The choline binding site of phospholipase C (Bacillus cereus): insights into substrate specificity

The phosphatidylcholine-preferring phospholipase C from Bacillus cereus (PLC(Bc)) is a 28.5 kDa enzyme with three zinc ions in its active site. The roles that a number of amino acid residues play as zinc ligands and in binding and catalysis have been elucidated. Recent mechanistic studies indicate that the rate of the reaction is limited by a proton-transfer step during chemical hydrolysis and not substrate binding or product release. An X-ray structure of PLC(Bc) complexed with a phosphonate inhibitor related to phosphatidylcholine revealed that the three amino acid residues Glu4, Tyr56, and Phe66 comprise the choline binding pocket. However, because the contributions that these three residues make to substrate recognition and specificity were unknown, a series of site-specific mutants for Glu4, Tyr56, and Phe66 were constructed by PCR mutagenesis. On the basis of a comparison of their respective CD spectra and melting temperatures, it appears that the mutants adopt folded structures in solution that are virtually identical to that of wild-type PLC(Bc). The kinetic parameters k(cat) and K(m) for the hydrolysis of the three soluble substrates 1, 2-dihexanoyl-sn-glycero-3-phosphocholine (C6PC), 1, 2-dihexanoyl-sn-glycero-3-phosphoethanolamine (C6PE), and 1, 2-dihexanoyl-sn-glycero-3-phospho-L-serine (C6PS) at concentrations below their corresponding critical micelle concentration (cmc) values were determined for each mutant. Replacement of Phe66 with a nonaromatic residue dramatically decreased k(cat) (approximately 200-fold) and reduced PLC(Bc) activity toward C6PC, C6PE, and C6PS, whereas changes to Glu4 and Tyr56 typically led to much more modest losses in catalytic efficiencies. Mutations of Glu4 had relatively little effect upon k(cat) and K(m) for C6PS, but they significantly influenced K(m) for C6PC and C6PE. Replacing Tyr56 with nonaromatic residues also affects catalytic efficiency, albeit to a much lesser degree than the corresponding changes at position 66. However, the presence of an aromatic residue at position 56 seems to confer some substrate selectivity for C6PC and C6PE, which bear a positive charge on the headgroup, relative to C6PS, which has no net charge on the headgroup; this increase in specificity arises largely from a reduced k(cat) for C6PS.     

Structural studies examining the substrate specificity profiles of PC-PLC(Bc) protein variants

The phosphatidylcholine preferring phospholipase C from Bacillus cereus (PC-PLC(Bc)) catalyzes the hydrolysis of phospholipids in the following order of preference: phosphatidylcholine (PC)>phosphatidylethanolamine (PE)>phosphatidylserine (PS). In previous work, mutagenic, kinetic, and crystallographic experiments suggested that varying the amino acids at the 4th, 56th, and 66th positions had a significant influence upon the substrate specificity profile of PC-PLC(Bc). Here, we report the crystal structures of the native form of several PC-PLC(Bc) variants that exhibited altered substrate specificities for PC, PE, and PS at maximum resolutions of 1.90-2.05 Angstrom. Comparing the structures of these variants to the structure of the wild-type enzyme reveals only minor differences with respect to the number and location of active site water molecules and the side chain conformations of residues at the 4th and 56th positions. These results suggest that subtle changes in steric and electronic properties in the substrate binding site of PC-PLC(Bc) are responsible for the significant changes in substrate selectivity.     

B. cereus phospholipase C engineering for efficient degumming of vegetable oil

Enzymatic phospholipid removal (degumming) is a fast-growing and environmentally friendly process for vegetable oil refining. Type C phospholipases (PLC) are the preferred enzymes since they provide an extra yield in the oil recovery.Bacillus cereus PLC can hydrolyze phosphatidylcholine (PC) but has a limited efficiency at removing phosphatidylethanolamine (PE), which together represent ∼70% of the phospholipids present in crude soybean oil. In the present work, we show that the B. cereus PLC mutant F66Y can remove up to 90% of PE while retaining its efficiency at hydrolyzing PC. Oil treatment with the engineered enzyme provides an extra yield of 1.84% making the B. cereus PLC F66Y mutant an attractive candidate for its industrial use.     

