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I Rayment

Publications and source records attributed to I Rayment.

At least 37 records · Page 2Linked to original sources
PubMed1999

The structure of carbamoyl phosphate synthetase determined to 2.1 A resolution.

Carbamoyl phosphate synthetase catalyzes the formation of carbamoyl phosphate from one molecule of bicarbonate, two molecules of Mg2+ATP and one molecule of glutamine or ammonia depending upon the particular form of the enzyme under investigation. As isolated from Escherichia coli, the enzyme is an alpha,beta-heterodimer consisting of a small subunit that hydrolyzes glutamine and a large subunit that catalyzes the two required phosphorylation events. Here the three-dimensional structure of carbamoyl phosphate synthetase from E. coli refined to 2.1 A resolution with an R factor of 17.9% is described. The small subunit is distinctly bilobal with a catalytic triad (Cys269, His353 and Glu355) situated between the two structural domains. As observed in those enzymes belonging to the alpha/beta-hydrolase family, the active-site nucleophile, Cys269, is perched at the top of a tight turn. The large subunit consists of four structural units: the carboxyphosphate synthetic component, the oligomerization domain, the carbamoyl phosphate synthetic component and the allosteric domain. Both the carboxyphosphate and carbamoyl phosphate synthetic components bind Mn2+ADP. In the carboxyphosphate synthetic component, the two observed Mn2+ ions are both octahedrally coordinated by oxygen-containing ligands and are bridged by the carboxylate side chain of Glu299. Glu215 plays a key allosteric role by coordinating to the physiologically important potassium ion and hydrogen bonding to the ribose hydroxyl groups of ADP. In the carbamoyl phosphate synthetic component, the single observed Mn2+ ion is also octahedrally coordinated by oxygen-containing ligands and Glu761 plays a similar role to that of Glu215. The carboxyphosphate and carbamoyl phosphate synthetic components, while topologically equivalent, are structurally different, as would be expected in light of their separate biochemical functions.

Adenosine Triphosphate↗
PubMed1999

Channel gate! Tension, leak and disclosure.

The crystal structure of a bacterial MscL shows how this homopentameric channel protein is held tightly shut to prevent leakage whilst at rest. By inference, the structure also shows how a stretch force in the lipid bilayer causes the channel to open. We now have a concrete picture as to how a stimulus 'gates' an ion channel.

Amino Acid Sequence↗
PubMed1998

Evolution of enzymatic activities in the enolase superfamily: crystal structure of (D)-glucarate dehydratase from Pseudomonas putida.

The structure of (D)-glucarate dehydratase from Pseudomonas putida (GlucD) has been solved at 2.3 A resolution by multiple isomorphous replacement and refined to a final R-factor of 19.0%. The protein crystallizes in the space group I222 with one subunit in the asymmetric unit. The unit cell dimensions are a = 69.6 A, b = 108.8 A, and c = 122.6 A. The crystals were grown using the batch method where the primary precipitant was poly(ethylene glycol) 1000. The structure reveals that GlucD is a tetramer of four identical polypeptides, each containing 451 residues. The structure was determined without a bound substrate or substrate analogue. Three disordered regions are noted: the N-terminus through residue 11, a loop containing residues 99 through 110, and the C-terminus from residue 423. On the basis of primary sequence alignments, we previously concluded that GlucD is a member of the mandelate racemase (MR) subfamily of the enolase superfamily [Babbitt, P. C., Hasson, M. S., Wedekind, J. E., Palmer, D. R. J., Barrett, W. C., Reed, G. J., Rayment, I., Ringe, D., Kenyon, G. L., and Gerlt, J. A. (1996) Biochemistry 35, 16489-16501]. This prediction is now verified, since the overall fold of GlucD is strikingly similar to those of MR, muconate lactonizing enzyme I, and enolase. Also, many of the active site residues of GlucD can be superimposed on those found in the active site of MR. The implications of this structure on the evolution of catalysis in the enolase superfamily are discussed.

