The Transition of Human Estrogen Sulfotransferase from Generalist to Specialist Using Directed Enzyme Evolution

Amar, Dotan; Fridman, Ilana Berger; Amara, Neri; Tafa, Gemechu; Meijler, Michaël M.; Aharoni, Amir

doi:10.1016/j.jmb.2011.12.013

Cited by 24 publications

(18 citation statements)

References 38 publications

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“…S5 in the supplemental material), followed by separation of class I and class II PPK2s. Broad specificity is believed to be a property of primordial enzymes, which later evolved into highly specific and efficient enzymes (28). Both the phylogenetic analysis and the broad substrate specificity of class III PPK2 suggested that class III is probably closest to a PPK2 ancestor.…”

Section: Resultsmentioning

confidence: 99%

A New Subfamily of Polyphosphate Kinase 2 (Class III PPK2) Catalyzes both Nucleoside Monophosphate Phosphorylation and Nucleoside Diphosphate Phosphorylation

Motomura

Hirota

Okada

et al. 2014

Appl Environ Microbiol

100

117

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Inorganic polyphosphate (polyP) is a linear polymer of tens to hundreds of phosphate (P i ) residues linked by "high-energy" phosphoanhydride bonds as in ATP. PolyP kinases, responsible for the synthesis and utilization of polyP, are divided into two families (PPK1 and PPK2) due to differences in amino acid sequence and kinetic properties. PPK2 catalyzes preferentially polyPdriven nucleotide phosphorylation (utilization of polyP), which is important for the survival of microbial cells under conditions of stress or pathogenesis. Phylogenetic analysis suggested that the PPK2 family could be divided into three subfamilies (classes I, II, and III). Class I and II PPK2s catalyze nucleoside diphosphate and nucleoside monophosphate phosphorylation, respectively. Here, we demonstrated that class III PPK2 catalyzes both nucleoside monophosphate and nucleoside diphosphate phosphorylation, thereby enabling us to synthesize ATP from AMP by a single enzyme. Moreover, class III PPK2 showed broad substrate specificity over purine and pyrimidine bases. This is the first demonstration that class III PPK2 possesses both class I and II activities. Inorganic polyphosphate (polyP), a linear polymer of tens to hundreds of phosphate (P i ) residues, has been found in all living organisms from bacteria to higher eukaryotes (1). PolyP has numerous biological functions that include serving as a means of storing energy (1, 2), a reservoir for P i (1, 2), a chelator of metal ions (3), a buffer against alkali ions (4), a channel for DNA entry (5), a regulator of stress and survival (6), and a supportive component in gene regulation (7) and enzyme function (8).Polyphosphate kinase 1 (PPK1) is an enzyme that catalyzes the transfer of the terminal P i residue of ATP to short-chain polyP, generating long-chain polyP (9). PPK1 is responsible for the synthesis of most of the cellular polyP. PPK1 also catalyzes polyPdriven ATP synthesis by its reverse reaction. In the case of Escherichia coli PPK1, the order of substrate specificity is ADP Ͼ GDP Ͼ UDP, CDP (10). Another widely distributed polyphosphate kinase (PPK2), which shows no sequence similarity to PPK1, has been found in Pseudomonas aeruginosa as an enzyme catalyzing GTP synthesis from GDP and polyP. In contrast to PPK1, PPK2 preferentially catalyzes the reverse reaction. The expression of PPK2 increases 100 times in P. aeruginosa during the stationary growth phase, suggesting that PPK2 functions in the generation of GTP to support the synthesis of alginate, an exopolysaccharide essential for its virulence (11).Many microbial genomes encode 2 or 3 PPK2 paralogs. Metagenomic analysis of Accumulibacter phosphatis, a dominant polyP-accumulating microorganism in the enhanced biological phosphorus removal (EBPR) system, revealed the presence of five paralogs of PPK2 (12), suggesting that metabolism of polyP by PPK2 is important for its survival in the EBPR system. Nocek et al. found that most of the PPK2 enzymes contain a single domain of ϳ230 amino acids in length (1-domain PPK2), while some co...