Determination of the substrate specificity of the phospholipase D from Streptomyces chromofuscus via an inorganic phosphate quantitation assay

The substrate specificity for phospholipase D from Streptomyces chromofuscus (PLD(Sc)) has been determined utilizing an assay based on the quantitation of inorganic phosphate. 1,2-Di-n-hexanoyl phosphatidylcholine (C6PC), phosphatidylethanolamine (C6PE), phosphatidylserine (C6PS), phosphatidylglycerol (C6PG), and an unnatural phospholipid bearing a neohexyl headgroup (C6PDB) were examined as substrates. The assay relies on the quenching of the PLD(Sc)-catalyzed hydrolysis of the phospholipid substrates with EDTA followed by the hydrolysis of the phosphatidic acid product with alkaline phosphatase. The inorganic phosphate thus released is quantitated through the formation of a complex with ammonium molybdate, which has an absorbance maximum at 700 nm. To minimize the time involved and the reagents consumed, the assay is conducted in 96-well plates. The results of this study indicate that the catalytic efficiency for PLD(Sc) on the substrates is C6PC > C6PS approximately C6PE > C6PG > C6PDB.     

Selective 31P(1H) nuclear Overhauser effect study on the polar headgroup conformation of phospholipids in micelles in organic solvents

The polar headgroup structure of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) in inverted micelles in chloroform or benzene was investigated by the selective 31P(H) nuclear Overhauser effect (NOE). In the frequency dependence of the 31P(1H) NOE, PC micelles in CDCl3 showed two maxima. The larger maximum was located at the resonance of the glycerol-CH2OP protons and the smaller at the resonance of the N-methyl protons. In PC/PE mixed micelles in C6D6, both PC and PE showed three maxima which were located at the resonance of the CH2OP protons, the N-methyl protons and the amino protons in the frequency dependence of the 31P-NOE. The N-methyl protons of PC and the amino protons of PE were closely spaced to the phosphate groups of neighboring lipid molecules. The polar headgroups of PC and PE in the mixed micelles were concluded to lie in the plane perpendicular to the molecular axes. The frequency dependence of the 31P(H) NOE for PS micelles in C6D6 showed the maxima at the resonances of the amino protons and the CH2OP protons. The polar headgroups of PS molecules were not extended parallel to the molecular axes in the inverted micelles.     

Differential interfacial and substrate binding modes of mammalian pancreatic phospholipases A2: a comparison among human, bovine, and porcine enzymes.

To identify the residues essential for interfacial binding and substrate binding of human pancreatic phospholipase A2 (hpPLA2), several ionic residues in the putative interfacial binding surface (R6E, K7E, K10E, and K116E) and substrate binding site (D53K and K56E) were mutated. Interfacial affinity of these mutants was measured using anionic polymerized liposomes, and their enzymatic activity was measured using various substrates including phospholipid monomers, zwitterionic and anionic micelles, and anionic polymerized mixed liposomes. Similar mutations (R6E, K10E, K56E, and K116E) were made to porcine pancreatic phospholipase A2 (ppPLA2), and the properties of mutants were measured by the same methods. Results indicate that hpPLA2 and ppPLA2 have similar interfacial binding mechanisms in which cationic residues in the amino terminus and Lys-116 in the carboxy terminus are involved in binding to anionic lipid surfaces. Small but definite differences between the two enzymes were observed in overall interfacial affinity and activity and the effects of the mutations on interfacial enzyme activity. The interfacial binding of hpPLA2 and ppPLA2 is distinct from that of bovine pancreatic phospholipase A2 in that Lys-56 is involved in the interfacial binding of the latter enzyme. The unique phospholipid headgroup specificity of hpPLA2 derives from the presence of Asp-53 in the substrate binding site. This residue appears to participate in stabilizing electrostatic interactions with the cationic ethanolamine headgroup, hence the phosphatidylethanolamine preference of hpPLA2. Taken together, these studies reveal the similarities and the differences in the mechanisms by which mammalian pancreatic phospholipases A2 interact with lipid aggregates and perform interfacial catalysis.     

