Modulate Substrate Specificity Research Articles

Substrate displacement of CK1 C-termini regulates kinase specificity.

CK1 kinases participate in many signaling pathways, and their regulation is of meaningful biological consequence. CK1s autophosphorylate their C-terminal noncatalytic tails, and eliminating these tails increases substrate phosphorylation in vitro, suggesting that the autophosphorylated C-termini act as inhibitory pseudosubstrates. To test this prediction, we comprehensively identified the autophosphorylation sites on Schizosaccharomyces pombe Hhp1 and human CK1ε. Phosphoablating mutations increased Hhp1 and CK1ε activity toward substrates. Peptides corresponding to the C-termini interacted with the kinase domains only when phosphorylated, and substrates competitively inhibited binding of the autophosphorylated tails to the substrate binding grooves. Tail autophosphorylation influenced the catalytic efficiency with which CK1s targeted different substrates, and truncating the tail of CK1δ broadened its linear peptide substrate motif, indicating that tails contribute to substrate specificity as well. Considering autophosphorylation of both T220 in the catalytic domain and C-terminal sites, we propose a displacement specificity model to describe how autophosphorylation modulates substrate specificity for the CK1 family.

The PPP1R15 Family of eIF2-alpha Phosphatase Targeting Subunits (GADD34 and CReP).

The vertebrate PPP1R15 family consists of the proteins GADD34 (growth arrest and DNA damage-inducible protein 34, the product of the PPP1R15A gene) and CReP (constitutive repressor of eIF2α phosphorylation, the product of the PPP1R15B gene), both of which function as targeting/regulatory subunits for protein phosphatase 1 (PP1) by regulating subcellular localization, modulating substrate specificity and assembling complexes with target proteins. The primary cellular function of these proteins is to facilitate the dephosphorylation of eukaryotic initiation factor 2-alpha (eIF2α) by PP1 during cell stress. In this review, we will provide a comprehensive overview of the cellular function, biochemistry and pharmacology of GADD34 and CReP, starting with a brief introduction of eIF2α phosphorylation via the integrated protein response (ISR). We discuss the roles GADD34 and CReP play as feedback inhibitors of the unfolded protein response (UPR) and highlight the critical function they serve as inhibitors of the PERK-dependent branch, which is particularly important since it can mediate cell survival or cell death, depending on how long the stressful stimuli lasts, and GADD34 and CReP play key roles in fine-tuning this cellular decision. We briefly discuss the roles of GADD34 and CReP homologs in model systems and then focus on what we have learned about their function from knockout mice and human patients, followed by a brief review of several diseases in which GADD34 and CReP have been implicated, including cancer, diabetes and especially neurodegenerative disease. Because of the potential importance of GADD34 and CReP in aspects of human health and disease, we will discuss several pharmacological inhibitors of GADD34 and/or CReP that show promise as treatments and the controversies as to their mechanism of action. This review will finish with a discussion of the biochemical properties of GADD34 and CReP, their regulation and the additional interacting partners that may provide insight into the roles these proteins may play in other cellular pathways. We will conclude with a brief outline of critical areas for future study.

Modulating Substrate Specificity of Rhizobium sp. Histamine Dehydrogenase through Protein Engineering for Food Quality Applications.

Histamine is a biogenic amine found in fish-derived and fermented food products with physiological relevance since its concentration is proportional to food spoilage and health risk for sensitive consumers. There are various analytical methods for histamine quantification from food samples; however, a simple and quick enzymatic detection and quantification method is highly desirable. Histamine dehydrogenase (HDH) is a candidate for enzymatic histamine detection; however, other biogenic amines can change its activity or produce false positive results with an observed substrate inhibition at higher concentrations. In this work, we studied the effect of site saturation mutagenesis in Rhizobium sp. Histamine Dehydrogenase (Rsp HDH) in nine amino acid positions selected through structural alignment analysis, substrate docking, and proximity to the proposed histamine-binding site. The resulting libraries were screened for histamine and agmatine activity. Variants from two libraries (positions 72 and 110) showed improved histamine/agmatine activity ratio, decreased substrate inhibition, and maintained thermal resistance. In addition, activity characterization of the identified Phe72Thr and Asn110Val HDH variants showed a clear substrate inhibition curve for histamine and modified kinetic parameters. The observed maximum velocity (Vmax) increased for variant Phe72Thr at the cost of an increased value for the Michaelis-Menten constant (Km) for histamine. The increased Km value, decreased substrate inhibition, and biogenic amine interference observed for variant Phe72Thr support a tradeoff between substrate affinity and substrate inhibition in the catalytic mechanism of HDHs. Considering this tradeoff for future enzyme engineering of HDH could lead to breakthroughs in performance increases and understanding of this enzyme class.

