Expression of the Clifednamide Biosynthetic Pathway in Streptomyces Generates 27,28-seco-Derivatives.

Polycyclic tetramate macrolactams (PoTeMs) are a group of hybrid PK-NRP natural products having a variable set of carbocyclic rings, a conserved assembly pathway, and diverse bioactivities. We report here the identification of seven new PoTeMs, clifednamides D-J (3-9), along with the known clifednamides A (1) and B (2) through rational pathway refactoring and heterologous expression. Remarkably, clifednamides D (3), G (6), and H (7) feature an unprecedented 27,28-seco skeleton. The cytotoxic activities of compounds 1-9 indicated that the hydroxy group of C-25, the methyl group of C-30, the inner five-membered ring, and the intact macrocycle are all critical for the activities. Meanwhile, the cytochrome P450 enzyme CftS023A and the hydroxylase CftS023E involved in oxidative tailoring of clifednamides were found to decorate the fused 5-6 bicyclic intermediates. Accordingly, the biosynthetic pathway for clifednamides was proposed.

Structural and molecular basis for the novel catalytic mechanism and evolution of DddP, an abundant peptidase-like bacterial Dimethylsulfoniopropionate lyase: a new enzyme from an old fold.

The microbial cleavage of dimethylsulfoniopropionate (DMSP) generates volatile dimethyl sulfide (DMS) and is an important step in global sulfur and carbon cycles. DddP is a DMSP lyase in marine bacteria, and the deduced dddP gene product is abundant in marine metagenomic data sets. However, DddP belongs to the M24 peptidase family according to sequence alignment. Peptidases hydrolyze C-N bonds, but DddP is deduced to cleave C-S bonds. Mechanisms responsible for this striking functional shift are currently unknown. We determined the structures of DMSP lyase RlDddP (the DddP from Ruegeria lacuscaerulensis ITI_1157) bound to inhibitory 2-(N-morpholino) ethanesulfonic acid or PO4 (3-) and of two mutants of RlDddP bound to acrylate. Based on structural, mutational and biochemical analyses, we characterized a new ion-shift catalytic mechanism of RlDddP for DMSP cleavage. Furthermore, we suggested the structural mechanism leading to the loss of peptidase activity and the subsequent development of DMSP lyase activity in DddP. This study sheds light on the catalytic mechanism and the divergent evolution of DddP, leading to a better understanding of marine bacterial DMSP catabolism and global DMS production.

Identification and catalytic characterization of a nonribosomal peptide synthetase-like (NRPS-like) enzyme involved in the biosynthesis of echosides from Streptomyces sp. LZ35.

Echosides, isolated from Streptomyces sp. LZ35, represent a class of para-terphenyl natural products that display DNA topoisomerase I and IIα inhibitory activities. By analyzing the genome draft of strain LZ35, the ech gene cluster was identified to be responsible for the biosynthesis of echosides, which was further confirmed by gene disruption and HPLC analysis. Meanwhile, the biosynthetic pathway for echosides was proposed. Furthermore, the echA-gene, encoding a tri-domain nonribosomal peptide synthetase (NRPS)-like enzyme, was identified as a polyporic acid synthetase and biochemically characterized in vitro. This is the first study to our knowledge on the biochemical characterization of an Actinobacteria quinone synthetase, which accepts phenylpyruvic acid as a native substrate. Therefore, our results may help investigate the function of other NRPS-like enzymes in Actinobacteria.

Crystallization and preliminary x-ray diffraction analysis of a novel mannose-binding lectin with antiretroviral properties from Polygonatum cyrtonema hua.

A novel antiretroviral protein Polygonatum cyrtonema lectin (PCL) belonging to the monocot mannose-binding lectin (MMBL) superfamily has been crystallized using hanging-drop vapor-diffusion method. The crystals diffract to 2.0 A resolution and belong to space group P2(1), with unit-cell parameters of a=39.308 A, b=48.317 A, c=112.221 A, and beta=90.12 degrees . Preliminary analysis indicates that the asymmetric unit contains four PCL molecules with a solvent content of about 45%. A set of X-ray data has been collected for the crystal structure determination.

Crystal structure of Mabinlin II: a novel structural type of sweet proteins and the main structural basis for its sweetness.

