D‐3‐phosphoglycerate dehydrogenase from the silkworm Bombyx mori: Identification,functional characterization,and expression

D‐3‐phosphoglycerate dehydrogenase (PHGDH) is a key enzyme involved in the synthesis of l ‐serine. Despite the high serine content in silk proteins and the crucial role of PHGDH in serine biosynthesis, PHGDH has not been described in silkworms to date. Here, we identified PHGDH in the silkworm Bombyx mori and evaluated its biochemical properties. On the basis of the amino acid sequence and phylogenetic tree, this PHGDH has been categorized as a new type and designated as bmPHGDH. The recombinant bmPHGDH was overexpressed and purified to homogeneity. Kinetic studies revealed that PHGDH uses NADH as a coenzyme to reduce phosphohydroxypyruvate. High expression levels of bmphgdh messenger RNA (mRNA) were observed in the middle part of the silk gland and midgut in a standard strain of silkworm. Moreover, a sericin‐deficient silkworm strain displayed reduced expression of bmphgdh mRNA. These findings indicate that bmPHGDH might play a crucial role in the provision of l ‐serine in the larva of B. mori.     

Identification of a α‐helical molten globule intermediate and structural characterization of β‐cardiotoxin,an all β‐sheet protein isolated from the venom of Ophiophagus hannah (king cobra)

β‐Cardiotoxin is a novel member of the snake venom three‐finger toxin (3FTX) family. This is the first exogenous protein to antagonize β‐adrenergic receptors and thereby causing reduction in heart rates (bradycardia) when administered into animals, unlike the conventional cardiotoxins as reported earlier. 3FTXs are stable all β‐sheet peptides with 60–80 amino acid residues. Here, we describe the three‐dimensional crystal structure of β‐cardiotoxin together with the identification of a molten globule intermediate in the unfolding pathway of this protein. In spite of the overall structural similarity of this protein with conventional cardiotoxins, there are notable differences observed at the loop region and in the charge distribution on the surface, which are known to be critical for cytolytic activity of cardiotoxins. The molten globule intermediate state present in the thermal unfolding pathway of β‐cardiotoxin was however not observed during the chemical denaturation of the protein. Interestingly, circular dichroism (CD) and NMR studies revealed the presence of α‐helical secondary structure in the molten globule intermediate. These results point to substantial conformational plasticity of β‐cardiotoxin, which might aid the protein in responding to the sometimes conflicting demands of structure, stability, and function during its biological lifetime.     

Characterization of a type of β‐bend ribbon spiral generated by the repeating (Xaa–Yaa–Aib–Pro) motif: The solution structure of harzianin HC IX,a 14‐residue peptaibol forming voltage‐dependent ion channels

The three‐dimensional solution structure of harzianin HC IX, a peptaibol antibiotic isolated from the fungus Trichoderma harzianum, was determined using CD, homonuclear, and heteronuclear two‐dimensional nmr spectroscopy combined with molecular modeling. This 14‐residue peptide, Ac Aib¹ Asn² Leu³ Aib⁴ Pro⁵ Ala⁶ Ile⁷ Aib⁸ Pro⁹ Iva¹⁰ Leu¹¹ Aib¹² Pro¹³ Leuol¹⁴ (Aib, α‐aminoisobutyric acid; Iva, isovaline; Leuol, leucinol), is a main representative of a short‐sequence peptaibol class characterized by an acetylated N‐terminus, a C‐terminal amino alcohol, and the presence of three Aib‐L ‐Pro motifs at positions 4–5, 8–9, and 12–13, separated by two dipeptide units. In spite of a lower number of residues, compared to the 18/20‐residue peptaibols such as alamethicin, harzianin HC IX exhibits remarkable membrane‐perturbing properties. It interacts with phospholipid bilayers, increasing their permeability and forming voltage‐gated ion channels through a mechanism slightly differing from that proposed for alamethicin. Sequence‐specific ¹H‐ and ¹³C‐nmr assignments and conformational nmr parameters (³J_NHCαH coupling constants, quantitative nuclear Overhauser enhancement data, temperature coefficients of amide and carbonyl groups, NH–ND exchange rates) were obtained in methanol solution. Sixty structures were calculated based on 98 interproton distance restraints and 6 Φ dihedral angle restraints, using high temperature restrained molecular dynamics and energy minimization. Thirty‐seven out of the sixty generated structures were consistent with the nmr data and were convergent. The peptide backbone consists in a ribbon of overlapping β‐turns twisted into a continuous spiral from Asn² to Leuol¹⁴ and forming a 26 Å long helix‐like structure. This structure is slightly amphipathic, with the three Aib–Pro motifs aligned on the less hydrophobic face of the spiral where the Asn² side chain is also present, while the more hydrophobic bulky side chains of leucines, isoleucine, isovaline, and leucinol are located on the concave side. The repetitive (Xaa–Yaa–Aib–Pro) tetrapeptide subunit, making up the peptide sequence, is characterized by four sets of (Φ,Ψ) torsional angles, with the following mean values: Φ_i = −90°, Ψ_i = −27°; Φ_i+1 = −98°, Ψ_i+1 = −17°; Φ_i+2 = −49°, Ψ_i+2 = −50°; Φ_i+3 = −78°, Ψ_i+3 = +3°. We term this particular structure, specifically occurring in the case of (Xaa–Yaa–Aib–Pro)_n sequences, the (Xaa–Yaa–Aib–Pro)‐β‐bend ribbon spiral. It is stabilized by 4 → 1 intramolecular hydrogen bonds and differs from both the canonical 3₁₀‐helix made of a succession of type III β‐turns and from the β‐bend ribbon spiral that has been described in the case of (Aib–Pro)n peptide segments. © 1999 John Wiley & Sons, Inc. Biopoly 50: 71–85, 1999     

