Expression, purification and characterization of recombinant (E)-beta-farnesene synthase from Artemisia annua

A cDNA clone (GenBank Accession No. AY835398) encoding a sesquiterpene synthase, (E)-β-farnesene synthase, has been isolated from Artemisia annua L. It contains a 1746-bp open reading frame coding for 574 amino acids (66.9 kDa) with a calculated pI = 5.03. The deduced amino acid sequence is 30-50% identical with sequences of other sesquiterpene synthases from angiosperms. The recombinant enzyme, produced in Escherichia coli, catalyzed the formation of a single product, β-farnesene, from farnesyl diphosphate. The pH optimum for the recombinant enzyme is around 6.5 and the K_m- and k_cat-values for farnesyl diphosphate, is 2.1 μM and 9.5 × 10⁻³ s⁻¹, respectively resulting in the efficiency 4.5 × 10⁻³ M⁻¹ s⁻¹. The enzyme exhibits substantial activity in the presence of Mg²⁺, Mn²⁺ or Co²⁺ but essentially no activity when Zn²⁺, Ni²⁺ or Cu²⁺ is used as cofactor. The concentration required for maximum activity are estimated to 5 mM, 0.5 mM and <10 μM for Mg²⁺, Co²⁺ or Mn²⁺, respectively. Geranyl diphosphate is not a substrate for the recombinant enzyme.     

Structural and biochemical characterization of the therapeutic Anabaena variabilis phenylalanine ammonia lyase

We have recently observed promising success in a mouse model for treating the metabolic disorder phenylketonuria with phenylalanine ammonia lyase (PAL) from Rhodosporidium toruloides and Anabaena variabilis. Both molecules, however, required further optimization in order to overcome problems with protease susceptibility, thermal stability, and aggregation. Previously, we optimized PAL from R. toruloides, and in this case we reduced aggregation of the A. variabilis PAL by mutating two surface cysteine residues (C503 and C565) to serines. Additionally, we report the structural and biochemical characterization of the A. variabilis PAL C503S/C565S double mutant and carefully compare this molecule with the R. toruloides engineered PAL molecule. Unlike previously published PAL structures, significant electron density is observed for the two active-site loops in the A. variabilis C503S/C565S double mutant, yielding a complete view of the active site. Docking studies and N-hydroxysuccinimide-biotin binding studies support a proposed mechanism in which the amino group of the phenylalanine substrate is attacked directly by the 4-methylidene-imidazole-5-one prosthetic group. We propose a helix-to-loop conformational switch in the helices flanking the inner active-site loop that regulates accessibility of the active site. Differences in loop stability among PAL homologs may explain the observed variation in enzyme efficiency, despite the highly conserved structure of the active site. A. variabilis C503S/C565S PAL is shown to be both more thermally stable and more resistant to proteolytic cleavage than R. toruloides PAL. Additional increases in thermal stability and protease resistance upon ligand binding may be due to enhanced interactions among the residues of the active site, possibly locking the active-site structure in place and stabilizing the tetramer. Examination of the A. variabilis C503S/C565S PAL structure, combined with analysis of its physical properties, provides a structural basis for further engineering of residues that could result in a better therapeutic molecule.     

Biochemical characterization of an ABC transporter LptBFGC complex required for the outer membrane sorting of lipopolysaccharides

Seven Lpt proteins (A through G) are thought to be involved in lipopolysaccharide transport from the inner to outer membrane of Escherichia coli. LptB belongs to the ATP-binding cassette transporter superfamily. Although the lptB gene lacks neighboring genes encoding membrane subunits, bioinformatic analyses recently indicated that two distantly located consecutive genes, lptF and lptG, could encode membrane subunits. To examine this possibility, LptB was expressed with LptF and LptG. We report here that both LptF and LptG formed a complex with LptB. Furthermore, an inner membrane protein, LptC, which had been implicated in lipopolysaccharide transport, was also included in this complex.

Structured summary

MINT-7137021: lptb (uniprotkb:P0A9V1) physically interacts (MI:0914) with lptc (uniprotkb:P0ADV9), lptg (uniprotkb:P0ADC6) and lptf (uniprotkb:P0AF98) by pull down (MI:0096)MINT-7137160: lptb (uniprotkb:P0A9V1) physically interacts (MI:0914) with lptf (uniprotkb:P0AF98) and lptg (uniprotkb:P0ADC6) by pull down (MI:0096)