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

Kinesin-2 KIF3AC and KIF3AB Can Drive Long-Range Transport along Microtubules

AbstractMammalian KIF3AC is classified as a heterotrimeric kinesin-2 that is best known for organelle transport in neurons, yet in vitro studies to characterize its single molecule behavior are lacking. The results presented show that a KIF3AC motor that includes the native helix α7 sequence for coiled-coil formation is highly processive with run lengths of ∼1.23 μm and matching those exhibited by conventional kinesin-1. This result was unexpected because KIF3AC exhibits the canonical kinesin-2 neck-linker sequence that has been reported to be responsible for shorter run lengths observed for another heterotrimeric kinesin-2, KIF3AB. However, KIF3AB with its native neck linker and helix α7 is also highly processive with run lengths of ∼1.62 μm and exceeding those of KIF3AC and kinesin-1. Loop L11, a component of the microtubule-motor interface and implicated in activating ADP release upon microtubule collision, is significantly extended in KIF3C as compared with other kinesins. A KIF3AC encoding a truncation in KIF3C loop L11 (KIF3ACΔL11) exhibited longer run lengths at ∼1.55 μm than wild-type KIF3AC and were more similar to KIF3AB run lengths, suggesting that L11 also contributes to tuning motor processivity. The steady-state ATPase results show that shortening L11 does not alter kcat, consistent with the observation that single molecule velocities are not affected by this truncation. However, shortening loop L11 of KIF3C significantly increases the microtubule affinity of KIF3ACΔL11, revealing another structural and mechanistic property that can modulate processivity. The results presented provide new, to our knowledge, insights to understand structure-function relationships governing processivity and a better understanding of the potential of KIF3AC for long-distance transport in neurons

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

Threedimensional structure of myosin subfragment-1 from electron microscopy of sectioned crystals

Abstract. Image analysis of electron micrographs of thin-sectioned myosin subfragment-1 (Sl) crystals has been used to determine the structure of the myosin head at ti25-A resolution. Previous work established that the unit cell of type I crystals of myosin Sl contains eight molecules arranged with orthorhombic space group symmetry P2 12,2, and provided preliminary information on the size and shape of the myosin hea