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Research has been conducted on adenosine 5'-phosphosulfate kinase from the filamentous fungus Penicillium chrysogenum. The crystallization of this kinase has been carried out in the presence of ADP and adenosine 5'-phosphosulfate in investigating the structural basis of the substrate inhibition and the residues involved in substrate binding and the details are reported.

Research has been conducted on the dialkylglycine decarboxylase. The kinetics of dialkylglycine decarboxylase inhibition by aminophosphonate inhibitors has been investigated and the X-ray crystal structures of the inhibitors and of the dialkylglycine decarboxylase have been determined and are described.

Research has been conducted on the enzyme ATP sulfurylase producing ATP and inorganic sulfate from adenylyl sulfate and inorganic pyrophosphate. The crystal structure of the ATP sulfurylase from the giant tubeworm Riftia pachyptila is discussed.

Research has been conducted on the ATP sulfurylase from Penicillium chrysogenum. The ATP sufurylase structure, the active site region, and the functions of its conserved residues have been determined.

Research has been conducted on the adenosine 5'-phosphosulfate (APS) kinase. The crystal structure of the filamentous fungus's ligand-free APS kinase is investigated.

Structural analysis of Dictyostelium discoideum Mg.ADP complexes of motor segment of myosin II in the presence of Beryllium and Aluminium between 2.0-2.6 angstrong resolution suggests that MgADP.AlF4(super-) complex is similar to the transition state of hydrolysis and the MgADP.BeF(sub x) complex is analogous to the ATP bound state. The tertiary structure of truncated myosin head is identical to the three-dimensional structure of chicken skeletal myosin. The narrow cleft breaks the 5 kDa segment and mediates conformational changes during power stroke.

The first molecular model of a bioluminiscent enzyme was formulated, using a combination of multiple isomorphous replacement and molecular averaging between the two heterodimers in the asymmetric unit. Luciferase from all bacterial species analyzed so far is comprised of two subunits, alpha and beta. Both subunits fold into a single-domain eight-stranded beta/alpha barrel motif and assemble around a parallel four-helix bundle centered on a pseudo twofold axis that relates the structurally similar alpha and beta subunits.

The high-resolution crystal structures of substrate-loaded Asp105Asn and His88Gln mutants of Synechocystis sp. were examined to gain better insight into the structural stabilization and overall catalytic mechanism related to interconversion of biliverdin IX[alpha] to phycocyanobilin. The findings for protonation state of histidine residues proposed a new mechanistic scheme for phycocyanobilin:ferredoxin oxidoreductase (PcyA)-mediated reduction of both vinyl groups of biliverdin wherein an axial water molecule, which prematurely binds and ejects from both mutants upon one electron reduction, is required for catalytic turnover of the semireduced state.

A high-field electron paramagnetic resonance (EPR) spectroscopy of frozen solutions and single crystals of the one-electron reduced protein-substrate complex of two PcyA mutants D105N from the cyanobacteria Synechocystis sp. PCC6803 and Nostoc sp. PCC7120 is presented. The results obtained provide insight into protonation states of the biliverdin radical which have the carbonyl oxygen atoms on both the A and the D pyrrole rings.

The crystal structure of the yeast PAPS reductase binary complex with product PAP bound is described. The structure has shown that the complete intact C-terminal tail bound over the active site pocket and has provided additional clues to the catalytic mechanism.

Binary x-ray crystal structures of a multifunctional Pasteurella multocida sialyltransferase with a donor analogue are determined. This represents the first sialyltransferase structure and the first GT-B-type glycosyltransferase structure that is bound to both a donor analogue and an acceptor simultaneously.

A combination of the X-ray crystallography technique and several biochemical measurements is employed to study the radical mechanism of phycocyanobilin (PCB)-ferredoxin oxidoreductase (PcyA) from the cyanobacterium Nostoc. The analysis of the homology model provides a new insight into the catalytic mechanisms of various other ferredoxin-dependent reductase families, which are commonly found in oxygenic photosynthetic organisms.

The roles of the active site residues V241,E211, and 150 in the GABA-AT mechanism have been probed by site-directed mutagenesis. The structure of GABA-AT is similar in overall fold and active site structure to that of dialkylglycine decarboxylase, which catalyzes both transamination and decarboxylation half-reactions in its normal catalytic cycle.