PASylation Enhances the Stability,Potency, and Plasma Half‐Life of Interferon α‐2a: A Molecular Dynamics Simulation

In this study, the effectiveness of PASylation in enhancing the potency and plasma half‐life of pharmaceutical proteins has been accredited as an alternative technique to the conventional methods such as PEGylation. Proline, alanine, and serine (PAS) chain has shown some advantages including biodegradability improvement and plasma half‐life enhancement while lacking immunogenicity or toxicity. Although some experimental studies have been performed to find the mechanism behind PASylation, the detailed mechanism of PAS effects on the pharmaceutical proteins has remained obscure, especially at the molecular level. In this study, the interaction of interferon α‐2a (IFN) and PAS chain is investigated using molecular dynamics simulation method. Several important parameters including secondary structure, root‐mean‐square distance, and solvent accessible surface area to investigate the stability, bioavailability, and bioactivity of the PASylated protein are studied. The results demonstrate that IFN conformation is not affected critically through PASylation while it results in improvement of the protein stability and bioactivity. Therefore, PASylation can be considered as a proper biological alternative technique to increase the plasma half‐life of the biopharmaceutical proteins through enlarging apparent volume. The proposed simulation represents a computational approach that would provide a basis for the study of PASylated pharmaceutical proteins for different future applications.     

Raw starch–degrading α‐amylase from Bacillus aquimaris MKSC 6.2: isolation and expression of the gene,bioinformatics and biochemical characterization of the recombinant enzyme

Aims

The aims were to isolate a raw starch–degrading α‐amylase gene baqA from Bacillus aquimaris MKSC 6.2, and to characterize the gene product through in silico study and its expression in Escherichia coli.

Methods and Results

A 1539 complete open reading frame of a starch–degrading α‐amylase gene baqA from B. aquimaris MKSC 6·2 has been determined by employing PCR and inverse PCR techniques. Bioinformatics analysis revealed that B. aquimaris MKSC 6.2 α‐amylase (BaqA) has no starch‐binding domain, and together with a few putative α‐amylases from bacilli may establish a novel GH13 subfamily most closely related to GH13_1. Two consecutive tryptophans (Trp201 and Trp202, BaqA numbering) were identified as a sequence fingerprint of this novel GH13 subfamily. Escherichia coli cells produced the recombinant BaqA protein as inclusion bodies. The refolded recombinant BaqA protein degraded raw cassava and corn starches, but exhibited no activity with soluble starch.

Conclusions

A novel raw starch–degrading B. aquimaris MKSC 6.2 α‐amylase BaqA is proposed to be a member of new GH13 subfamily.

Significance and Impact of the Study

This study has contributed to the overall knowledge and understanding of amylolytic enzymes that are able to bind and digest raw starch directly.