Edward Hough

The first crystal structure of a phospholipase D

BACKGROUND The phospholipase D (PLD) superfamily includes enzymes that are involved in phospholipid metabolism, nucleases, toxins and virus envelope proteins of unknown function. PLD hydrolyzes the terminal phosphodiester bond of phospholipids to phosphatidic acid and a hydrophilic constituent. Phosphatidic acid is a compound that is heavily involved in signal transduction. PLD also catalyses a transphosphatidylation reaction in the presence of phosphatidylcholine and a short-chained primary or secondary alcohol. RESULTS The first crystal structure of a 54 kDa PLD has been determined to 1.9 A resolution using the multiwavelength anomalous dispersion (MAD) method on a single WO(4) ion and refined to 1.4 A resolution. PLD from the bacterial source Streptomyces sp. strain PMF consists of a single polypeptide chain that is folded into two domains. An active site is located at the interface between these domains. The presented structure supports the proposed superfamily relationship with the published structure of the 16 kDa endonuclease from Salmonella typhimurium. CONCLUSIONS The structure of PLD provides insight into the structure and mode of action of not only bacterial, plant and mammalian PLDs, but also of a variety of enzymes as diverse as cardiolipin synthases, phosphatidylserine synthases, toxins, endonucleases, as well as poxvirus envelope proteins having a so far unknown function. The common features of these enzymes are that they can bind to a phosphodiester moiety, and that most of these enzymes are active as bi-lobed monomers or dimers.

The 1.9 A Crystal Structure of Heat-Labile Shrimp Alkaline Phosphatase

Alkaline phosphatases are non-specific phosphomonoesterases that are distributed widely in species ranging from bacteria to man. This study has concentrated on the tissue-nonspecific alkaline phosphatase from arctic shrimps (shrimp alkaline phosphatase, SAP). Originating from a cold-active species, SAP is thermolabile and is used widely in vitro, e.g. to dephosphorylate DNA or dNTPs, since it can be inactivated by a short rise in temperature. Since alkaline phosphatases are zinc-containing enzymes, a multiwavelength anomalous dispersion (MAD) experiment was performed on the zinc K edge, which led to the determination of the structure to a resolution of 1.9 A. Anomalous data clearly showed the presence of a zinc triad in the active site, whereas alkaline phosphatases usually contain two zinc and one magnesium ion per monomer. SAP shares the core, an extended beta-sheet flanked by alpha-helices, and a metal triad with the currently known alkaline phosphatase structures (Escherichia coli structures and a human placental structure). Although SAP lacks some features specific for the mammalian enzyme, their backbones are very similar and may therefore be typical for other higher organisms. Furthermore, SAP possesses a striking feature that the other structures lack: surface potential representations show that the enzymes net charge of -80 is distributed such that the surface is predominantly negatively charged, except for the positively charged active site. The negatively charged substrate must therefore be directed strongly towards the active site. It is generally accepted that optimization of the electrostatics is one of the characteristics related to cold-adaptation. SAP demonstrates this principle very clearly.

Crystal structure of the ternary complex of the catalytic domain of human phenylalanine hydroxylase with tetrahydrobiopterin and 3-(2-thienyl)-L-alanine, and its implications for the mechanism of catalysis and substrate activation.