Fatty Acid Allosteric Regulation of C-H Activation in Plant and Animal Lipoxygenases.

Lipoxygenases (LOXs) catalyze the (per) oxidation of fatty acids that serve as important mediators for cell signaling and inflammation. These reactions are initiated by a C-H activation step that is allosterically regulated in plant and animal enzymes. LOXs from higher eukaryotes are equipped with an N-terminal PLAT (Polycystin-1, Lipoxygenase, Alpha-Toxin) domain that has been implicated to bind to small molecule allosteric effectors, which in turn modulate substrate specificity and the rate-limiting steps of catalysis. Herein, the kinetic and structural evidence that describes the allosteric regulation of plant and animal lipoxygenase chemistry by fatty acids and their derivatives are summarized.

Modulating D-amino acid oxidase (DAAO) substrate specificity through facilitated solvent access.

D-amino acid oxidase (DAAO) degrades D-amino acids to produce α-ketoacids, hydrogen peroxide and ammonia. DAAO has often been investigated and engineered for industrial and clinical applications. We combined information from literature with a detailed analysis of the structure to engineer mammalian DAAOs. The structural analysis was complemented with molecular dynamics simulations to characterize solvent accessibility and product release mechanisms. We identified non-obvious residues located on the loops on the border between the active site and the secondary binding pocket essential for pig and human DAAO substrate specificity and activity. We engineered DAAOs by mutating such critical residues and characterised the biochemical activity of the resulting variants. The results highlight the importance of the selected residues in modulating substrate specificity, product egress and enzyme activity, suggesting further steps of DAAO re-engineering towards desired clinical and industrial applications.

Impact of Redox Modifications on ERK2 Substrate Phosphorylation

Extracellular regulated kinase 2 (ERK2), which is the terminal serine/threonine protein kinase in the Ras-Raf-MEK-ERK signaling axis, is involved in the regulation of many cellular processes, including cell proliferation, differentiation, survival, and cell cycle progression. Recently, ERK2 was shown to undergo reversible oxidation, inhibiting reactivity toward Elk1 substrate, following growth factor stimulation in a number of cell types (Keyes et al., FRBM, 2017). Interestingly, one of the residues exhibiting oxidation sensitivity (C159), is located within the D-recruitment site (DRS). Since the DRS is involved in binding a subset of ERK2 substrates, we compared the ability of wild-type ERK2 and ERK2(C159S) to phosphorylate a model DRS peptide substrate, Sub-D, following treatment with various concentrations of H2O2. Kinetic analysis demonstrated that, while both wild-type ERK2 and ERK2(C159S) exhibit similar apparent kcat and Km values for Sub-D in the absence of H2O2, treatment with H2O2 causes more significant changes in kcat,app and Km,app for Sub-D in wild-type compared with the C159S mutant of ERK2. Together, these data suggest that redox modification of ERK2 may alter its ability to phosphorylate substrates recognized by the DRS. This raised the intriguing possibility that oxidation could alter ERK2’s activity toward some, but not all, of its downstream substrates. To explore this possibility further, we investigated the impact of redox modification on ERK2’s global substrate selectivity using functional protein microarrays. Consistent with our hypothesis, ERK2-mediated phosphorylation of several substrates was unaffected by H2O2 treatment while others exhibited H2O2-dependent changes in their phosphorylation status, indicating that C159 oxidation controls ERK activity by modulating substrate specificity. Together, these studies offer important insights into redox regulation of ERK2 and provide potential points of signal integration between kinase- and redox-dependent signaling pathways.

Insights into Substrate Specificity of NlpC/P60 Cell Wall Hydrolases Containing Bacterial SH3 Domains

ABSTRACTBacterial SH3 (SH3b) domains are commonly fused with papain-like Nlp/P60 cell wall hydrolase domains. To understand how the modular architecture of SH3b and NlpC/P60 affects the activity of the catalytic domain, three putative NlpC/P60 cell wall hydrolases were biochemically and structurally characterized. These enzymes all have γ-d-Glu-A2pm (A2pm is diaminopimelic acid) cysteine amidase (or dl-endopeptidase) activities but with different substrate specificities. One enzyme is a cell wall lysin that cleaves peptidoglycan (PG), while the other two are cell wall recycling enzymes that only cleave stem peptides with an N-terminal l-Ala. Their crystal structures revealed a highly conserved structure consisting of two SH3b domains and a C-terminal NlpC/P60 catalytic domain, despite very low sequence identity. Interestingly, loops from the first SH3b domain dock into the ends of the active site groove of the catalytic domain, remodel the substrate binding site, and modulate substrate specificity. Two amino acid differences at the domain interface alter the substrate binding specificity in favor of stem peptides in recycling enzymes, whereas the SH3b domain may extend the peptidoglycan binding surface in the cell wall lysins. Remarkably, the cell wall lysin can be converted into a recycling enzyme with a single mutation.