Amino Acid Sequence↗
PubMed1998

Three-dimensional structure of adenosylcobinamide kinase/adenosylcobinamide phosphate guanylyltransferase from Salmonella typhimurium determined to 2.3 A resolution,.

The X-ray structure of adenosylcobinamide kinase/adenosylcobinamide phosphate guanylyltransferase (CobU) from Salmonella typhimurium has been determined to 2.3 A resolution. This enzyme of subunit molecular weight 19 770 plays a central role in the assembly of the nucleotide loop for adenosylcobalamin where it catalyzes both the phosphorylation of the 1-amino-2-propanol side chain of the corrin ring and the subsequent attachment of GMP to form the product adenosylcobinamide-GDP. The kinase activity is believed to be associated with a P-loop motif, whereas the transferase activity proceeds at a different site on the enzyme via a guanylyl intermediate. The enzyme was crystallized in the space group C2221 with unit cell dimensions of a = 96.4 A, b = 114.4 A, and c = 106.7 A, with three subunits per asymmetric unit. The structure reveals that the enzyme is a molecular trimer and appears somewhat like a propeller with overall molecular dimensions of approximately 64 A x 77 A x 131 A. Each subunit consists of a single domain that is dominated by a seven-stranded mixed beta-sheet flanked on either side by a total of five alpha-helices and one helical turn. Six of the seven beta-strands run parallel. The C-terminal strand lies at the edge of the sheet and runs antiparallel to the others. Interestingly, CobU displays a remarkable structural and topological similarity to the central domain of the RecA protein, although the reason for this observation is unclear. The structure contains a P-loop motif located at the base of a prominent cleft formed by the association of two subunits and is most likely the kinase active site. Each subunit of CobU contains a cis peptide bond between Glu80 and Cys81 where Glu80 faces the P-loop and might serve to coordinate the magnesium ion of the triphosphate substrate. Interestingly, His46, which is the putative site for guanylylation, lies approximately 21 A from the P-loop and is solvent-exposed. This suggests that the enzyme undergoes a conformational change when the substrates bind to bring these two active sites into closer proximity.

Adenosine Triphosphate↗
PubMed1998

Structure of the bis(Mg2+)-ATP-oxalate complex of the rabbit muscle pyruvate kinase at 2.1 A resolution: ATP binding over a barrel.

Pyruvate kinase from rabbit muscle has been cocrystallized as a complex with MgIIATP, oxalate, Mg2+, and either K+ or Na+. Crystals with either Na+ or K+ belong to the space group P2(1)2(1)2(1), and the asymmetric units contain two tetramers. The structures were solved by molecular replacement and refined to 2.1 (K+) and 2.35 A (Na+) resolution. The structures of the Na+ and K+ complexes are virtually isomorphous. Each of the eight subunits within the asymmetric unit contains MgIIoxalate as a bidentate complex linked to the protein through coordination of Mg2+ to the carboxylates of Glu 271 and Asp 295. Six of the subunits also contain an alpha,beta,gamma-tridentate complex of MgIIATP, and the active-site cleft, located between domains A and B, is closed in these subunits. In the remaining two subunits MgIIATP is missing, and the active-site cleft is open. Closure of the active-site cleft in the fully liganded subunits includes a rotation of 41 degrees of the B domain relative to the A domain. alpha-Carbons of residues in the B domain undergo movements of up to 17.8 A (Lys 124) in the cleft closure. Lys 206, Arg 119, and Asp 177 from the B domain move several angstroms from their positions in the open conformation to contact the MgIIATP complex in the active site. The gamma-phosphate of ATP coordinates to both magnesium ions and to the monovalent cation, K+ or Na+. A Mg2+-coordinated oxygen from the MgIIoxalate complex lies 3.0 A from Pgamma of ATP, and this oxygen is positioned for an in-line attack on the phosphorus. The side chains of Lys 269 and Arg 119 are positioned to provide leaving-group activation in the forward and reverse directions. There is no obvious candidate for the acid/base catalyst near the 2-si face of the prospective enolate of the normal substrate. A functional group linked through solvent and side-chain hydroxyls may function in a proton relay.