Insights into the Functional Architecture of the Catalytic Center of a Maize β-Glucosidase Zm-p60.1

The maize (Zea mays) beta-glucosidase Zm-p60.1 has been implicated in regulation of plant development by the targeted release of free cytokinins from cytokinin-O-glucosides, their inactive storage forms. The crystal structure of the wild-type enzyme was solved at 2.05-A resolution, allowing molecular docking analysis to be conducted. This indicated that the enzyme specificity toward substrates with aryl aglycones is determined by aglycone aromatic system stacking with W373, and interactions with edges of F193, F200, and F461 located opposite W373 in a slot-like aglycone-binding site. These aglycone-active site interactions recently were hypothesized to determine substrate specificity in inactive enzyme substrate complexes of ZM-Glu1, an allozyme of Zm-p60.1. Here, we test this hypothesis by kinetic analysis of F193I/Y/W mutants. The decreased K(m) of all mutants confirmed the involvement of F193 in determining enzyme affinity toward substrates with an aromatic aglycone. It was unexpected that a 30-fold decrease in k(cat) was found in F193I mutant compared with the wild type. Kinetic analysis and computer modeling demonstrated that the F193-aglycone-W373 interaction not only contributes to aglycone recognition as hypothesized previously but also codetermines catalytic rate by fixing the glucosidic bond in an orientation favorable for attack by the catalytic pair, E186 and E401. The catalytic pair, assigned initially by their location in the structure, was confirmed by kinetic analysis of E186D/Q and E401D/Q mutants. It was unexpected that the E401D as well as C205S and C211S mutations dramatically impaired the assembly of a catalysis-competent homodimer, suggesting novel links between the active site structure and dimer formation.     

Structural transitions in short-chain lipid assemblies studied by (31)P-NMR spectroscopy

The self-assembled supramolecular structures of diacylphosphatidylcholine (diC(n)PC), diacylphosphatidylethanolamine (diC(n)PE), diacylphosphatidyglycerol (diC(n)PG), and diacylphosphatidylserine (diC(n)PS) were investigated by (31)P nuclear magnetic resonance (NMR) spectroscopy as a function of the hydrophobic acyl chain length. Short-chain homologs of these lipids formed micelles, and longer-chain homologs formed bilayers. The shortest acyl chain lengths that supported bilayer structures depended on the headgroup of the lipids. They increased in the order PE (C(6)) < PC (C(9)) < or = PS (C(9) or C(10)) < PG (C(11) or C(12)). This order correlated with the effective headgroup area, which is a function of the physical size, charge, hydration, and hydrogen-bonding capacity of the four headgroups. Electrostatic screening of the headgroup charge with NaCl reduced the effective headgroup area of PS and PG and thereby decreased the micelle-to-bilayer transition of these lipid classes to shorter chain lengths. The experimentally determined supramolecular structures were compared to the assembly states predicted by packing constraints that were calculated from the hydrocarbon-chain volume and effective headgroup area of each lipid. The model accurately predicted the chain-length threshold for bilayer formation if the relative displacement of the acyl chains of the phospholipid were taken into account. The model also predicted cylindrical rather than spherical micelles for all four diacylphospholipid classes and the (31)P-NMR spectra provided evidence for a tubular network that appeared as an intermediate phase at the micelle-to-bilayer transition. The free energy of micellization per methylene group was independent of the structure of the supramolecular assembly, but was -0.95 kJ/mol (-0.23 kcal/mol) for the PGs compared to -2.5 kJ/mol (-0.60 kcal/mol) for the PCs. The integral membrane protein OmpA did not change the bilayer structure of thin (diC(10)PC) bilayers.     

