Yunge Li

CRYSTAL STRUCTURE OF PROTEUS VULGARIS CHONDROITIN SULFATE ABC LYASE I AT 1.9A RESOLUTION.

Chondroitin Sulfate ABC lyase I from Proteus vulgaris is an endolytic, broad-specificity glycosaminoglycan lyase, which degrades chondroitin, chondroitin-4-sulfate, dermatan sulfate, chondroitin-6-sulfate, and hyaluronan by beta-elimination of 1,4-hexosaminidic bond to unsaturated disaccharides and tetrasaccharides. Its structure revealed three domains. The N-terminal domain has a fold similar to that of carbohydrate-binding domains of xylanases and some lectins, the middle and C-terminal domains are similar to the structures of the two-domain chondroitin lyase AC and bacterial hyaluronidases. Although the middle domain shows a very low level of sequence identity with the catalytic domains of chondroitinase AC and hyaluronidase, the residues implicated in catalysis of the latter enzymes are present in chondroitinase ABC I. The substrate-binding site in chondroitinase ABC I is in a wide-open cleft, consistent with the endolytic action pattern of this enzyme. The tryptophan residues crucial for substrate binding in chondroitinase AC and hyaluronidases are lacking in chondroitinase ABC I. The structure of chondroitinase ABC I provides a framework for probing specific functions of active-site residues for understanding the remarkably broad specificity of this enzyme and perhaps engineering a desired specificity. The electron density map showed clearly that the deposited DNA sequence for residues 495-530 of chondroitin ABC lyase I, the segment containing two putative active-site residues, contains a frame-shift error resulting in an incorrectly translated amino acid sequence.

The Structure of the RlmB 23S rRNA Methyltransferase Reveals a New Methyltransferase Fold with a Unique Knot

In Escherichia coli, RlmB catalyzes the methylation of guanosine 2251, a modification conserved in the peptidyltransferase domain of 23S rRNA. The crystal structure of this 2O-methyltransferase has been determined at 2.5 A resolution. RlmB consists of an N-terminal domain connected by a flexible extended linker to a catalytic C-terminal domain and forms a dimer in solution. The C-terminal domain displays a divergent methyltransferase fold with a unique knotted region, and lacks the classic AdoMet binding site features. The N-terminal domain is similar to ribosomal proteins L7 and L30, suggesting a role in 23S rRNA recognition. The conserved residues in this novel family of 2O-methyltransferases cluster in the knotted region, suggesting the location of the catalytic and AdoMet binding sites.

Crystal structure of a dodecameric FMN‐dependent UbiX‐like decarboxylase (Pad1) from Escherichia coli O157: H7

The crystal structure of the flavoprotein Pad1 from Escherichia coli O157:H7 complexed with the cofactor FMN has been determined by the multiple anomalous diffraction method and refined at 2.0 Å resolution. This protein is a paralog of UbiX (3‐octaprenyl‐4‐hydroxybenzoate carboxylyase, 51% sequence identity) that catalyzes the third step in ubiquinone biosynthesis and to Saccharomyces cerevisiae Pad1 (54% identity), an enzyme that confers resistance to the antimicrobial compounds phenylacrylic acids through decarbox‐ylation of these compounds. Each Pad1 monomer consists of a typical Rossmann fold containing a non–covalently bound molecule of FMN. The fold of Pad1 is similar to MrsD, an enzyme associated with lantibiotic synthesis; EpiD, a peptidyl‐cysteine decarboxylase; and AtHAL3a, the enzyme, which decarboxylates 4′‐phosphopantothenoylcysteine to 4′‐phosphopantetheine during coenzyme A biosynthesis, all with a similar location of the FMN binding site at the interface between two monomers, yet each having little sequence similarity to one another. All of these proteins associate into oligomers, with a trimer forming the common structural unit in each case. In MrsD and EpiD, which belong to the homo‐dodecameric flavin‐containing cysteine decarboxylase (HFCD) family, these trimers associate further into dodecamers. Pad1 also forms dodecamers, although the association of the trimers is completely different, resulting in exposure of a different side of the trimer unit to the solvent. This exposure affects the location of the substrate binding site and, specifically, its access to the FMN cofactor. Therefore, Pad1 forms a separate family, distinguishable from the HFCD family.

Structure-function analysis of Escherichia coli MnmG (GidA), a highly conserved tRNA-modifying enzyme.

The MnmE-MnmG complex is involved in tRNA modification. We have determined the crystal structure of Escherichia coli MnmG at 2.4-A resolution, mutated highly conserved residues with putative roles in flavin adenine dinucleotide (FAD) or tRNA binding and MnmE interaction, and analyzed the effects of these mutations in vivo and in vitro. Limited trypsinolysis of MnmG suggests significant conformational changes upon FAD binding.