Adenosine Triphosphate↗
PubMed1998

X-ray crystal structure of the yeast Kar3 motor domain complexed with Mg.ADP to 2.3 A resolution.

The kinesin family of motor proteins, which contain a conserved motor domain of approximately 350 amino acids, generate movement against microtubules. Over 90 members of this family have been identified, including motors that move toward the minus or plus end of microtubules. The Kar3 protein from Saccharomyces cerevisiae is a minus end-directed kinesin family member that is involved in both nuclear fusion, or karyogamy, and mitosis. The Kar3 protein is 729 residues in length with the motor domain located in the C-terminal 347 residues. Recently, the three-dimensional structures of two kinesin family members have been reported. These structures include the motor domains of the plus end-directed kinesin heavy chain [Kull, F. J., et al. (1996) Nature 380, 550-555] and the minus end-directed Ncd [Sablin, E. P., et al. (1996) Nature 380, 555-559]. We now report the structure of the Kar3 protein complexed with Mg.ADP obtained from crystallographic data to 2.3 A. The structure is similar to those of the earlier kinesin family members, but shows differences as well, most notably in the length of helix alpha 4, a helix which is believed to be involved in conformational changes during the hydrolysis cycle.

Adenosine Diphosphate↗
PubMed1997

X-ray crystal structure and solution fluorescence characterization of Mg.2'(3')-O-(N-methylanthraniloyl) nucleotides bound to the Dictyostelium discoideum myosin motor domain.

Mant (2'(3')-O-(N-methylanthraniloyl)) labeled nucleotides have proven to be useful tools in the study of the kinetic mechanism of the myosin ATPase by fluorescence spectroscopy. The sensitivity of the mant fluorophore to its local environment also makes it suitable to investigate the exposure of bound nucleotides to solvent from collisional quenching measurements. Here we present the crystal structure of mant-ADP and beryllium fluoride complexed with Dictyostelium discoideum myosin motor domain (S1dC) at 1.9 A resolution. We complement the structural approach with an investigation of the accessibility of the mant moiety to solvent using acrylamide quenching of fluorescence emission. In contrast to rabbit skeletal myosin subfragment 1, where the mant group is protected from acrylamide (Ksv=0.2 M-1), the fluorophore is relatively exposed when bound to Dictyostelium myosin motor domain (Ksv= 1.4 M-1). Differences between the Dictyostelium structure and that of vertebrate skeletal subfragment 1, in the region of the nucleotide binding pocket, are proposed as an explanation for the differences observed in the solvent accessibility of complexed mant-nucleotides. We conclude that protection of the mant group from acrylamide quenching does not report on overall closure of the nucleotide binding pocket but reflects more local structural changes.

Acrylamide↗
PubMed1997

X-ray structures of the MgADP, MgATPgammaS, and MgAMPPNP complexes of the Dictyostelium discoideum myosin motor domain.