A mutational analysis of the two motifs common to adenine methyltransferases.

All methyltransferases that use S-adenosyl methionine as the methyl group donor contain a sequence similar to (D/E/S)XFXGXG which has been postulated to form part of the cofactor binding site. In N6-adenine DNA methyltransferases there is a second motif, (D/N)PP(Y/F), which has been proposed to play a role similar to the catalytically essential PC motif conserved in all C5-cytosine DNA methyltransferases. We have made a series of amino acid changes in these two motifs in the EcoKI N6-adenine DNA methyltransferase. The mutant enzymes have been purified to homogeneity and characterized by physical biochemical methods. The first G is the most conserved residue in motif I. Changing this G to D completely abolished S-adenosyl methionine binding, but left enzyme structure and DNA target recognition unaltered, thus documenting the S-adenosyl methionine binding function of motif I in N6-adenine methyltransferases. Substitution of the N with D, or F with either G or C, in motif II abolished enzyme activity, but left S-adenosyl methionine and DNA binding unaltered. Changes of F to Y or W resulted in partial enzyme activity, implying that an aromatic residue is important for methylation. The substitution of W for F greatly enhanced UV-induced cross-linking between the enzyme and S-adenosyl methionine, suggesting that the aromatic residue is close in space to the methyl-group donor.     

Regulation of phospholipase C delta activity by sphingomyelin and sphingosine.

Phospholipase C delta (PLC delta) is strongly inhibited by sphingomyelin (SM). The inhibition occurs in both the presence and the absence of spermine, an activator of PLC delta. Phosphatidylethanolamine (PE), phosphatidylcholine (PC), phosphatidylserine (PS), and phosphatidylinositol (PI) also inhibit PLC delta in the presence of spermine but are much less effective than SM. PE and PC activate and PS and PI inhibit PLC delta in the absence of spermine. Again, the inhibition by PS and PI is much weaker than the inhibition observed with SM. Similar or identical effects are observed in detergent micelle and liposome assays. Comparisons of physiological concentrations of SM with concentrations yielding 50% inhibition of PLC delta in vitro indicate that SM is likely to be a major factor in regulating the activity of PLC delta by inhibition. It is proposed that, in vivo, sphingomyelin acts as an inhibitor of PLC delta, which enables the enzyme to be regulated by activation. In certain circumstances, there is a substantial decline in SM and this may lead to a partial relief of the inhibition. PLC delta is activated by sphingosine in the absence of spermine. However, this activation occurs at unphysiologically high concentrations of sphingosine. The effects of SM and sphingosine on PLC delta in marked contrast to those observed with protein kinase C, which is unaffected by sphingomyelin and inhibited by sphingosine.     

Human opioid peptide Met-enkephalin binds to anionic phosphatidylserine in high preference to zwitterionic phosphatidylcholine: natural-abundance 13C NMR study on the binding state in large unilamellar vesicles

