between the two AceK domains. An AMP-mediated conformational change exposes and shields ATP, acting as a switch between AceK kinase and phosphatase activities, and ICDH-binding induces further conformational change for AceK activation. The substrate recognition loop of AceK binds to the ICDH dimer, allowing higher-order substrate recognition and interaction, and inducing critical conformational change at the phosphorylation site of ICDH.

AlkB recognition of a bulky DNA base adduct stabilized by chemical cross-linking

E. coli AlkB is a direct DNA/RNA repair protein that oxidatively reverses N1 alkylated purines and N3 alkylated pyrimidines to regular bases. Previous crystal structures have revealed N1-methyl adenine (1-meA) recognition by AlkB and a unique base flipping mechanism, but how the AlkB active site can accommodate bulky base adducts is largely unknown. Employing a previously developed chemical cross-linking technique, we crystallized AlkB with a duplex DNA containing a caged thymine base (cagedT). The structure revealed a flexible hairpin lid and a reorganized substrate recognition loop used by AlkB to accommodate cagedT. These observations demonstrate, at the molecular level, how bulky DNA adducts may be recognized and processed by AlkB.

Residues Distant from the Active Site Influence Protein-tyrosine Phosphatase 1B Inhibitor Binding

Regions of protein-tyrosine phosphatase (PTP) 1B that are distant from the active site yet affect inhibitor binding were identified by a novel library screen. This screen was based on the observation that expression of v-Src in yeast leads to lethality, which can be rescued by the coexpression of PTP1B. However, this rescue is lost when yeast are grown in the presence of PTP1B inhibitors. To identify regions of PTP1B (amino acids 1-400, catalytic domain plus 80-amino acid C-terminal tail) that can affect the binding of the difluoromethyl phosphonate (DFMP) inhibitor 7-bromo-6-difluoromethylphosphonate 3-naphthalenenitrile, a library coexpressing PTP1B mutants and v-Src was generated, and the ability of yeast to grow in the presence of the inhibitor was evaluated. PTP1B inhibitor-resistant mutations were found to concentrate on helix alpha7 and its surrounding region, but not in the active site. No resistant amino acid substitutions were found to occur in the C-terminal tail, suggesting that this region has little effect on active-site inhibitor binding. An in-depth characterization of a resistant substitution localizing to region alpha7 (S295F) revealed that this change minimally affected enzyme catalytic activity, but significantly reduced the potency of a panel of structurally diverse DFMP PTP1B inhibitors. This loss of inhibitor potency was found to be due to the difluoro moiety of these inhibitors because only the difluoro inhibitors were shifted. For example, the inhibitor potency of a monofluorinated or non-fluorinated analog of one of these DFMP inhibitors was only minimally affected. Using this type of library screen, which can scan the nearly full-length PTP1B sequence (catalytic domain and C-terminal tail) for effects on inhibitor binding, we have been able to identify novel regions of PTP1B that specifically affect the binding of DFMP inhibitors.

Molecular Determinants of Substrate Recognition in Hematopoietic Protein-tyrosine Phosphatase

The extracellular signal-regulated protein kinase 2 (ERK2) plays a central role in cellular proliferation and differentiation. Full activation of ERK2 requires dual phosphorylation of Thr183 and Tyr185 in the activation loop. Tyr185 dephosphorylation by the hematopoietic protein-tyrosine phosphatase (HePTP) represents an important mechanism for down-regulating ERK2 activity. The bisphosphorylated ERK2 is a highly efficient substrate for HePTP with a kcat/Km of 2.6 x 10(6) m(-1) s(-1). In contrast, the kcat/Km values for the HePTP-catalyzed hydrolysis of Tyr(P) peptides are 3 orders of magnitude lower. To gain insight into the molecular basis for HePTP substrate specificity, we analyzed the effects of altering structural features unique to HePTP on the HePTP-catalyzed hydrolysis of p-nitrophenyl phosphate, Tyr(P) peptides, and its physiological substrate ERK2. Our results suggest that substrate specificity is conferred upon HePTP by both negative and positive selections. To avoid nonspecific tyrosine dephosphorylation, HePTP employs Thr106 in the substrate recognition loop as a key negative determinant to restrain its protein-tyrosine phosphatase activity. The extremely high efficiency and fidelity of ERK2 dephosphorylation by HePTP is achieved by a bipartite protein-protein interaction mechanism, in which docking interactions between the kinase interaction motif in HePTP and the common docking site in ERK2 promote the HePTP-catalyzed ERK2 dephosphorylation (approximately 20-fold increase in kcat/Km) by increasing the local substrate concentration, and second site interactions between the HePTP catalytic site and the ERK2 substrate-binding region enhance catalysis (approximately 20-fold increase in kcat/Km) by organizing the catalytic residues with respect to Tyr(P)185 for optimal phosphoryl transfer.