The three-dimensional structures of the truncated myosin head from Dictyostelium discoideum myosin II (S1dC) complexed with MgAMPPNP, MgATPgammaS, and MgADP are reported at 2.1, 1.9, and 2.1 A resolution, respectively. Crystals were obtained by cocrystallization and were isomorphous with respect to those of S1dC. MgADP.BeFx [Fisher, A. J., et al. (1995) Biochemistry 34, 8960-8972]. In all three structures, the electron density for the entire nucleotide was clearly discernible. The overall structures of all three complexes are very similar to that of the beryllium fluoride complex which suggests that the differences in the physiological effects of ATPgammaS and AMPPNP are due to the changes in the equilibrium between the actin-bound and actin-free states of myosin caused by the lower affinity of AMPPNP for myosin. In S1dC.MgAMPPNP, the presence of the bridging nitrogen prompts the side chain of Asn233 to rotate which disrupts the hydrogen bonding pattern in the nucleotide binding pocket and alters the water structure surrounding the ribose hydroxyl groups. It appears that this change is responsible for the reduced affinity of AMPPNP for myosin relative to ATPgammaS. In contrast to the G-proteins, there is no major change in the conformation of the ligands that coordinate the nucleotide in S1dC.MgADP. This is due to three water molecules that adopt the approximate positions of the three oxygens on the gamma-phosphate and maintain the interactions with the Mg2+ ion and protein molecule. Interestingly, the thiophosphate group is evident in S1dC.MgATPgammaS even though it is slowly hydrolyzed by myosin. This suggests that the conformation observed here and in chicken skeletal myosin subfragment-1 [Rayment, I., et al. (1993) Science 261, 50-58] is unable to hydrolyze ATP and represents the structure of the prehydrolysis weak binding state of myosin.

Adenosine Diphosphate↗
PubMed1997

Ligand-induced domain movement in pyruvate kinase: structure of the enzyme from rabbit muscle with Mg2+, K+, and L-phospholactate at 2.7 A resolution.

The structure of rabbit muscle pyruvate kinase crystallized as a complex with Mg2+, K+, and L-phospholactate (L-P-lactate) has been solved and refined to 2.7 A resolution. The crystals, grown from solutions of polyethylene glycol 8000 at pH 7.5, belong to the space group P2(1) and have unit cell parameters a = 144.4 A, b = 112.6 A, c = 171.2 A, and beta = 93.7 degrees. The asymmetric unit contains two tetramers. The crystal structure reveals that the eight subunits within the asymmetric unit adopt several different conformations. These conformations are characterized by differences in the relative positions of protein domains A and B, resulting in different degrees of closure of the active site cleft that occupies the interface between these two domains. The global conformational differences may be described as rotations of the B domain with respect to the (beta/alpha)8-barrel of the A domain. Carbon atoms of the backbone in domain B rotate >20 degrees from the most open to the most closed subunit. The different conformations among subunits within the asymmetric unit are accompanied by 3-3.8 A shifts in the position of Mg2+ and a significant change in the orientation of the phenyl ring of Phe 243. In all of the subunits, Mg2+ coordinates to the protein through the carboxylate side chains of Glu 271 and Asp 295. In the subunit having the most closed conformation, Mg2+ also coordinates to the carboxylate oxygen, the bridging ester oxygen, and a nonbridging phosphoryl oxygen of L-P-lactate. Mg2+ to L-P-lactate coordination is missing in subunits exhibiting a more open conformation. K+ coordinates to four protein ligands and to a phosphoryl oxygen of the L-P-lactate. The position and liganding of K+ are unaffected by the different conformations of the subunits. The side chain of Arg 72, Mg2+, and K+ provides a locus of positive charge for the phosphate moiety of the analog in the closed subunit.

Animals↗
PubMed1997

Structure of carbamoyl phosphate synthetase: a journey of 96 A from substrate to product.

Carbamoyl phosphate synthetase catalyzes the production of carbamoyl phosphate from bicarbonate, glutamine, and two molecules of MgATP. As isolated from Escherichia coli, the enzyme has a total molecular weight of approximately 160K and consists of two polypeptide chains referred to as the large and small subunits. Here we describe the X-ray crystal structure of this enzyme determined to 2.8 A resolution in the presence of ADP, Mn2+, phosphate, and ornithine. The small subunit is distinctly bilobal with the active site residues located in the interface formed by the NH2- and COOH-terminal domains. Interestingly, the structure of the COOH-terminal half is similar to that observed in the trpG-type amidotransferase family. The large subunit can be envisioned as two halves referred to as the carboxyphosphate and carbamoyl phosphate synthetic components. Each component contains four distinct domains. Strikingly, the two halves of the large subunit are related by a nearly exact 2-fold rotational axis, thus suggesting that this polypeptide chain evolved from a homodimeric precursor. The molecular motifs of the first three domains observed in each synthetic component are similar to those observed in biotin carboxylase. A linear distance of approximately 80 A separates the binding sites for the hydrolysis of glutamine in the small subunit and the ATP-dependent phosphorylations of bicarbonate and carbamate in the large subunit. The reactive and unstable enzyme intermediates must therefore be sequentially channeled from one active site to the next through the interior of the protein.