Phenylalanine hydroxylase catalyzes the stereospecific hydroxylation of L-phenylalanine, the committed step in the degradation of this amino acid. We have solved the crystal structure of the ternary complex (hPheOH-Fe(II).BH(4).THA) of the catalytically active Fe(II) form of a truncated form (DeltaN1-102/DeltaC428-452) of human phenylalanine hydroxylase (hPheOH), using the catalytically active reduced cofactor 6(R)-L-erythro-5,6,7,8-tetrahydrobiopterin (BH(4)) and 3-(2-thienyl)-L-alanine (THA) as a substrate analogue. The analogue is bound in the second coordination sphere of the catalytic iron atom with the thiophene ring stacking against the imidazole group of His285 (average interplanar distance 3.8A) and with a network of hydrogen bonds and hydrophobic contacts. Binding of the analogue to the binary complex hPheOH-Fe(II).BH(4) triggers structural changes throughout the entire molecule, which adopts a slightly more compact structure. The largest change occurs in the loop region comprising residues 131-155, where the maximum r.m.s. displacement (9.6A) is at Tyr138. This loop is refolded, bringing the hydroxyl oxygen atom of Tyr138 18.5A closer to the iron atom and into the active site. The iron geometry is highly distorted square pyramidal, and Glu330 adopts a conformation different from that observed in the hPheOH-Fe(II).BH(4) structure, with bidentate iron coordination. BH(4) binds in the second coordination sphere of the catalytic iron atom, and is displaced 2.6A in the direction of Glu286 and the iron atom, relative to the hPheOH-Fe(II).BH(4) structure, thus changing its hydrogen bonding network. The active-site structure of the ternary complex gives new insight into the substrate specificity of the enzyme, notably the low affinity for L-tyrosine. Furthermore, the structure has implications both for the catalytic mechanism and the molecular basis for the activation of the full-length tetrameric enzyme by its substrate. The large conformational change, moving Tyr138 from a surface position into the active site, may reflect a possible functional role for this residue.

Protein Engineering the Surface of Enzymes

The protein surface is the interface through which a protein senses the external world. Its composition of charged, polar and hydrophobic residues is crucial for the stability and activity of the protein. The charge state of seven of the twenty naturally occurring amino acids is pH dependent. A total of 95% of all titratable residues are located on the surface of soluble proteins. In evolutionary related families of proteins such residues are particularly prone to substitutions, insertions and deletions. We present here an analysis of the residue composition of 4038 proteins, selected from 125 protein families with < 25% identity between core members of each family. Whereas only 16.8% of the residues were truly buried, 40.7% were > 30% exposed on the surface and the remainder were < 30% exposed. The individual residue types show distinct differences. The data presented provides an important new approach to protein engineering of protein surfaces. Guidelines for the optimization of solvent exposure for a given residue are given. The cutinase family of enzymes has been investigated. The stability of native cutinase has been studied as a function of pH, and has been compared with the cutinase activity towards tributyrin. Whereas the onset of enzymatic activity is linked with the deprotonation of the active site HIS188, destabilization of the 3D structure as determined by differential scanning calorimetry is coupled with the loss of activity at very basic pH values. A modeling investigation of the pH dependence of the electrostatic potentials reveals that the activity range is accompanied by the development of a highly significant negative potential in the active site cleft. The 3D structures of three mutants of the Fusarium solani pisi cutinase have been solved to high resolution using X-ray diffraction analysis. Preliminary X-ray data are presented.

Crystal structures of phosphate, iodide and iodate-inhibited phospholipase C from Bacillus cereus and structural investigations of the binding of reaction products and a substrate analogue

The crystal structure of the complex formed between phospholipase C (PLC) from Bacillus cereus and inorganic phosphate (Pi), which is an inhibitor, has been determined and refined to 2.1 A resolution. The final R-factor is 19.7%. We have also studied the binding of two other inhibitors, iodide and iodate, to PLC. X-ray data for these two complexes were collected to 2.8 A resolution during the search for heavy-atom derivatives. A series of screening experiments where PLC crystals have been treated with several reaction products and a substrate analogue were carried out to clarify the question of substrate binding. The results have so far been ambiguous but are discussed briefly. Phosphate and iodate are both found to bind to the three metal ions in the protein molecule, suggesting that these ions are involved directly in the catalytic process and thereby identifying the active site. PLC also binds nine iodide ions, eight of which are on the surface of the molecule and of lower occupancy. The ninth blocks the entrance to the active site cleft and is of higher occupancy. Altogether, these results suggest that the substrate, a phospholipid, is associated directly with the metal ions during catalysis.

Structure and proposed amino-acid sequence of a pepsin from atlantic cod (Gadus morhua).