The x-ray crystal structures of Escherichia coil gamma-aminobutyrate aminotransferase unbound and bound to the inhibitor aminooxyacetate are described. The binding of aminooxyacetate causes remarkably small changes in the active site structure with no large domain movements and the active site structure comparisons with pig gamma-aminobutyrate aminotransferase and dialkylglycine decarboxylase are discussed.

The kinetic characteristics of the individual sulfurylase and kinase activities of 3'-phosphoadenosine 5'-phosphosulfate synthetase (PAPSS1) and the assumption of adenosine 5'-phosphosulfate (ATP) channeling are examined. Isoform 1 of human bifunctional PAPS synthetase was expressed in Escherichia coli and purified to near-homogeneity, and the component reactions were kinetically characterized.

A time-dependent wave packet method is used to study the associative desorption of H2(nu) on a graphite (0001) surface via an Eley-Rideal mechanism. The presence of other (nonreactive) hydrogen atoms does not have an effect on the formation probabilities of H2(nu), especially at low translational energies of the incoming H atom and at resonances.

The crystal structures of antibody 2D12.5 Fab bound to an yttrium-DOTA analogue and separately to a gadolinium-DOTA analogue is reported. The binding interactions between the antibody and metal-DOTA complex are indirectly affected by changing the metal, because there are no direct protein-metal interactions.

An antibody that binds rare earth complexes selectively is used as a docking station for a set of probe molecules, of particular interest for medical imaging and therapy. The monoclonal antibody 2D12.5, developed against the DOTA analogue Y-BAD conjugated to the immunogenic protein KLH is examined.

Time-dependent quantum mechanical calculations are performed on the surface-catalyzed formation of H2 on a graphite(0001) surface via the Eley-Rideal mechanism. The 3D calculations reveal that less vibrational excitation for the product molecules than in calculations that used only two degree of freedom.

The 1.8 Å resolution crystal structures of Escherichia coli aspartate aminotransferase reconstituted with 1-deazapyridoxal 5′-phosphate (deazaPLP; 2-formyl-3-hydroxy-4-methylbenzyl phosphate) in the internal aldimine and l-aspartate external aldimine forms are reported. The 햫-aspartate∙deazaPLP external aldimine is extraordinarily stable (half-life of >20 days), allowing crystals of this intermediate to be grown by cocrystallization with l-aspartate. This structure is compared to that of the α-methyl-햫-aspartate∙PLP external aldimine. Overlays with the corresponding pyridoxal 5′-phosphate (PLP) aldimines show very similar orientations of deazaPLP with respect to PLP. The lack of a hydrogen bond between Asp222 and deazaPLP, which serves to "anchor" PLP in the active site, releases strain in the deazaPLP internal aldimine that is enforced in the PLP internal aldimine [Hayashi, H., Mizuguchi, H., Miyahara, I., Islam, M. M., Ikushiro, H., Nakajima, Y., Hirotsu, K., and Kagamiyama, H. (2003) Biochim. Biophys. Acta1647, 103] as evidenced by the planarity of the pyridine ring and the Schiff base linkage with Lys258. Additionally, loss of this anchor causes a 10° greater tilt of deazaPLP toward the substrate in the external aldimine. An important mechanistic difference between the 햫-aspartate∙deazaPLP and α-methyl-l-aspartate∙PLP external aldimines is a hydrogen bond between Gly38 and Lys258 in the former, positioning the catalytic base above and approximately equidistant between Cα and C4′. In contrast, in the α-methyl-햫-aspartate∙PLP external aldimine, the ϵ-amino group of Lys258 is rotated ∼70° to form a hydrogen bond to Tyr70 because of the steric bulk of the methyl group. [ABSTRACT FROM AUTHOR]

