Three-dimensional structure of galactose-1-phosphate uridyltransferase from Escherichia coli at 1.8 Å resolution

ABSTRACT: Galactose-1-phosphate uridylyltransferase catalyzes the reversible transfer of the uridine 5'-monophosphoryl moiety of UDP-glucose to the phosphate group of galactose 1-phosphate to form UDPgalactose. This enzyme participates in the Leloir pathway of galactose metabolism, and its absence is the primary cause of the potentially lethal disease galactosemia. The three-dimensional structure of the dimeric enzyme from Escherichia coli complexed with uridine 5'-diphosphate is reported here. The structure was solved by multi le isomorphous replacement and electron density modification techniques and has "half-barrel". The barrel staves are formed by nine strands of antiparallel P-sheet. The barrel axis is approximately parallel to the local dyad that relates each subunit. Two amphipathic helices fill the halfbarrel sequestering its hydrophobic interior. An iron atom resides on the outside of the barrel, centered in the subunit interface. Intrasubunit coordination to iron resembles a distorted square pyramid formed by the equatorial ligation of two histidines and a bidentate carboxylate group and a single axial histidine. The subunit interface is stabilized by this coordination and is further characterized by the formation of two intermolecular "mini-sheets" distinct from the strands of the half-barrel. Loops that connect the mini-sheet strands contribute to the formation of the active site, which resides on the external surface of the barrel rim. Loops of the barrel strands are tethered together by a structural zinc atom that orients the local fold in a manner essential for catalysis. In one of the latter loops, Sy of a cysteine is modified by P-mercaptoethanol, which prevents the a-phosphorus of the nucleotide from access to the nucleophile His166. This conformation does not appear to perturb the interactions to the uracil and ribose moieties as mediated through the side chains of Led4, Phe75, Am7', Asp78, Phe79, and Va11°8. Several of the latter residues have been implicated in human galactosemia. The present structure explains the deleterious effects of many of those mutations. been refined to 1.8 x resolution. Enzyme subunits consist of a single domain with the topology of a Galactose-1 -phosphate uridylyltransferase (hexose-1 -phosphate uridylyltransferase, EC 2.7.7.12) catalyzes the nucleotide exchange between UDP-hexoses and hexose 1-phosphates. This provides an essential balance of UDPglc' and UDP-gal for the cell. Such activated hexose sugars are consumed in the synthesis of disaccharides, glycoproteins, glycolipids, and glycoge

Phosphate coordination and movement of DNA in the Tn5 synaptic complex: role of the (R)YREK motif

Bacterial DNA transposition is an important model system for studying DNA recombination events such as HIV-1 DNA integration and RAG-1-mediated V(D)J recombination. This communication focuses on the role of protein–phosphate contacts in manipulating DNA structure as a requirement for transposition catalysis. In particular, the participation of the nontransferred strand (NTS) 5′ phosphate in Tn5 transposition strand transfer is analyzed. The 5′ phosphate plays no direct catalytic role, nonetheless its presence stimulates strand transfer ∼30-fold. X-ray crystallography indicates that transposase–DNA complexes formed with NTS 5′ phosphorylated DNA have two properties that contrast with structures formed with complexes lacking the 5′ phosphate or complexes generated from in-crystal hairpin cleavage. Transposase residues R210, Y319 and R322 of the (R)YREK motif coordinate the 5′ phosphate rather than the subterminal NTS phosphate, and the 5′ NTS end is moved away from the 3′ transferred strand end. Mutation R210A impairs the 5′ phosphate stimulation. It is posited that DNA phosphate coordination by R210, Y319 and R322 results in movement of the 5′ NTS DNA away from the 3′-end thus allowing efficient target DNA binding. It is likely that this role for the newly identified RYR triad is utilized by other transposase-related proteins

Small-Scale Batch Crystallization of Proteins Revisited An Underutilized Way to Grow Large Protein Crystals

AbstractGrowth of high-quality crystals is a major obstacle in many structural investigations. In recent years, the techniques for screening crystals have improved dramatically, whereas the methods for obtaining large crystals have progressed more slowly. This is an important issue since, although many structures can be solved from small crystals with synchrotron radiation, it is far easier to solve and refine structures when strong data is recorded from large crystals. In an effort to improve the size of crystals, a strategy for a small-scale batch method has been developed that in many cases yields far larger crystals than attainable by vapor diffusion

Kinesin and myosin: molecular motors with similar engines

AbstractStructure determination of the catalytic domains of two members of the kinesin superfamily reveals that this class of molecular motor exhibits the same architecture as myosin and suggests that these microtubule-  and actin-based motors arose from a common ancestor