The crystal structure of a pepsin from the gastric mucosa of Atlantic cod has been determined to 2.16 A resolution. Data were collected on orthorhombic crystals with cell dimensions a = 35.98, b = 75.40 and c = 108.10 A, on a FAST area-detector system. The phase problem was solved by the molecular-replacement method using porcine pepsin (PDB entry 5PEP) as a search model. The structure has been refined to a crystallographic R factor of 20.8% using all reflections between 8.0 and 2.16 A, without prior knowledge of the primary sequence. The resulting crystal structure is very similar to the porcine enzyme, consisting of two domains with predominantly beta-sheet structure in the same sequential positions as the enzyme from pig. In the course of the model building, 122 residues were substituted and two residues deleted from the starting model to give a polypeptide chain of 324 amino acids and a sequence identity of 57.7% with the pig pepsin. No carbohydrate residues were located. Sequence alignment with available aspartic proteinases, indicates that the fish enzyme seems to be more related to mammalian gastric pepsins than to the mammalian gastricsins and chymosins, lysosomal cathepsin Ds and a pepsin from tuna fish. The amino-acid composition of the cod enzyme, however, is more in accordance with the cathepsin Ds.

Crystallization and preliminary diffraction analysis of a truncated homodimer of human phenylalanine hydroxylase

A recombinant truncated form (Δ1‐102/Δ428‐452) of the non‐heme iron‐dependent metalloenzyme human phenylalanine hydroxylase (hPAH, phenylalanine 4‐monooxygenase; EC 1.14.16.1) was expressed in E. coli, purified to homogeneity as a homodimer (70 kDa) and crystallized using the hanging drop vapour diffusion method. The crystals are orthorhombic, space group C222 with cell dimensions of a=66.6 Å, b=108.4 Å, c=125.7 Å. The calculated packing parameter (V m) is 3.24 Å3/Da with four 2‐fold symmetric dimers (or eight momomers) in the unit cell. Data have been collected to 2.0 Å resolution.

The 2.1 A Structure of Aerococcus Viridans L-Lactate Oxidase (Lox).

The crystal structure of L-lactate oxidase (LOX) from Aerococcus viridans has been determined at 2.1 A resolution. LOX catalyzes the flavin mononucleotide (FMN) dependent oxidation of lactate to pyruvate and hydrogen peroxide. LOX belongs to the alpha-hydroxy-acid oxidase flavoenzyme family; members of which bind similar substrates and to some extent have conserved catalytic properties and structural motifs. LOX crystallized as two tightly packed tetramers in the asymmetric unit, each having fourfold symmetry. The present structure shows a conserved FMN coordination, but also reveals novel residues involved in substrate binding compared with other family members.

The Origin of Trypsin: Evidence for Multiple Gene Duplications in Trypsins

Abstract. The trypsin family of serine proteases is one of the most studied protein families, with a wealth of amino acid sequence information available in public databases. Since trypsin-like enzymes are widely distributed in living organisms in nature, likely evolutionary scenarios have been proposed. A novel methodology for Fourier transformation of biological sequences (FOTOBIS) is presented. The methodology is well suited for the identification of the size and extent of short repeats in protein sequences. In the present paper the trypsin family of enzymes is analyzed with FOTOBIS and strong evidence for tandem gene duplication is found. A likely evolutionary path for the development of present-day trypsins involved an intrinsic extensive tandem gene duplication of a small DNA fragment of 15–18 nucleotides, corresponding to five or six amino acids. This ancestral trypsin gene was subsequently duplicated, leading to the earliest version of a full-sized trypsin, from which the contemporary trypsins have developed.

Crystallization and preliminary X-ray crystallographic studies of benzamidine-inhibited trypsin from the north atlantic salmon (Salmo salar)

Crystals of benzamidine-inhibited trypsin from the North Atlantic salmon (Salmo salar) have been grown from ammonium sulphate solution at pH 5.0. Two crystal forms suitable for X-ray structure analysis, obtained from a hanging-drop experiment, have been characterized. Both belong to space-group P22(1)2(1) with cell dimensions a = 39.2 A, b = 62.4 A, c = 84.6 A and a = 31.4 A, b = 74.8 A, c = 83.5 A, for forms I and II, respectively. Intensity data to 1.82 A have been collected for crystal form I on a CAD4 diffractometer, and initial phases have been obtained by molecular replacement methods. The conventional R-factor after two rounds of model building and subsequent refinement is 0.25 for data between 6.0 and 2.0 A. So far no water molecules have been included in the model.