The cyanobacterial enzyme phycocyanobilin :ferredoxin oxidoreductase (PcyA) catalyzes the two- step four-electron reduction of biliverdin IXα to phycocyanobilin, the precursor of biliprotein chromophores found in phycobilisonies. It is known that catalysis proceeds via paramagnetic radical intermediates, but the structure of these intermediates and the transfer pathways for the four protons involved are not known. In this study, high-field electron paramagnetic resonance (EPR) spectroscopy of frozen solutions and single crystals of the one-electron reduced protein-substrate complex of two PcyA mutants D1O5N from the cyanobacteria Synechocystis sp. PCC6803 and Nostoc sp. PCC71 20 are examined. Detailed analysis of Synechocystis D105N mutant spectra at 130 and 406 GHz reveals a biliverdin radical with a very narrow g tensor with principal values 2.00359(5), 2.00341(5), and 2.00218(5). Using density-functional theory (DFT) computations to explore the possible protonation states of the biliverdin radical, it is shown that this g tensor is consistent with a biliverdin radical where the carbonyl oxygen atoms on both the A and the D pyrrole rings are protonated. This experimentally confirms the reaction mechanism recently proposed (Tu, et al. Biochemistry 2007, 46, 1484). [ABSTRACT FROM AUTHOR]

The G-protein-coupled receptor BT-R 1 in the moth Manduca sexta represents a class of single-membrane-spanning α-helical proteins within the cadherin family that regulate intercellular adhesion and contribute to important signaling activities that control cellular homeostasis. The Cry1A toxins, Cry1Aa, Cry1Ab, and Cry1Ac, produced by Bacillus thuringiensis bind BT-R 1 very tightly ( K d = 1.1 nM) and trigger a Mg 2+ -dependent signaling pathway that involves the stimulation of G-protein α-subunit, which subsequently launches a coordinated signaling cascade, resulting in insect death. The three Cry1A toxins compete for the same binding site on BT-R 1 , and the pattern of inhibition of insecticidal activity against M. sexta is strikingly similar for all three toxins. The binding domain is localized in the 12th cadherin repeat (EC12: Asp1349 to Arg1460, 1349 DR 1460 ) in BT-R 1 and to various truncation fragments derived therefrom. Fine mapping of EC12 revealed that the smallest fragment capable of binding is a highly conserved 94-amino acid polypeptide bounded by Ile1363 and Ser1456 ( 1363 IS 1456 ), designated as the toxin-binding site (TBS). Logistical regression analysis revealed that binding of an EC12 truncation fragment containing the TBS is antagonistic to each of the Cry1A toxins and completely inhibits the insecticidal activity of all three. Elucidation of the EC12 motif of the TBS by X-ray crystallography at a 1.9 Å resolution combined with results of competitive binding analyses, live cell experiments, and whole insect bioassays substantiate the exclusive involvement of BT-R 1 in initiating insect cell death and demonstrate that the natural receptor BT-R 1 contains a single TBS.

Adenosine Deaminases Acting on RNA (ADARs) convert adenosine to inosine in double stranded RNA. Human ADARs can be directed to predetermined target sites in the transcriptome by complementary guide strands, allowing for the correction of disease-causing mutations at the RNA level. Here we use structural information available for ADAR2-RNA complexes to guide the design of nucleoside analogs for the position in the guide strand that contacts a conserved glutamic acid residue in ADARs (E488 in human ADAR2), which flips the adenosine into the ADAR active site for deamination. Mutating this residue to glutamine (E488Q) results in higher activity because of the hydrogen bond donating ability of Q488 to N3 of the orphan cytidine on the guide strand. We describe the evaluation of cytidine analogs for this position that stabilize an activated conformation of the enzyme-RNA complex and increase catalytic rate for deamination by the wild-type enzyme. A new crystal structure of ADAR2 bound to duplex RNA bearing a cytidine analog revealed a close contact between E488, stabilized by an additional hydrogen bond and altered charge distribution when compared to cytidine. In human cells and mouse primary liver fibroblasts, this single nucleotide modification increased directed editing yields when compared to an otherwise identical guide oligonucleotide. Our results show that modification of the guide RNA can mimic the effect of hyperactive mutants and advance the approach of recruiting endogenous ADARs for site-directed RNA editing.