Allosteric Site↗
PubMed1997

Structural analysis of the H166G site-directed mutant of galactose-1-phosphate uridylyltransferase complexed with either UDP-glucose or UDP-galactose: detailed description of the nucleotide sugar binding site.

Galactose-1-phosphate uridylyltransferase plays a key role in galactose metabolism by catalyzing the transfer of a uridine 5'-phosphoryl group from UDP-glucose to galactose 1-phosphate. The enzyme from Escherichia coli is composed of two identical subunits. The structures of the enzyme/UDP-glucose and UDP-galactose complexes, in which the catalytic nucleophile His 166 has been replaced with a glycine residue, have been determined and refined to 1.8 A resolution by single crystal X-ray diffraction analysis. Crystals employed in the investigation belonged to the space group P2(1) with unit cell dimensions of a = 68 A, b = 58 A, c = 189 A, and beta = 100 degrees and two dimers in the asymmetric unit. The models for these enzyme/substrate complexes have demonstrated that the active site of the uridylyltransferase is formed by amino acid residues contributed from both subunits in the dimer. Those amino acid residues critically involved in sugar binding include Asn 153 and Gly 159 from the first subunit and Lys 311, Phe 312, Val 314, Tyr 316, Glu 317, and Gln 323 from the second subunit. The uridylyltransferase is able to accommodate both UDP-galactose and UDP-glucose substrates by simple movements of the side chains of Glu 317 and Gln 323 and by a change in the backbone dihedral angles of Val 314. The removal of the imidazole group at position 166 results in little structural perturbation of the polypeptide chain backbone when compared to the previously determined structure for the wild-type enzyme. Instead, the cavity created by the mutation is partially compensated for by the presence of a potassium ion and its accompanying coordination sphere. As such, the mutant protein structures presented here represent valid models for understanding substrate recognition and binding in the native galactose-1-phosphate uridylyltransferase.

Binding Sites↗
PubMed1997

Structural studies on myosin II: communication between distant protein domains.

Understanding how chemical energy is converted into directed movement is a fundamental problem in biology. In higher organisms this is accomplished through the hydrolysis of ATP by three families of motor proteins: myosin, dynein and kinesin. The most abundant of these is myosin, which operates against actin and plays a central role in muscle contraction. As summarized here, great progress has been made towards understanding the molecular basis of movement through the determination of the three-dimensional structures of myosin and actin and through the establishment of systems for site-directed mutagenesis of this motor protein. It now appears that the generation of movement is coupled to ATP hydrolysis by a series of domain movements within myosin.

Adenosine Triphosphate↗
PubMed1997

Structure of the beta 2 homodimer of bacterial luciferase from Vibrio harveyi: X-ray analysis of a kinetic protein folding trap.