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Section: Resultsmentioning

confidence: 99%

A New Subfamily of Polyphosphate Kinase 2 (Class III PPK2) Catalyzes both Nucleoside Monophosphate Phosphorylation and Nucleoside Diphosphate Phosphorylation

Motomura

Hirota

Okada

et al. 2014

Appl Environ Microbiol

100

117

View full text Add to dashboard Cite

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“…A promising direction is provided by considering the evolution from phosphotriesterase (with a paraoxon substrate) to arylesterase (with a 2-naphthylhexanoate substrate [67]). The observed trend presents a functional tradeoff, which seems to be a general phenomenon [68–71]. In exploring the above trend we examined a strategy of evaluating the catalytic effect for a whole set of different mutants for three possible mechanisms (MJL Mills et al ., unpublished), where the first involves an attack from a − OH nucleophile that is situated between the two Zn ions, the second involves an attack by a − OH nucleophile that is bound to one Zn ion, and the third involves a proton transfer from the attacking − OH to an Asp residue that ligates one of the Zn ions.…”

Section: Computer Aided Enzyme Designmentioning

confidence: 99%

Computer aided enzyme design and catalytic concepts

Frushicheva

Mills

Schöpf

et al. 2014

Current Opinion in Chemical Biology

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Gaining a deeper understanding of enzyme catalysis is of great practical and fundamental importance. Over the years it has become clear that despite advances made in experimental mutational studies, a quantitative understanding of enzyme catalysis will not be possible without the use of computer modeling approaches. While we believe that electrostatic preorganization is by far the most important catalytic factor, convincing the wider scientific community of this may require the demonstration of effective rational enzyme design. Here we make the point that the main current advances in enzyme design are basically advances in directed evolution and that computer aided enzyme design must involve approaches that can reproduce catalysis in well-defined test cases. Such an approach is provided by the empirical valence bond method.

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“…We then generated a back-to-consensus DR3 library using sequence alignment of different DR3 ECD orthologues (S1 Fig) [25,26]. Previously, focused gene libraries for directed evolution were generated by integrating structural, functional and evolutionary information available for the protein of interest [27].…”

Section: Resultsmentioning

confidence: 99%

“…Previously, focused gene libraries for directed evolution were generated by integrating structural, functional and evolutionary information available for the protein of interest [27]. Mutagenesis of residues that are different from the consensus sequence of products of the gene family was previously used to generate a back-to-consensus library [25,26]. Screening of these libraries allowed for isolation of mutants with enhanced stability and activity, relative to the native protein [28,29].…”

Section: Resultsmentioning

confidence: 99%

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Directed evolution of a soluble human DR3 receptor for the inhibition of TL1A induced cytokine secretion

2017

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TNF-like 1A (TL1A) is a cytokine belonging to the TNF superfamily that promotes inflammation in autoimmune diseases. Inhibiting the interaction of TL1A with the endogenous death-domain receptor 3 (DR3) offers a therapeutic approach for treating TL1A-induced autoimmune diseases. Here, we generated improved DR3 variants showing increased TL1A binding affinity and stability using a directed evolution approach. Given the high cysteine content and post-translational modification of DR3, we employed yeast surface display and expression in mammalian cell lines for screening, expression and characterization of improved DR3 variants. A cell-based assay performed with the human TF-1 cell line and CD4+ T cells showed that two improved DR3 mutants efficiently inhibited TL1A-induced cell death and secretion of IFN-γ, respectively. These DR3 mutants can be used as drug candidates for the treatment of inflammatory bowel diseases and for other autoimmune diseases, including rheumatic arthritis and asthma.

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The Transition of Human Estrogen Sulfotransferase from Generalist to Specialist Using Directed Enzyme Evolution

Cited by 24 publications

References 38 publications

A New Subfamily of Polyphosphate Kinase 2 (Class III PPK2) Catalyzes both Nucleoside Monophosphate Phosphorylation and Nucleoside Diphosphate Phosphorylation

A New Subfamily of Polyphosphate Kinase 2 (Class III PPK2) Catalyzes both Nucleoside Monophosphate Phosphorylation and Nucleoside Diphosphate Phosphorylation

Computer aided enzyme design and catalytic concepts

Directed evolution of a soluble human DR3 receptor for the inhibition of TL1A induced cytokine secretion

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