Calmodulin (CaM) regulates the activity of a Ca 2+ channel known as the cardiac ryanodine receptor (RyR2), which facilitates the release of Ca 2+ from the sarcoplasmic reticulum during excitation-contraction coupling in cardiomyocytes. Mutations that disrupt this CaM-dependent channel inactivation result in cardiac arrhythmias. RyR2 contains three different CaM binding sites: CaMBD1 (residues 1940-1965), CaMBD2 (residues 3580-3611), and CaMBD3 (residues 4246-4275). Here, we report a crystal structure of Ca 2+ -bound CaM bound to RyR2 CaMBD3. The structure reveals Ca 2+ bound to the four EF-hands of CaM as well as a fifth Ca 2+ bound to CaM in the interdomain linker region involving Asp81 and Glu85. The CaM mutant E85A abolishes the binding of the fifth Ca 2+ and weakens the binding of CaMBD3 to Ca 2+ -bound CaM. Thus, the binding of the fifth Ca 2+ is important for stabilizing the complex in solution and is not a crystalline artifact. The CaMBD3 peptide in the complex adopts an α-helix (between Phe4246 and Val4271) that interacts with both lobes of CaM. Hydrophobic residues in the CaMBD3 helix (Leu4255 and Leu4259) form intermolecular contacts with the CaM N-lobe, and the CaMBD3 mutations (L4255A and L4259A) each weaken the binding of CaM to RyR2. Aromatic residues on the opposite side of the CaMBD3 helix (Phe4246 and Tyr4250) interact with the CaM C-lobe, but the mutants (F4246A and Y4250A) have no detectable effect on CaM binding in solution. We suggest that the binding of CaM to CaMBD3 and the binding of a fifth Ca 2+ to CaM may contribute to the regulation of RyR2 channel function.

Cytidine 5'-monophosphate (CMP)-sialic acid synthetase (CSS) is an essential enzyme involved in the biosynthesis of carbohydrates and glycoconjugates containing sialic acids, a class of α-keto acids that are generally terminal key recognition residues by many proteins that play important biological and pathological roles. The CSS from Neisseria meningitidis (NmCSS) has been commonly used with other enzymes such as sialic acid aldolase and/or sialyltransferase in synthesizing a diverse array of compounds containing sialic acid or its naturally occurring and non-natural derivatives. To better understand its catalytic mechanism and substrate promiscuity, four NmCSS crystal structures trapped at various stages of the catalytic cycle with bound substrates, substrate analogues, and products have been obtained and are presented here. These structures suggest a mechanism for an "open" and "closed" conformational transition that occurs as sialic acid binds to the NmCSS/cytidine-5'-triphosphate (CTP) complex. The closed conformation positions critical residues to help facilitate the nucleophilic attack of sialic acid C2-OH to the α-phosphate of CTP, which is also aided by two observed divalent cations. Product formation drives the active site opening, promoting the release of products.

The lack of α2-6-linkage specific sialidases limits the structural and functional studies of sialic-acid-containing molecules. Photobacterium damselae α2-6-sialyltransferase (Pd2,6ST) was shown previously to have α2-6-specific, but weak, sialidase activity. Here, we develop a high-throughput blue-white colony screening method to identify Pd2,6ST mutants with improved α2-6-sialidase activity from mutant libraries generated by sequential saturation mutagenesis. A triple mutant (Pd2,6ST S232L/T356S/W361F) has been identified with 100-fold improved activity, high α2-6-sialyl linkage selectivity, and ability to cleave two common sialic acid forms, N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). It is a valuable tool for sialoglycan structural analysis and functional characterization. The sequential saturation mutagenesis and screening strategy developed here can be explored to evolve other linkage-specific neoglycosidases from the corresponding glycosyltransferases.

Ribonucleoside analogues bearing terminal alkynes, including 7-ethynyl-8-aza-7-deazaadenosine (7-EAA), are useful for RNA modification applications. However, although alkyne- and triazole-bearing ribonucleosides are in widespread use, very little information is available on the impact of these modifications on RNA structure. By solving crystal structures for RNA duplexes containing these analogues, we show that, like adenosine, 7-EAA and a triazole derived from 7-EAA base pair with uridine and are well-accommodated within an A-form helix. We show that copper-catalyzed azide/alkyne cycloaddition (CuAAC) reactions with 7-EAA are sensitive to the RNA secondary structure context, with single-stranded sites reacting faster than duplex sites. 7-EAA and its triazole products are recognized in RNA template strands as adenosine by avian myoblastosis virus reverse transcriptase. In addition, 7-EAA in RNA is a substrate for an active site mutant of the RNA editing adenosine deaminase, ADAR2. These studies extend our understanding of the impact of these novel nucleobase analogues and set the stage for their use in probing RNA structure and metabolism.