Luciferase, as isolated from Vibrio harveyi, is an alpha beta heterodimer. When allowed to fold in the absence of the alpha subunit, either in vitro or in vivo, the beta subunit of enzyme will form a kinetically stable homodimer that does not unfold even after prolonged incubation in 5 M urea at pH 7.0 and 18 degrees C. This form of the beta subunit, arising via kinetic partitioning on the folding pathway, appears to constitute a kinetically trapped alternative to the heterodimeric enzyme (Sinclair JF, Ziegler MM, Baldwin TO. 1994. Kinetic partitioning during protein folding yields multiple native states. Nature Struct Biol 1: 320-326). Here we describe the X-ray crystal structure of the beta 2 homodimer of luciferase from V. harveyi determined and refined at 1.95 A resolution. Crystals employed in the investigational belonged to the orthorhombic space group P2(1)2(1)2(1) with unit cell dimensions of a = 58.8 A, b = 62.0 A, and c = 218.2 A and contained one dimer per asymmetric unit. Like that observed in the functional luciferase alpha beta heterodimer, the major tertiary structural motif of each beta subunit consists of an (alpha/beta)8 barrel (Fisher AJ, Raushel FM, Baldwin TO, Rayment I. 1995. Three-dimensional structure of bacterial luciferase from Vibrio harveyi at 2.4 A resolution. Biochemistry 34: 6581-6586). The root-mean-square deviation of the alpha-carbon coordinates between the beta subunits of the hetero- and homodimers is 0.7 A. This high resolution X-ray analysis demonstrated that "domain" or "loop" swapping has not occurred upon formation of the beta 2 homodimer and thus the stability of the beta 2 species to denaturation cannot be explained in such simple terms. In fact, the subunit:subunit interfaces observed in both the beta 2 homodimer and alpha beta heterodimer are remarkably similar in hydrogen-bonding patterns and buried surface areas.

Biopolymers↗
PubMed1996

The enolase superfamily: a general strategy for enzyme-catalyzed abstraction of the alpha-protons of carboxylic acids.

We have discovered a superfamily of enzymes related by their ability to catalyze the abstraction of the alpha-proton of a carboxylic acid to form an enolic intermediate. Although each reaction catalyzed by these enzymes is initiated by this common step, their overall reactions (including racemization, beta-elimination of water, beta-elimination of ammonia, and cycloisomerization) as well as the stereochemical consequences (syn vs anti) of the beta-elimination reactions are diverse. Analysis of sequence and structural similarities among these proteins suggests that all of their chemical reactions are mediated by a common active site architecture modified through evolution to allow the enolic intermediates to partition to different products in their respective active sites via different overall mechanisms. All of these enzymes retain the ability to catalyze the thermodynamically difficult step of proton abstraction. These homologous proteins, designated the "enolase superfamily", include enolase as well as more metabolically specialized enzymes: mandelate racemase, galactonate dehydratase, glucarate dehydratase, muconate-lactonizing enzymes, N-acylamino acid racemase, beta-methylaspartate ammonia-lyase, and o-succinylbenzoate synthase. Comparative analysis of structure-function relationships within the superfamily suggests that carboxyphosphonoenolpyruvate synthase, another member of the superfamily, does not catalyze the reaction proposed in the literature but catalyzes an enolase-like reaction instead. The established and deduced structure-function relationships in the superfamily allow the prediction that other apparent members of the family for which no catalytic functions have yet been assigned will also perform chemistry involving abstraction of the alpha-protons of carboxylic acids.

Amino Acid Sequence↗
PubMed1996

Molecular structure of a proteolytic fragment of TLP20.

Myosin light-chain kinase is responsible for the phosphorylation of myosin in smooth muscle cells. In some tissue types, the C-terminal portion of this large enzyme is expressed as an independent protein and has been given the name telokin. Recently, an antibody directed against telokin was found to interact with a protein derived from the baculovirus Autographa californica nuclear polyhedrosis virus. This protein was biochemically characterized and given the name TLP20 for telokin-like protein of 20 000 molecular weight. The amino-acid sequence of TLP20 was determined on the basis of a cDNA clone and subsequent alignment searches failed to reveal any homology to telokin or to other known proteins. The three-dimensional structure of a proteolytic portion of TLP20 is reported here. Crystals employed in the investigation were grown from ammonium sulfate solutions at pH 6.0 and belonged to the space group P2(1)3 with unit-cell dimensions of a = b = c = 76.3 A and one molecule per asymmetric unit. The structure was determined by multiple isomorphous replacement with three heavy-atom derivatives. Least-squares refinement of the model reduced the crystallographic R factor to 18.1% for all measured X-ray data from 30.0 to 2.2 A. The overall fold of the molecule may be described as a seven-stranded antiparallel beta-barrel flanked on the bottom by two additional beta-strands and on the top by an alpha-helix. Quite surprisingly, the three-dimensional structure of this beta-barrel is not similar to telokin or to any other known protein.