Structure of Hydrogenase Maturation Protein HypF with Reaction Intermediates Shows Two Active Sites

[NiFe]-hydrogenases are multimeric proteins. Thexa0large subunit contains the NiFe(CN)(2)CO bimetallic active center and the small subunit contains Fe-S clusters. Biosynthesis and assembly of the NiFe(CN)(2)CO active center requires six Hyp accessory proteins. The synthesis of the CN(-) ligands isxa0catalyzed by the combined actions of HypF andxa0HypE using carbamoylphosphate as a substrate.xa0We report the structure of Escherichia coli HypF(92-750) lacking the N-terminal acylphosphatase domain. HypF(92-750) comprises the novel Zn-finger domain, the nucleotide-binding YrdC-like domain, and the Kae1-like universal domain, also binding a nucleotide and a Zn(2+) ion. The two nucleotide-binding sites are sequestered in an internal cavity, facing each other and separated by ∼14xa0Å. The YrdC-like domain converts carbamoyl moiety to a carbamoyl adenylate intermediate, which is channeled to the Kae1-like domain. Mutations within either nucleotide-binding site compromise hydrogenase maturation but do not affect the carbamoylphosphate phosphatase activity.

Crystal Structure of a Trimeric Form of Dephosphocoenzyme A Kinase from Escherichia coli

Coenzyme A (CoA) is an essential cofactor used in a wide variety of biochemical pathways. The final step in the biosynthesis of CoA is catalyzed by dephosphocoenzyme A kinase (DPCK, E.C. 2.7.1.24). Here we report the crystal structure of DPCK from Escherichia coli at 1.8 Å resolution. This enzyme forms a tightly packed trimer in its crystal state, in contrast to its observed monomeric structure in solution and to the monomeric, homologous DPCK structure from Haemophilus influenzae. We have confirmed the existence of the trimeric form of the enzyme in solution using gel filtration chromatography measurements. Dephospho‐CoA kinase is structurally similar to many nucleoside kinases and other P‐loop‐containing nucleotide triphosphate hydrolases, despite having negligible sequence similarity to these enzymes. Each monomer consists of five parallel β‐strands flanked by α‐helices, with an ATP‐binding site formed by a P‐loop motif. Orthologs of the E. coli DPCK sequence exist in a wide range of organisms, including humans. Multiple alignment of orthologous DPCK sequences reveals a set of highly conserved residues in the vicinity of the nucleotide/CoA binding site.

Crystallization and preliminary X-ray diffraction studies of human procathepsin L.

Human procathepsin L has been expressed in the yeast Pichia pastoris and its inactive (Cys25Ser) and unglycosylated (Thr110Ala) mutant purified, concentrated to 4 mg/ml, and crystallized by vapor diffusion against solution containing 1.4 M (Na, K)PO4 buffer, pH 7.8. Crystal size was Increased by multiple macroseeding. The crystals are orthorhombic, of space group P212121, with cell dimensions of a = 40.2 Å, b = 88.4 Å, and c = 94.9 Å. A 2.2 Å native data set was collected using synchrotron radiation. Although molecular replacement solution for the mature portion of the enzyme was easily found, the resulting maps could not be interpreted in the proregion. Heavy‐atom derivative search is in progress.

Multiple crystal forms of lipases from Geotrichum candidum

Multiple stable crystal forms of two lipases from the fungus Geotrichum candidum have been obtained. The diffraction pattern extends to beyond 2.0 A resolution. Similarity of the cell dimensions of various forms suggested similar packing of molecules in different crystals. This was confirmed by rotation function results. Four heavy-atoms derivatives have been identified.

Molecules of Escherichia coli MobB assemble into densely packed hollow cylinders in a crystal lattice with 75% solvent content

The crystal structure of Escherichia coli MobB, an enzyme involved in the final step of molybdenum-cofactor biosynthesis, forms intertwined dimers. Each molecule consists of two segments and requires the second monomer for stable folding. Dimerization buries a quarter of the solvent-accessible area of the monomer. These dimers assemble into a hexagonal lattice with P6(4)22 symmetry and occupy only approximately 25% of the unit-cell volume. The symmetry-related dimers associate tightly into a helical structure with a diameter of 250 A and a pitch of 98 A. Two such helices are intertwined, shifted by 49 A along the sixfold axis. Within the crystal, these helices form thin-walled cylinders with an external diameter of 250 A and an internal diameter of 190 A. Their center is filled with solvent. These cylinders pack closely together, forming a hexagonal lattice with the highest possible packing density. This arrangement of dimers allows extensive intermolecular contacts with 75% solvent content in the crystal.