N-Acetylneuraminate lyases (NALs) or sialic acid aldolases catalyze the reversible aldol cleavage of N-acetylneuraminic acid (Neu5Ac, the most common form of sialic acid) to form pyruvate and N-acetyl-d-mannosamine. Although equilibrium favors sialic acid cleavage, these enzymes can be used for high-yield chemoenzymatic synthesis of structurally diverse sialic acids in the presence of excess pyruvate. Engineering these enzymes to synthesize structurally modified natural sialic acids and their non-natural derivatives holds promise in creating novel therapeutic agents. Atomic-resolution structures of these enzymes will greatly assist in guiding mutagenic and modeling studies to engineer enzymes with altered substrate specificity. We report here the crystal structures of wild-type Pasteurella multocida N-acetylneuraminate lyase and its K164A mutant. Like other bacterial lyases, it assembles into a homotetramer with each monomer folding into a classic (β/α)₈ TIM barrel. Two wild-type structures were determined, in the absence of substrates, and trapped in a Schiff base intermediate between Lys164 and pyruvate, respectively. Three structures of the K164A variant were determined: one in the absence of substrates and two binary complexes with N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). Both sialic acids bind to the active site in the open-chain ketone form of the monosaccharide. The structures reveal that every hydroxyl group of the linear sugars makes hydrogen bond interactions with the enzyme, and the residues that determine specificity were identified. Additionally, the structures provide some clues for explaining the natural discrimination of sialic acid substrates between the P. multocida and Escherichia coli NALs.

Nanostructure and molecular orientation play a crucial role in determining the functionality of organic thin films. In practical devices, such as organic solar cells consisting of donor-acceptor mixtures, crystallinity is poor and these qualities cannot be readily determined by conventional diffraction techniques, while common microscopy only reveals surface morphology. Using a simple nondestructive technique, namely, continuous-wave electron paramagnetic resonance spectroscopy, which exploits the well-understood angular dependence of the g-factor and hyperfine tensors, we show that in the solar cell blend of C(60) and copper phthalocyanine (CuPc)-for which X-ray diffraction gives no information-the CuPc, and by implication the C(60), molecules form nanoclusters, with the planes of the CuPc molecules oriented perpendicular to the film surface. This information demonstrates that the current nanostructure in CuPc:C(60) solar cells is far from optimal and suggests that their efficiency could be considerably increased by alternative film growth algorithms.

Glycosyltransferases are important catalysts for enzymatic and chemoenzymatic synthesis of complex carbohydrates and glycoconjugates. The glycosylation efficiencies of wild-type glycosyltransferases vary considerably when different acceptor substrates are used. Using a multifunctional Pasteurella multocida sialyltransferase 1 (PmST1) as an example, we show here that the sugar nucleotide donor hydrolysis activity of glycosyltransferases contributes significantly to the low yield of glycosylation when a poor acceptor substrate is used. With a protein crystal structure-based rational design, we generated a single mutant (PmST1 M144D) with decreased donor hydrolysis activity without significantly affecting its α2-3-sialylation activity when a poor fucose-containing acceptor substrate was used. The single mutant also has a drastically decreased α2-3-sialidase activity. X-ray and NMR structural studies revealed that unlike the wild-type PmST1, which changes to a closed conformation once a donor binds, the M144D mutant structure adopts an open conformation even in the presence of the donor substrate. The PmST1 M144D mutant with decreased donor hydrolysis and reduced sialidase activity has been used as a powerful catalyst for efficient chemoenzymatic synthesis of complex sialyl Lewis(x) antigens containing different sialic acid forms. This work sheds new light on the effect of donor hydrolysis activity of glycosyltransferases on glycosyltransferase-catalyzed reactions and provides a novel strategy to improve glycosyltransferase substrate promiscuity by decreasing its donor hydrolysis activity.