A human opioid neuropeptide, Met-enkephalin (M-Enk: Tyr1-Gly2-Gly3-Phe4-Met5), having no net charge binds to anionic phosphatidylserine (PS) in high preference to zwitterionic phosphatidylcholine (PC). The binding mechanism in the PS and PC bilayers was studied on the basis of the inter- and intramolecular interaction data obtained by natural-abundance 13C nuclear magnetic resonance (NMR) of the peptide. Prominent upfield changes of the 13C resonance were observed in the C-terminal residue upon binding to PS, whereas no such marked change was observed upon binding to PC. The upfield chemical shift changes with their characteristic carbon site dependence are ascribed to the electrostatic binding between the peptide C-terminal CO2- and the PS headgroup NH3+. Despite the net negative charge of the PS bilayer surface, M-Enk thus anchors the negatively charged C-terminus. In the N-terminal residue, on the other hand, marked downfield chemical shift changes are observed upon binding to both the PS and PC bilayers, the magnitude of the changes being much larger in the PS system. The downfield changes with their characteristic carbon site dependence are ascribed to the electrostatic binding between the peptide N-terminal NH3+ and the lipid headgroup negative charge(s) (CO2- or PO4- in PS, PO4- in PC). Perturbation on the signal half-widths due to membrane binding also indicates the preferential and deeper binding of M-Enk on the PS membrane surface than on the PC membrane surface. Local charge cancellation takes place efficiently between M-Enk termini and the PS headgroups and compensates for the strong electrostatic hydration of the ionic groups. Distribution of the charged (positive and negative) and uncharged sites in the headgroups along the bilayer normal is responsible for the marked difference between PS and PC headgroups in controlling the binding state of the zwitterionic M-Enk.     

Human ferrochelatase: characterization of substrate-iron binding and proton-abstracting residues

The terminal step in heme biosynthesis, the insertion of ferrous iron into protoporphyrin IX to form protoheme, is catalyzed by the enzyme ferrochelatase (EC 4.99.1.1). A number of highly conserved residues identified from the crystal structure of human ferrochelatase as being in the active site were examined by site-directed mutagenesis. The mutants Y123F, Y165F, Y191H, and R164L each had an increased K(m) for iron without an altered K(m) for porphyrin. The double mutant R164L/Y165F had a 6-fold increased K(m) for iron and a 10-fold decreased V(max). The double mutant Y123F/Y191F had low activity with an elevated K(m) for iron, and Y123F/Y165F had no measurable activity. The mutants H263A/C/N, D340N, E343Q, E343H, and E343K had no measurable enzyme activity, while E343D, E347Q, and H341C had decreased V(max)s without significant alteration of the K(m)s for either substrate. D340E had near-normal kinetic parameters, while D383A and H231A had increased K(m)s for iron. On the basis of these data and the crystal structure of human ferrochelatase, it is proposed that residues E343, H341, and D340 form a conduit from H263 in the active site to the protein exterior and function in proton extraction from the porphyrin macrocycle. The role of H263 as the porphyrin proton-accepting residue is central to catalysis since metalation only occurs in conjunction with proton abstraction. It is suggested that iron is transported from the exterior of the enzyme at D383/H231 via residues W227 and Y191 to the site of metalation at residues R164 and Y165 which are on the opposite side of the active site pocket from H263. This model should be general for mitochondrial membrane-associated eucaryotic ferrochelatases but may differ for bacterial ferrochelatases since the spatial orientation of the enzyme within prokaryotic cells may differ.     

Thermodynamic interrogation of a folding disease. Mutant mapping of position 107 in human carbonic anhydrase II linked to marble brain disease

Marble brain disease (MBD) also known as Guibaud-Vainsel syndrome is caused by autosomal recessive mutations in the human carbonic anhydrase II (HCA II) gene. HCA II is a 259 amino acid single domain enzyme and is dominated by a 10-stranded beta-sheet. One mutation associated with MBD entails the H107Y substitution where H107 is a highly conserved residue in the carbonic anhydrase protein family. We have previously demonstrated that the H107Y mutation is a remarkably destabilizing folding mutation [Almstedt et al. (2004) J. Mol. Biol. 342, 619-633]. Here, the exceptional destabilization by the H107Y mutation has been further investigated. A mutational survey of position H107 and a neighboring conserved position E117 has been performed entailing the mutants H107A, H107F, H107N, E117A and the double mutants H107A/E117A and H107N/E117A. All mutants were severely destabilized versus GuHCl and heat denaturation. Thermal denaturation and GuHCl phase diagram and ANS analyses showed that the mutants shifted HCA II toward populating ensembles of intermediates of molten globule type under physiological conditions. The native state stability of the mutants was in the following order: wt > H107N > E117A > H107A > H107F > H107Y > H107N/E117A > H107A/E117A. In conclusion: (i) H107N is least destabilizing likely due to compensatory H-bonding ability of the introduced Asn residue. (ii) Double mutant cycles surprisingly reveal additive destabilization of H107N and E117A showing that H107 and E117 are independently stabilizing the folded protein. (iii) H107Y and H107F are exceptionally destabilizing due to bulkiness of the side chains whereas H107A is more accommodating, indicating long-range destabilizing effects of the natural pathogenic H107Y mutation.     