Identification of residues which regulate activity of the STE20-related kinase hMINK

Activity of the STE20-related kinase hMINK was investigated. hMINK was expressed widely, though not ubiquitously, in human tissues; highest levels being found in haematopoietic tissues but also in brain, placenta, and lung. Mutagenesis revealed that T 191 and Y 193 in the substrate recognition loop of the catalytic domain were critical for kinase activity against exogenous substrates and autophosphorylation. A mutation on T 187 showed reduced enzymatic activity against exogenous substrates but retained autophosphorylation activity. Phosphorylation was confirmed by the use of a phospho-specific T 187 antibody. hMINK activated the JNK signal transduction pathway and optimal JNK activation occurred when the C-terminus was deleted. In addition, overexpression of the C-terminal domain devoid of kinase activity also resulted in significant activation of the JNK pathway. These data suggest that hMINK requires an activation step that dissociates the C terminal, thereby freeing the catalytic domain to interact with substrates. Models for receptor-mediated activation of hMINK are discussed.

The Putative Substrate Recognition Loop of Escherichia coli Ribonuclease H Is Not Essential for Activity

The RNase H family of enzymes catalyzes the hydrolysis of RNA from RNA DNA hybrids in a divalent metal-dependent fashion. To date, structure/function studies have focused on two members of this family: Escherichia coli RNase HI, a small monomeric protein; and human immunodeficiency virus, type I (HIV) RNase H, a domain of HIV reverse transcriptase. The isolated RNase H domain from HIV reverse transcriptase can be expressed independently and shares significant structural homology with its E. coli homologue; however, unlike the bacterial protein, it is inactive. The most notable difference between the inactive domain from HIV and the active E. coli protein is a basic helix/loop sequence, present in E. coli but absent from the HIV homologue. Substitution of this basic region into the HIV domain partially restores its activity and increases its thermodynamic stability. By deleting the basic helix/loop region, we have modeled the structural difference between these two polypeptides onto the E. coli homologue. Surprisingly, the resulting mutant protein is active in Mn2+-dependent fashion. Therefore, the basic helix/loop is not required for RNase H activity.

Functional domains of human aromatase cytochrome P450 characterized by linear alignment and site-directed mutagenesis.

The relationship of function to structure of aromatase cytochrome P450 (P450arom; the product of the CYP19 gene) has been examined by means of sequence alignment and site-directed mutagenesis. Comparison has been made between the sequence of P450arom and the two soluble bacterial cytochrome P450 isoforms, whose three-dimensional structure has been determined (P450BM3 and P450cam). From this comparison, it appears that although there is a similarity of overall structure in cytochromes P450, there is enough significant difference in the regions involved in substrate recognition and substrate binding that residues believed to be involved, even in the known structures, must be tested. With this in mind, we have generated a detailed alignment of P450arom, including the definition of putative alpha-helices and beta-sheets based on comparison of the alignments of P450BM3 and P450cam, generated from their three-dimensional structure, and have made mutations in regions we believe to be involved in substrate recognition at the solvent surface and orientation in the heme pocket. We have mutated F116 and F134 to determine if they are present in the heme pocket, and Q225 and L228 to determine if they are a part of the substrate recognition loop. Although F116E is essentially inactive and may be a folding mutant or may inhibit reductase binding, F134E is more active than the wild type and may be located in the heme pocket facilitating the hydrogen abstraction from C2 of androstenedione. Mutations at Q225 and L228 also result in the anticipated changes in the apparent Km and maximum velocity.(ABSTRACT TRUNCATED AT 250 WORDS)