The development of molecular nanostructures plays a major role in emerging organic electronic applications, as it leads to improved performance and is compatible with our increasing need for miniaturization. In particular, nanowires have been obtained from solution or vapor phase and have displayed high conductivity or large interfacial areas in solar cells. In all cases however, the crystal structure remains as in films or bulk, and the exploitation of wires requires extensive postgrowth manipulation as their orientations are random. Here we report copper phthalocyanine (CuPc) nanowires with diameters of 10-100 nm, high directionality, and unprecedented aspect ratios. We demonstrate that they adopt a new crystal phase, designated eta-CuPc, where the molecules stack along the long axis. The resulting high electronic overlap along the centimeter length stacks achieved in our wires mediates antiferromagnetic couplings and broadens the optical absorption spectrum. The ability to fabricate ultralong, flexible metal phthalocyanine nanowires opens new possibilities for applications of these simple molecules.

Most assimilatory bacteria, fungi, and plants species reduce sulfate (in the activated form of APS or PAPS) to produce reduced sulfur. In yeast, PAPS reductase reduces PAPS to sulfite and PAP. Despite the difference in substrate specificity and catalytic cofactor, PAPS reductase is homologous to APS reductase in both sequence and structure, and they are suggested to share the same catalytic mechanism. Metazoans do not possess the sulfate reduction pathway, which makes APS/PAPS reductases potential drug targets for human pathogens. Here, we present the 2.05 A resolution crystal structure of the yeast PAPS reductase binary complex with product PAP bound. The N-terminal region mediates dimeric interactions resulting in a unique homodimer assembly not seen in previous APS/PAPS reductase structures. The "pyrophosphate-binding" sequence (47)TTAFGLTG(54) defines the substrate 3'-phosphate binding pocket. In yeast, Gly54 replaces a conserved aspartate found in APS reductases vacating space and charge to accommodate the 3'-phosphate of PAPS, thus regulating substrate specificity. Also, for the first time, the complete C-terminal catalytic motif (244)ECGIH(248) is revealed in the active site. The catalytic residue Cys245 is ideally positioned for an in-line attack on the beta-sulfate of PAPS. In addition, the side chain of His248 is only 4.2 A from the Sgamma of Cys245 and may serve as a catalytic base to deprotonate the active site cysteine. A hydrophobic sequence (252)RFAQFL(257) at the end of the C-terminus may provide anchoring interactions preventing the tail from swinging away from the active site as seen in other APS/PAPS reductases.

Sialyltransferases are key enzymes involved in the biosynthesis of biologically and pathologically important sialic acid-containing molecules in nature. Binary X-ray crystal structures of a multifunctional Pasteurella multocida sialyltransferase (Delta24PmST1) with a donor analogue CMP-3F(a)Neu5Ac or CMP-3F(e)Neu5Ac were determined at 2.0 and 1.9 A resolutions, respectively. Ternary X-ray structures of the protein in complex with CMP or a donor analogue CMP-3F(a)Neu5Ac and an acceptor lactose have been determined at 2.0 and 2.27 A resolutions, respectively. This represents the first sialyltransferase structure and the first GT-B-type glycosyltransferase structure that is bound to both a donor analogue and an acceptor simultaneously. The four structures presented here reveal that binding of the nucleotide-activated donor sugar causes a buried tryptophan to flip out of the protein core to interact with the donor sugar and helps define the acceptor sugar binding site. Additionally, key amino acid residues involved in the catalysis have been identified. Structural and kinetic data support a direct displacement mechanism involving an oxocarbenium ion-like transition state assisted with Asp141 serving as a general base to activate the acceptor hydroxyl group.

The X-ray crystal structure of the substrate-free form of phycocyanobilin (PCB)-ferredoxin oxidoreductase (PcyA; EC 1.3.7.5) from the cyanobacterium Nostoc sp. PCC7120 has been solved at 2.5 A resolution. A comparative analysis of this structure with those recently reported for substrate-bound and substrate-free forms of PcyA from the cyanobacterium Synechocystis sp. PCC6803 (Hagiwara et al. (2006) Proc. Natl. Acad. Sci. U.S.A. 103, 27-32; Hagiwara et al. (2006) FEBS Lett. 580, 3823-3828) provides a compelling picture of substrate-induced changes in the PcyA enzyme and the chemical basis of PcyA's catalytic activity. On the basis of these structures and the biochemical analysis of site-directed mutants of Nostoc PcyA, including mutants reported in recent studies (Tu et al. (2006) J. Biol. Chem. 281, 3127-3136) as well as mutants described in this study, a revised mechanism for the PcyA-mediated four-electron reduction of biliverdin IXalpha to 3E/3Z-phycocyanobilin via enzyme-bound bilin radical intermediates is proposed. The mechanistic insight of these studies, along with homology modeling, have provided new insight into the catalytic mechanisms of other members of the ferredoxin-dependent bilin reductase family that are widespread in oxygenic photosynthetic organisms.