Insights into the structural determinants of substrate specificity and activity in mouse aldehyde oxidases.

In this work, a combination of homology modeling and molecular dynamics (MD) simulations was used to investigate the factors that modulate substrate specificity and activity of the mouse AOX isoforms: mAOX1, mAOX2 (previously mAOX3l1), mAOX3 and mAOX4. The results indicate that the AOX isoform structures are highly preserved and even more conserved than the corresponding amino acid sequences. The only differences are at the protein surface and substrate-binding site region. The substrate-binding site of all isoforms consists of two regions: the active site, which is highly conserved among all isoforms, and a isoform-specific region located above. We predict that mAOX1 accepts a broader range of substrates of different shape, size and nature relative to the other isoforms. In contrast, mAOX4 appears to accept a more restricted range of substrates. Its narrow and hydrophobic binding site indicates that it only accepts small hydrophobic substrates. Although mAOX2 and mAOX3 are very similar to each other, we propose the following pairs of overlapping substrate specificities: mAOX2/mAOX4 and mAOX3/mAXO1. Based on these considerations, we propose that the catalytic activity between all isoforms should be similar but the differences observed in the binding site might influence the substrate specificity of each enzyme. These results also suggest that the presence of several AOX isoforms in mouse allows them to oxidize more efficiently a wider range of substrates. This contrasts with the same or other organisms that only express one isoform and are less efficient or incapable of oxidizing the same type of substrates.

Structural basis for the evolution of vancomycin resistance D,D-peptidases

Emergence of high-level resistance to the last resort glycopeptide antibiotic vancomycin in Enterococcus spp. and its spread to methicillin-resistant Staphylococcus aureus is a public health threat. Resistance to vancomycin is due to substitution of the D-Ala-D-Ala terminus of cell wall precursors, which forms the antibiotic target, by D-Ala-D-Lac or D-Ala-D-Ser of low binding affinities. Resistance also requires depletion of the normal precursors catalyzed by the zinc-dependent D,D-peptidases VanX and VanY acting on dipeptide (D-Ala-D-Ala) or pentapeptide (UDP-MurNac-L-Ala-D-γ-Glu-L-Lys-D-Ala-D-Ala), respectively. Some resistance operons encode VanXY D,D-peptidase acting on both substrates. Van D,D-peptidases represent attractive targets for combinational antimicrobial therapies to curb resistance; however, the molecular basis of their specificity remains poorly understood, hindering development of potent inhibitors. Therefore we undertook detailed structure-function analysis of VanXY and VanY enzymes. Obtained structural information revealed the substrate-binding site of VanXYC as an extended cavity suitable for binding of di- or pentapeptides, contrasting with previous results showing that VanX contains a small, shallow active site. Biochemical and mutagenesis analysis identified a mobile cap over the catalytic site of VanXYC as the key structural element involved in a switch between di- and pentapeptide hydrolysis. The structures also provided the molecular basis for selectivity towards Van-susceptible peptidoglycan precursors. Overall, this study illustrates the adaptability of the D,D-peptidase fold in response to antibiotic pressure via evolution of particular structural elements that modulate substrate specificity. The results open new opportunities for structure-guided development of Van D,D-peptidases specific inhibitors as glycopeptides adjuvants. This project has been funded by NIAID under Contracts No. HHSN272200700058C and HHSN272201200026C.