Serine utilization as a precursor of phosphatidylserine and alkenyl-(plasmenyl)-, alkyl-, and acylethanolamine phosphoglycerides in cultured glioma cells

In several tissues and cell lines, serine utilized for phosphatidylserine (PS) synthesis is an eventual precursor of the base moiety of ethanolamine phosphoglycerides (PE). We investigated the biosynthesis and decarboxylation of PS in cultured C6 glioma cells, with particular attention to 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine (plasmenylethanolamine) biosynthesis. Incorporation of [3H]serine into PS reached a maximum within 4-8 h, and label in nonplasmenylethanolamine phosphoglyceride (NP-PE) and plasmenylethanolamine was maximal by 12-24 h and 48 h, respectively. After 8 h, label in PS decreased even though 40-60% of initial label remained in the culture medium. Serial additions of fresh [3H]serine restored PS synthesis to higher levels of labeled PS accumulation followed by a subsequent decrease in 4-8 h. High performance liquid chromatographic analyses confirmed that medium serine was depleted by 8 h, and thereafter metabolites, including acetate and formate, accounted for radioactivity in the medium. The rapid but transient appearance of labeled glycine and ATP inside the cells indicated conversion of serine by hydroxymethyltransferase. 78-85% of label from serine was in headgroup of PS or of PE formed by decarboxylation. A precursor-product relationship was suggested for label from [3H]serine appearing in the headgroup of diacyl, alkylacyl, and alkenylacyl subclasses of PE. By 48 h, a constant specific activity, ratio of approximately 1:1 was reached between plasmenylethanolamine and NP-PE, similar to the molar distribution of these lipids. In contrast, equilibrium was not achieved in cells incubated with [1,2-14C]ethanolamine; plasmenylethanolamine had 2-fold greater specific activity than labeled NP-PE by 72-96 h. These observations indicate that in cultured glioma cells 1) serine serves as a precursor of the head group of PS and of both plasmenyl and non-plasmenyl species of PE; 2) exchange of headgroup between NP-PE and plasmenylethanolamine may involve different donor pools of PE depending on whether the headgroup originates with exogenous serine or ethanolamine; 3) serine is rapidly converted to other metabolites, which limits exogenous serine as a direct phospholipid precursor.     

Role of electrostatic interactions for the stability and folding behavior of cold shock protein

The impacts of three charged‐residue‐involved mutations, E46A, R3E, and R3E/L66E, on the thermostability and folding behavior of the cold shock protein from the themophile Bacillus caldolyticus (Bc‐Csp) were investigated by using a modified Gō‐like model, in which the nonspecific electrostatic interactions of charged residues were taken into account. Our simulation results show that the wild‐type Bc‐Csp and its three mutants are all two‐sate folders, which is consistent with the experimental observations. It is found that these three mutations all lead to a decrease of protein thermodynamical stability, and the effect of R3E mutation is the strongest. The lower stability of these three mutants is due to the increase of the enthalpy of the folded state and the entropy of the unfolded state. Using this model, we also studied the folding kinetics and the folding/unfolding pathway of the wild‐type Bc‐Csp as well as its three mutants and then discussed the effects of electrostatic interactions on the folding kinetics. The results indicate that the substitutions at positions 3 and 46 largely decrease the folding kinetics, whereas the mutation of residue 66 only slightly decreases the folding rate. This result agrees well with the experimental observations. It is also found that these mutations have little effects on the folding transition state and the folding pathway, in which the N‐terminal β sheet folds earlier than the C‐terminal region. We also investigated the detailed unfolding pathway and found that it is really the reverse of the folding pathway, providing the validity of our simulation results. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