Sialyltransferases catalyze reactions that transfer a sialic acid from CMP-sialic acid to an acceptor (a structure terminated with galactose, N-acetylgalactosamine, or sialic acid). They are key enzymes that catalyze the synthesis of sialic acid-containing oligosaccharides, polysaccharides, and glycoconjugates that play pivotal roles in many critical physiological and pathological processes. The structures of a truncated multifunctional Pasteurella multocida sialyltransferase (Delta24PmST1), in the absence and presence of CMP, have been determined by X-ray crystallography at 1.65 and 2.0 A resolutions, respectively. The Delta24PmST1 exists as a monomer in solution and in crystals. Different from the reported crystal structure of a bifunctional sialyltransferase CstII that has only one Rossmann domain, the overall structure of the Delta24PmST1 consists of two separate Rossmann nucleotide-binding domains. The Delta24PmST1 structure, thus, represents the first sialyltransferase structure that belongs to the glycosyltransferase-B (GT-B) structural group. Unlike all other known GT-B structures, however, there is no C-terminal extension that interacts with the N-terminal domain in the Delta24PmST1 structure. The CMP binding site is located in the deep cleft between the two Rossmann domains. Nevertheless, the CMP only forms interactions with residues in the C-terminal domain. The binding of CMP to the protein causes a large closure movement of the N-terminal Rossmann domain toward the C-terminal nucleotide-binding domain. Ser 143 of the N-terminal domain moves up to hydrogen-bond to Tyr 388 of the C-terminal domain. Both Ser 143 and Tyr 388 form hydrogen bonds to a water molecule, which in turn hydrogen-bonds to the terminal phosphate oxygen of CMP. These interactions may trigger the closure between the two domains. Additionally, a short helix near the active site seen in the apo structure becomes disordered upon binding to CMP. This helix may swing down upon binding to donor CMP-sialic acid to form the binding pocket for an acceptor.

The X-ray crystal structures of Escherichia coli gamma-aminobutyrate aminotransferase unbound and bound to the inhibitor aminooxyacetate are reported. The enzyme crystallizes from ammonium sulfate solutions in the P3(2)21 space group with a tetramer in the asymmetric unit. Diffraction data were collected to 2.4 A resolution for the unliganded enzyme and 1.9 A resolution for the aminooxyacetate complex. The overall structure of the enzyme is similar to those of other aminotransferase subgroup II enzymes. The ability of gamma-aminobutyrate aminotransferase to act on primary amine substrates (gamma-aminobutyrate) in the first half-reaction and alpha-amino acids in the second is proposed to be enabled by the presence of Glu211, whose side chain carboxylate alternates between interactions with Arg398 in the primary amine half-reaction and an alternative binding site in the alpha-amino acid half-reaction, in which Arg398 binds the substrate alpha-carboxylate. The specificity for a carboxylate group on the substrate side chain is due primarily to the presence of Arg141, but also requires substantial local main chain rearrangements relative to the structurally homologous enzyme dialkylglycine decarboxylase, which is specific for small alkyl side chains. No iron-sulfur cluster is found in the bacterial enzyme as was found in the pig enzyme [Storici, P., De Biase, D., Bossa, F., Bruno, S., Mozzarelli, A., Peneff, C., Silverman, R. B., and Schirmer, T. (2004) J. Biol. Chem. 279, 363-73.]. The binding of aminooxyacetate causes remarkably small changes in the active site structure, and no large domain movements are observed. Active site structure comparisons with pig gamma-aminobutyrate aminotransferase and dialkylglycine decarboxylase are discussed.