Functional Properties and Structural Requirements of the Plasmid pMV158-Encoded MobM Relaxase Domain

A crucial element in the horizontal transfer of mobilizable and conjugative plasmids is the relaxase, a single-stranded endonuclease that nicks the origin of transfer (oriT) of the plasmid DNA. The relaxase of the pMV158 mobilizable plasmid is MobM (494 residues). In solution, MobM forms a dimer through its C-terminal domain, which is proposed to anchor the protein to the cell membrane and to participate in type 4 secretion system (T4SS) protein-protein interactions. In order to gain a deeper insight into the structural MobM requirements for efficient DNA catalysis, we studied two endonuclease domain variants that include the first 199 or 243 amino acid residues (MobMN199 and MobMN243, respectively). Our results confirmed that the two proteins behaved as monomers in solution. Interestingly, MobMN243 relaxed supercoiled DNA and cleaved single-stranded oligonucleotides harboring oriTpMV158, whereas MobMN199 was active only on supercoiled DNA. Protein stability studies using gel electrophoresis and mass spectrometry showed increased susceptibility to degradation at the domain boundary between the N- and C-terminal domains, suggesting that the domains change their relative orientation upon DNA binding. Overall, these results demonstrate that MobMN243 is capable of nicking the DNA substrate independently of its topology and that the amino acids 200 to 243 modulate substrate specificity but not the nicking activity per se. These findings suggest that these amino acids are involved in positioning the DNA for the nuclease reaction rather than in the nicking mechanism itself.

Functional Impact of a Single Mutation within the Transmembrane Domain of the Multidrug ABC Transporter Pdr5

The pleiotropic drug resistance network in budding yeast presents a first line of defense against xenobiotics, which is formed by primary and secondary active membrane transporters. Among these transporters, the ABC transporter Pdr5 is a key component, because it confers resistance against a broad spectrum of such cytotoxic agents. Furthermore, it represents a model system for homologous transporters from pathogenic fungi and has been intensively studied in the past. In addition to other mutational studies, the S1360F mutation of Pdr5 was found to modulate substrate specificity and resistance. Notably, in the S1360F background, the resistance against the immunosuppressant FK506 is drastically increased. We present a detailed analysis of this mutation that is located in the predicted cytosolic part of transmembrane helix 11. Our data demonstrate that kinetic and thermodynamic parameters of the S1360F mutant are similar to those of the wild-type protein, except for FK506-inhibited ATPase activity and the degree of competitive inhibition. In summary, our results indicate that the S1360F mutation within the transmembrane domain interferes drastically with the ability of the nucleotide-binding domains to hydrolyze ATP by interfering with interdomain crosstalk.

Engineering the cytokinin-glucoside specificity of the maize β-d-glucosidase Zm-p60.1 using site-directed random mutagenesis

The maize β-d-glucosidase Zm-p60.1 releases active cytokinins from their storage/transport forms, and its over-expression in tobacco disrupts zeatin metabolism. The role of the active-site microenvironment in fine-tuning Zm-p60.1 substrate specificity has been explored, particularly in the W373K mutant, using site-directed random mutagenesis to investigate the influence of amino acid changes around the 373 position. Two triple (P372T/W373K/M376L and P372S/W373K/M376L) and three double mutants (P372T/W373K, P372S/W373K and W373K/M376L) were prepared. Their catalytic parameters with two artificial substrates show tight interdependence between substrate catalysis and protein structure. P372T/W373K/M376L exhibited the most significant effect on natural substrate specificity: the ratio of hydrolysis of cis-zeatin-O-β-d-glucopyranoside versus the trans-zeatin-O-β-d-glucopyranoside shifted from 1.3 in wild-type to 9.4 in favor of the cis- isomer. The P372T and M376L mutations in P372T/W373K/M376L also significantly restored the hydrolytic velocity of the W373K mutant, up to 60% of wild-type velocity with cis-zeatin-O-β-d-glucopyranoside. These findings reveal complex relationships among amino acid residues that modulate substrate specificity and show the utility of site-directed random mutagenesis for changing and/or fine-tuning enzymes. Preferential cleavage of specific isomer-conjugates and the capacity to manipulate such preferences will allow the development of powerful tools for detailed probing and fine-tuning of cytokinin metabolism in planta.

Crystal Structure of α-Galactosidase from Lactobacillus acidophilus NCFM: Insight into Tetramer Formation and Substrate Binding