In vivo and in vitro characterization of Ser477X mutations in polyhydroxyalkanoate (PHA) synthase 1 from Pseudomonas sp. 61-3: effects of beneficial mutations on enzymatic activity, substrate specificity, and molecular weight of PHA

Evolutionary engineered polyhydroxyalkanoate (PHA) synthases from Pseudomonas sp. 61-3 enhance PHA accumulation and enable the monomer composition of PHAs to be regulated. We characterized a newly screened Ser477Arg (S477R) mutant of PHA synthase by in vivo analyses of P(3-hydroxybutyrate) [P(3HB)] homopolymer and P(3HB-co-3-hydroxyalkanoate) [P(3HB-co-3HA)] copolymer productions in the recombinants of Escherichia coli. The results indicated that the S477R mutation contributed to a shift in substrate specificity to smaller monomers containing a 3HB unit rather than to an enhancement in catalytic activity. Multiple mutations of S477R with other beneficial mutations, for example, Ser325Cys, exhibited synergistic effects on both an increase in PHA production (from 9 wt % to 21 wt %) and an alteration of substrate specificity. Furthermore, the effects of complete amino acid substitutions at position 477 were characterized in terms of in vivo PHA production and in vitro enzymatic activity. The five mutations, S477Ala(A)/Phe(F)/His(H)/Arg(R)/Tyr(Y), resulted in a shift in substrate specificity to smaller monomer units. The S477Gly(G) mutant greatly enhanced activity toward all different sizes of substrates with carbon numbers ranging from 4 to 12. These results indicated that the residue 477 contributes to both the catalytic activity and substrate specificity of PHA synthase. In recombinant E. coli, the S477A/F/G/H/R/Y mutations consistently led to increases (up to 6 times that of wild-type enzyme) in weight average molecular weights of P(3HB) homopolymers. On the basis of our studies, we created a structural feasibility accounting for the mutational effects on enzymatic activity and substrate specificity of PHA synthase.     

Effects of mutations at tyrosine 66 and asparagine 123 in the active site pocket of Escherichia coli uracil DNA glycosylase on uracil excision from synthetic DNA oligomers: evidence for the occurrence of long-range interactions between the enzyme and substrate

Uracil DNA glycosylase (UDG), a highly conserved DNA repair enzyme, excises uracil from DNA. Crystal structures of several UDGs have identified residues important for their exquisite specificity in detection and removal of uracil. Of these, Y66 and N123 in Escherichia coli UDG have been proposed to restrict the entry of non-uracil residues into the active site pocket. In this study, we show that the uracil excision activity of the Y66F mutant was similar to that of the wild-type protein, whereas the activities of the other mutants (Y66C, Y66S, N123D, N123E and N123Q) were compromised approximately 1000-fold. The latter class of mutants showed an increased dependence on the substrate chain length and suggested the existence of long-range interactions of the substrate with UDG. Investigation of the phosphate interactions by the ethylation interference assay reaffirmed the key importance of the -1, +1 and +2 phosphates (with respect to the scissile uracil) to the enzyme activity. Interestingly, this assay also revealed an additional interference at the -5 position phosphate, whose presence in the substrate had a positive effect on substrate utilisation by the mutants that do not possess a full complement of interactions in the active site pocket. Such long-range interactions may be crucial even for the wild-type enzyme under in vivo conditions. Further, our results suggest that the role of Y66 and N123 in UDG is not restricted merely to preventing the entry of non-uracil residues. We discuss their additional roles in conferring stability to the transition state enzyme-substrate complex and/or enhancing the leaving group quality of the uracilate anion during catalysis.     