Journal Article↗
PubMed1996

The structure of nucleotidylated histidine-166 of galactose-1-phosphate uridylyltransferase provides insight into phosphoryl group transfer.

Galactose-1-phosphate uridylyltransferase catalyzes the reaction of UDP-glucose with galactose 1-phosphate to form UDP-galactose and glucose 1-phosphate during normal cellular metabolism. The reaction proceeds through a double displacement mechanism characterized by the formation of a stable nucleotidylated histidine intermediate. This paper describes the preparation of the uridylyl-enzyme complex on the crystalline enzyme from Escherichia coli and its subsequent structure determination by X-ray crystallography. The refined structure has an R-factor of 19.6% (data between 65 and 1.86 A resolution) and reveals modest conformational changes at the active site compared to the inactive UMP/UDP-enzyme complex reported previously [Wedekind, J.E., Frey, P.A., & Rayment, I. (1995) Biochemistry 34, 11049-11061]. In particular, positions of the respective UMP alpha-phosphoryl groups differ by approximately 4 A. Well-defined electron density for the nucleotidylated imidazole supports the existence of a covalent bond between N epsilon 2 of the nucleophile and the alpha-phosphorus of UMP. A hydrogen bond that is conserved in both complexes between His 166 N delta 1 and the carbonyl O of His 164 serves to properly orient the nucleophile and electrostatically stabilize the positively charged imidazolium that results from nucleotidylation. Hydrogen bonds from side-chain Gln 168 to the nonbridging phosphoryl oxygens of the nucleotidyl intermediate appear crucial for the formation and reaction of the uridylyl-enzyme complex as well. The significance of the latter interaction is underscored by the fact that the predominant cause of the metabolic disease galactosemia is the mutation of the corresponding Gln (Gln 188 in humans) to Arg. A comparison to other phosphohistidyl enzymes is described, as well as a revised model for the mechanism of the uridylyltransferase.

Amino Acid Sequence↗
PubMed1996

The 1.5-A resolution crystal structure of bacterial luciferase in low salt conditions.

Bacterial luciferase is a flavin monooxygenase that catalyzes the oxidation of a long-chain aldehyde and releases energy in the form of visible light. A new crystal form of luciferase cloned from Vibrio harveyi has been grown under low-salt concentrations, which diffract x-rays beyond 1.5-A resolution. The x-ray structure of bacterial luciferase has been refined to a conventional R-factor of 18.2% for all recorded synchrotron data between 30.0 and 1.50-A resolution. Bacterial luciferase is an alpha-beta heterodimer, and the individual subunits fold into a single domain (beta/alpha)8 barrel. The high resolution structure reveals a non-prolyl cis peptide bond that forms between Ala74 and Ala75 in the alpha subunit near the putative active site. This cis peptide bond may have functional significance for creating a cavity at the active site. Bacterial luciferase employs reduced flavin as a substrate rather than a cofactor. The structure presented was determined in the absence of substrates. A comparison of the structural similarities between luciferase and a nonfluorescent flavoprotein, which is expressed in the lux operon of one genus of bioluminescent bacteria, suggests that the two proteins originated from a common ancestor. However, the flavin binding sites of the nonfluorescent protein are likely not representative of the flavin binding site on luciferase. The structure presented here will furnish a detailed molecular model for all bacterial luciferases.

Amino Acid Sequence↗