Lactobacillus acidophilus NCFM is a probiotic bacterium known for its beneficial effects on human health. The importance of α-galactosidases (α-Gals) for growth of probiotic organisms on oligosaccharides of the raffinose family present in many foods is increasingly recognized. Here, the crystal structure of α-Gal from L. acidophilus NCFM (LaMel36A) of glycoside hydrolase (GH) family 36 (GH36) is determined by single-wavelength anomalous dispersion. In addition, a 1.58-Å-resolution crystallographic complex with α-d-galactose at substrate binding subsite −1 was determined. LaMel36A has a large N-terminal twisted β-sandwich domain, connected by a long α-helix to the catalytic (β/α)8-barrel domain, and a C-terminal β-sheet domain. Four identical monomers form a tightly packed tetramer where three monomers contribute to the structural integrity of the active site in each monomer. Structural comparison of LaMel36A with the monomeric Thermotoga maritima α-Gal (TmGal36A) reveals that O2 of α-d-galactose in LaMel36A interacts with a backbone nitrogen in a glycine-rich loop of the catalytic domain, whereas the corresponding atom in TmGal36A is from a tryptophan side chain belonging to the N-terminal domain. Thus, two distinctly different structural motifs participate in substrate recognition. The tetrameric LaMel36A furthermore has a much deeper active site than the monomeric TmGal36A, which possibly modulates substrate specificity. Sequence analysis of GH36, inspired by the observed structural differences, results in four distinct subgroups having clearly different active-site sequence motifs. This novel subdivision incorporates functional and architectural features and may aid further biochemical and structural analyses within GH36.

Modulation of Nucleotide Specificity of Thermophilic FoF1-ATP Synthase by ϵ-Subunit

The C-terminal two α-helices of the ε-subunit of thermophilic Bacillus F(o)F(1)-ATP synthase (TF(o)F(1)) adopt two conformations: an extended long arm ("up-state") and a retracted hairpin ("down-state"). As ATP becomes poor, ε changes the conformation from the down-state to the up-state and suppresses further ATP hydrolysis. Using TF(o)F(1) expressed in Escherichia coli, we compared TF(o)F(1) with up- and down-state ε in the NTP (ATP, GTP, UTP, and CTP) synthesis reactions. TF(o)F(1) with the up-state ε was achieved by inclusion of hexokinase in the assay and TF(o)F(1) with the down-state ε was represented by εΔc-TF(o)F(1), in which ε lacks C-terminal helices and hence cannot adopt the up-state under any conditions. The results indicate that TF(o)F(1) with the down-state ε synthesizes GTP at the same rate of ATP, whereas TF(o)F(1) with the up-state ε synthesizes GTP at a half-rate. Though rates are slow, TF(o)F(1) with the down-state ε even catalyzes UTP and CTP synthesis. Authentic TF(o)F(1) from Bacillus cells also synthesizes ATP and GTP at the same rate in the presence of adenosine 5'-(β,γ-imino)triphosphate (AMP-PNP), an ATP analogue that has been known to stabilize the down-state. NTP hydrolysis and NTP-driven proton pumping activity of εΔc-TF(o)F(1) suggests similar modulation of nucleotide specificity in NTP hydrolysis. Thus, depending on its conformation, ε-subunit modulates substrate specificity of TF(o)F(1).

Regulation of AKT Phosphorylation at Ser473 and Thr308 by Endoplasmic Reticulum Stress Modulates Substrate Specificity in a Severity Dependent Manner

Endoplasmic reticulum (ER) stress is a common factor in the pathophysiology of diverse human diseases that are characterised by contrasting cellular behaviours, from proliferation in cancer to apoptosis in neurodegenerative disorders. Coincidently, dysregulation of AKT/PKB activity, which is the central regulator of cell growth, proliferation and survival, is often associated with the same diseases. Here, we demonstrate that ER stress modulates AKT substrate specificity in a severity-dependent manner, as shown by phospho-specific antibodies against known AKT targets. ER stress also reduces both total and phosphorylated AKT in a severity-dependent manner, without affecting activity of the upstream kinase PDK1. Normalisation to total AKT revealed that under ER stress phosphorylation of Thr308 is suppressed while that of Ser473 is increased. ER stress induces GRP78, and siRNA-mediated knock-down of GRP78 enhances phosphorylation at Ser473 by 3.6 fold, but not at Thr308. Substrate specificity is again altered. An in-situ proximity ligation assay revealed a physical interaction between GRP78 and AKT at the plasma membrane of cells following induction of ER stress. Staining was weak in cells with normal nuclear morphology but stronger in those displaying rounded, condensed nuclei. Co-immunoprecipitation of GRP78 and P-AKT(Ser473) confirmed the immuno-complex consists of non-phosphorylated AKT (Ser473 and Thr308). The interaction is likely specific as AKT did not bind to all molecular chaperones, and GRP78 did not bind to p70 S6 kinase. These findings provide one mechanistic explanation for how ER stress contributes to human pathologies demonstrating contrasting cell fates via modulation of AKT signalling.