Phospholipase C purification and specificity with respect to individual phospholipids and brain microsomal membrane phospholipids

Phospholipase C was purified from a crude preparation derived from Cl. perfringens utilizing a one-step polypreparative electrophoresis procedure. The purified enzyme has a molecular weight of 46,500 ± 500 and is essentially free of proteolytic and phospholipase A enzymatic activities. It exhibited the following substrate specificity: PC ≥ SM > PS > PI, lyso PC. PE was hydrolyzed when PC was present.Treatment of brain microsomes with purified phospholipase C reduced membrane phospholipids by 69%. All phospholipids were attacked including PE. PC was reduced to 4% and all other phospholipids to 23–43% of their control levels. Total fatty acid composition of brain microsomes was not affected by phospholipase C action.     

Mutational analysis of a feruloyl esterase from Aspergillus awamori involved in substrate discrimination and pH dependence

We cloned the feruloyl esterase A gene from Aspergillus awamori (AwfaeA) and engineered it to study substrate specificity and pH dependence of catalysis. Based on the crystal structures of two type-A feruloyl esterases (FAE-III and AnFAEA) from Aspergillus niger, residues located in the flap region of AwFAEA (Asp71, Thr72, Asp77, and Tyr80) were replaced with corresponding amino acid residues (Ile, Arg, Asn, and Phe), respectively, found in the lid of lipases from Rhizomucor miehei (RmLIP) and Humicola lanuginose (HlLIP). Furthermore, Asp77 of AwFAEA, which is conserved in Aspergillus FAEs and lipases, was replaced with a hydrophobic residue (Ile). Kinetic analysis of the mutant enzymes showed that the higher catalytic efficiency of the D77I and Y80F mutants toward alpha-naphthylbutyrate (C4) and alpha-naphthylcaprylate (C8), respectively, was due to a lower K(m) value. The higher catalytic efficiency of D77N toward C4 substrate was due to a combination of decreased K(m) and considerably increased k(cat). The D71I and Y80F mutants showed some activity toward long-acyl chain esters. On the other hand, the D77I mutant had no detectable activity toward phenolic acid methyl esters and feruloylated arabinoxylan. Moreover, the pH optima of the D77I, D77N, and Y80F mutants increased from 5.0 to 7.0-8.0, 7.0, and 6.0, respectively.     

Comparative structural analysis and substrate specificity engineering of the hyperthermostable beta-glucosidase CelB from Pyrococcus furiosus

The substrate specificity of the beta-glucosidase (CelB) from the hyperthermophilic archaeon Pyrococcus furiosus, a family 1 glycosyl hydrolase, has been studied at a molecular level. Following crystallization and X-ray diffraction of this enzyme, a 3.3 A resolution structural model has been obtained by molecular replacement. CelB shows a homo-tetramer configuration, with subunits having a typical (betaalpha)(8)-barrel fold. Its active site has been compared to the one of the previously determined 6-phospho-beta-glycosidase (LacG) from the mesophilic bacterium Lactococcus lactis. The overall design of the substrate binding pocket is very well conserved, with the exception of three residues that have been identified as a phosphate binding site in LacG. To verify the structural model and alter its substrate specificity, these three residues have been introduced at the corresponding positions in CelB (E417S, M424K, F426Y) in different combinations: single, double, and triple mutants. Characterization of the purified mutant CelB enzyme revealed that F426Y resulted in an increased affinity for galactosides, whereas M424K gave rise to a shifted pH optimum (from 5.0 to 6.0). Analysis of E417S revealed a 5-fold and a 3-fold increase of the efficiency of hydrolyzing o-nitrophenol-beta-D-galactopyranoside-6-phosphate, in the single and triple mutants, respectively. In contrast, their activity on nonphosphorylated sugars was largely reduced (30-300-fold). The residue at position E417 in CelB seems to be the determining factor for the difference in substrate specificity between the two types of family 1 glycosidases.