The crystal structure of a sweet protein Mabinlin II (Mab II) isolated from the mature seeds of Capparis masaikai Levl. grown in Southern China has been determined at 1.7A resolution by the SIRAS method. The Mab II 3D structure features in an "all alpha" fold mode consisting of A- and B-chains crosslinked by four disulfide bridges, which is distinct from all known sweet protein structures. The Mabinlin II molecule shows an amphiphilic surface, a cationic face (Face A) and a neutral face (Face B). A unique structural motif consisting of B54-B64 was found in Face B, which adopts a special sequence, NL-P-NI-C-NI-P-NI, featuring four [Asn-Leu/Ile] units connected by three conformational-constrained residues, thus is called the [NL/I] tetralet motif. The experiments for testing the possible interactions of separated A-chain and B-chain and the native Mabinlin II to the sweet-taste receptor were performed through the calcium imaging experiments with the HEK293E cells coexpressed hT1R2/T1R3. The result shows that hT1R2/T1R3 responds to both the integrated Mabinlin II and the individual B-chain in the same scale, but not to A-chain. The sweetness evaluation further identified that the separated B-chain can elicit the sweetness alone, but A-chain does not. All data in combination revealed that the sweet protein Mabinlin II can interact with the sweet-taste receptor hT1R2/T1R3 to elicit its sweet taste, and the B-chain with a unique [NL/I] tetralet motif is the essential structural element for the interaction with sweet-taste receptor to elicit the sweetness, while the A-chain may play a role in gaining a long aftertaste for the integrate Mabinlin II. The findings reported in this paper will be advantage for understanding the diversity of sweet proteins and engineering research for development of a unique sweetener for the food and agriculture based on the Mabinlin II structure as a native model.

A periplasmic glutamate/aspartate binding protein from Shigella flexneri: Gene cloning, over-expression, purification and preliminary crystallographic studies of the recombinant protein.

Periplasmic substrate binding proteins (PSBPs) are essential components of the bacterial periplasmic transport system, which transports a wide variety of nutrients from the periplasmic space to the cytoplasm. The glutamate/aspartate binding protein SfGlu/AspBP is a unique PSBP consisting of 279 amino acid residues. The SfGlu/AspBP gene was cloned, over-expressed, and purified by immobilized metal ion affinity chromatography and size-exclusion chromatography. The recombinant protein SfGlu/AspBP has been crystallized by the hanging-drop vapor-diffusion method and its X-ray diffraction data were collected at an atomic resolution of 1.15 A. The crystals belong to the space group P2(1) with unit cell parameters: a = 48.41 A, b = 68.18 A, c = 80.21 A and beta = 98.78 degrees. There are two molecules per asymmetric unit.

Crystallization and preliminary X-ray analysis of the highly thermostable sweet protein Mabinlin II.

Mabinlin II is a thermostable sweet protein isolated from the mature seeds of Capparis masaikai Levl. grown in the subtropical region of the South of China. The Mabinlin II has been crystallized and diffraction data were collected to 1.7 A resolution. The crystal belongs to space group C2 with unit cell parameters a = 80.11 A, b = 51.08 A, c = 47.34 A, beta = 122.77 degrees.

Purification and preliminary crystallographic studies of CutC, a novel copper homeostasis protein from Shigella flexneri.

CutC is a novel copper homeostasis protein containing 248 amino acids. Here we report the cloning, expression, purification, crystallization and preliminary X-ray crystallographic studies of CutC from Shigella flexneri 2a. Purification of CutC and its selenomethionine (SeMet) derivate were done using immobilized metal ion affinity chromatography, size-exclusion and ion-exchange chromatography. The purified proteins were crystallized using the hanging drop vapor diffusion method. The diffraction data for the native and SeMet CutC, respectively, have been collected with resolution of 1.7 A and 2.1 A. They belong to the space group C2221 and similar cell dimension. The native protein crystals have cell parameters: a=75.3267, b=97.6718, c=132.6910.

Optimizing protein crystal growth through dynamic seeding.

A dynamic seeding method that is different from the conventional method of seeding drops that have been equilibrated is described. The method basically consists of two steps. Firstly, microseeding was used in association with adjustment of the seeding-drop components, including buffer, additive and concentrations of the precipitants and protein, in order to screen suitable seeding conditions under which microseeds are seeded into a new non-equilibrated drop as the dynamic macroseed drop for the following step. Secondly, after being equilibrated for various times against the reservoir solution, the macroseed drops were used to prepare a dilution series with which the qualified crystals could be harvested using macroseeding. Compared with a conventional seeding technique, this method is distinct with a dynamic situation of macroseed drops before macroseeding and a non-equilibrium serial seeding where all the seeds are seeded into new non-equilibrated drops and the micro/macroseeding are efficiently combined into a whole system. The method simplifies control of the number of microseeds because an excess of microseeds has little effect on the final result. The method also simplifies the manipulation of macroseeds by optimizing the equilibration time and the dilution multiple of the macroseed drops before macroseeding. This dynamic seeding technique has been used in the crystallization of novel protein CutCm, which has a fast crystal-growth rate, and proved that the method is useful for optimizing protein crystallization.

Expression, purification and crystallization of a human protein SH3BGRL at atomic resolution.

The protein SH3BGRL, containing both SH3-binding and Homer EVH1-binding motifs, has been crystallized using the hanging-drop vapour-diffusion method. The crystals diffract to 0.88 A resolution and belong to space group P2(1)2(1)2(1), with unit-cell parameters a = 28.8886, b = 34.9676, c = 98.0016 A. Preliminary analysis indicates that the asymmetric unit contains one molecule and has a solvent content of about 34%.