Responsive hybrid block co-polymer conjugates of proteins–controlled architecture to modulate substrate specificity and solution behaviour

Responsive co-polymers based on polyethyleneglycol methacrylate (PEGMA) monomers have been grown by aqueous phase ATRP from a model protein, trypsin, to generate hybrid polymer-protein block conjugates. The conjugates (Hybrids I and II) both contained the same segment of grafted responsive co-polymer to afford a phase transition at 37 °C, Hybrid II however differed from Hybrid I by having a second block of hydrophilic pPEGMA monomer grown from the end of the responsive block. The resultant ‘diblock’ and ‘triblock’ hybrids were characterised in terms of their temperature-dependent behaviour in solution by dynamic light scattering, small-angle neutron scattering and pulsed-gradient spin-echo NMR, and their structures at surfaces examined by aqueous phase atomic force microscopy and cryo transmission electron microscopy. These data showed that Hybrids I and II differed in their solution behaviour with temperature, dependent on the arrangement of their grafted polymer blocks. Hybrid I self-assembled into higher-order structures above 37 °C before precipitating reversibly, whereas Hybrid II remained essentially constant in size across a similar temperature range even when its attached intermediate polymer block underwent a phase transition. The differences in polymer-protein hybrid behaviour were also manifest in enzyme activity assays with temperature-dependent hydrolysis of both peptide and protein substrates varying with hybrid architecture. Overall the data show that it is possible to grow responsive polymer-protein block co-polymers of varied structures, architectures and solution behaviour and that these can be used to control bioconjugate activity.

Modulating substrate specificity of histone acetyltransferase with unnatural amino acids

Controlling the substrate specificity of enzymes is a major challenge for protein engineers. Here we explore the effects of residue-specific incorporation of ortho-, meta- and para-fluorophenylalanine (oFF, mFF, pFF) on the selectivity of human histone acetyltransferase (HAT) protein, p300/CBP associated factor (PCAF). Varying the position of the fluorine group in the phenylalanine ring confers different effects on the ability of PCAF to acetylate target histone H3 as well as non-histone p53. Surprisingly, pFF-PCAF exhibits an increase in activity for non-histone p53, while mFF-PCAF is selective for histone H3. These results suggest that global incorporation of unnatural amino acids may be used to re-engineer protein specificity.

Angelman Syndrome: Finding the Lost Arc

Angelman syndrome is a neurodevelopmental disorder caused by mutations in the maternally inherited UBE3A gene, which encodes a ubiquitin ligase. Greer et al. (2010) now identify a UBE3A substrate called Arc that promotes endocytosis of neuronal AMPA receptors, providing insight into synaptic defects that may underlie the cognitive deficits in Angelman syndrome.

Drosophila MUS312 and the Vertebrate Ortholog BTBD12 Interact with DNA Structure-Specific Endonucleases in DNA Repair and Recombination

DNA recombination and repair pathways require structure-specific endonucleases to process DNA structures that include forks, flaps, and Holliday junctions. Previously, we determined that the Drosophila MEI-9-ERCC1 endonuclease interacts with the MUS312 protein to produce meiotic crossovers, and that MUS312 has a MEI-9-independent role in interstrand crosslink (ICL) repair. The importance of MUS312 to pathways crucial for maintaining genomic stability in Drosophila prompted us to search for orthologs in other organisms. Based on sequence, expression pattern, conserved protein-protein interactions, and ICL repair function, we determined that the mammalian ortholog of MUS312 is BTBD12. Orthology between these proteins and S. cerevisiae Slx4 helped identify a conserved interaction with a second structure-specific endonuclease, SLX1. Genetic and biochemical evidence described here and in related papers suggest that MUS312 and BTBD12 direct Holliday junction resolution by at least two distinct endonucleases in different recombination and repair contexts.

Modifications of human carboxylesterase for improved prodrug activation

Background: Carboxylesterases (CEs) are ubiquitous enzymes responsible for the hydrolysis of numerous clinically useful drugs. As ester moieties are frequently included in molecules to improve their water solubility and bioavailability, de facto they become substrates for CEs. Objective: In this review, we describe the properties of human CEs with regard to their ability to activate anticancer prodrugs and demonstrate how structure-based design can be used to modulate substrate specificity and to increase efficiency of hydrolysis. Methods: A specific example using CPT-11 and a human liver CE is discussed. However, these techniques can be applied to other enzymes and their associated prodrugs. Results: Structure-guided mutagenesis of CEs can be employed to alter substrate specificity and generate novel enzymes that are efficacious at anticancer prodrug activation.