Glycerol dehydrogenase. structure, specificity, and mechanism of a family III polyol dehydrogenase.

BACKGROUND: Bacillus stearothermophilus glycerol dehydrogenase (GlyDH) (glycerol:NAD(+) 2-oxidoreductase, EC 1.1.1.6) catalyzes the oxidation of glycerol to dihydroxyacetone (1,3-dihydroxypropanone) with concomitant reduction of NAD(+) to NADH. Analysis of the sequence of this enzyme indicates that it is a member of the so-called iron-containing alcohol dehydrogenase family. Despite this sequence similarity, GlyDH shows a strict dependence on zinc for activity. On the basis of this, we propose to rename this group the family III metal-dependent polyol dehydrogenases. To date, no structural data have been reported for any enzyme in this group. RESULTS: The crystal structure of B. stearothermophilus glycerol dehydrogenase has been determined at 1.7 A resolution to provide structural insights into the mechanistic features of this family. The enzyme has 370 amino acid residues, has a molecular mass of 39.5 kDa, and is a homooctamer in solution. CONCLUSIONS: Analysis of the crystal structures of the free enzyme and of the binary complexes with NAD(+) and glycerol show that the active site of GlyDH lies in the cleft between the enzyme's two domains, with the catalytic zinc ion playing a role in stabilizing an alkoxide intermediate. In addition, the specificity of this enzyme for a range of diols can be understood, as both hydroxyls of the glycerol form ligands to the enzyme-bound Zn(2+) ion at the active site. The structure further reveals a previously unsuspected similarity to dehydroquinate synthase, an enzyme whose more complex chemistry shares a common chemical step with that catalyzed by glycerol dehydrogenase, providing a striking example of divergent evolution. Finally, the structure suggests that the NAD(+) binding domain of GlyDH may be related to that of the classical Rossmann fold by switching the sequence order of the two mononucleotide binding folds that make up this domain.

Complex between Peptostreptococcus magnus protein L and a human antibody reveals structural convergence in the interaction modes of Fab binding proteins.

BACKGROUND: Peptostreptococcus magnus protein L (PpL) is a multidomain, bacterial surface protein whose presence correlates with virulence. It consists of up to five homologous immunoglobulin binding domains that interact with the variable (VL) regions of kappa light chains found on two thirds of mammalian antibodies. RESULTS: We refined the crystal structure of the complex between a human antibody Fab fragment (2A2) and a single PpL domain (61 residues) to 2.7 A. The asymmetric unit contains two Fab molecules sandwiching a single PpL domain, which contacts similar VL framework regions of two light chains via independent interfaces. The residues contacted on VL are remote from the hypervariable loops. One PpL-Vkappa interface agrees with previous biochemical data, while the second is novel. Site-directed mutagenesis and analytical-centrifugation studies suggest that the two PpL binding sites have markedly different affinities for VL. The PpL residues in both interactions are well conserved among different Peptostreptococcus magnus strains. The Fab contact positions identified in the complex explain the high specificity of PpL for antibodies with kappa rather than lambda chains. CONCLUSIONS: The PpL-Fab complex shows the first interaction of a bacterial virulence factor with a Fab light chain outside the conventional combining site. Structural comparison with two other bacterial proteins interacting with the Fab heavy chain shows that PpL, structurally homologous to streptococcal SpG domains, shares with the latter a similar binding mode. These two bacterial surface proteins interact with their respective immunoglobulin regions through a similar beta zipper interaction.

The identification of a structurally important cysteine residue in the glycerol dehydrogenase from Bacillus stearothermophilus.

Evidence is presented to demonstrate that the Zn2+ metallo-enzyme glycerol dehydrogenase from the thermophile Bacillus stearothermophilus has one cysteine residue per subunit which is only available for reaction with thiol reagents in the metal-depleted form of the enzyme. Modification of the metal-depleted enzyme by methyl methanethiosulphonate prevents the reactivation of the enzyme by Zn2+ ions and induces dissociation of the oligomer into subunits. The rate of reaction of the cysteine residue with the thiol reagent DTNB is limited by a factor other than reagent concentration and it is proposed that the reagent only reacts with the cysteine residue in dissociated monomers. The enzyme has been labelled at the single cysteine residue by radioactive iodo[2-3H]acetic acid. Two radiolabelled peptides have been isolated and sequenced; one peptide is a component of the other. Spectroscopic evidence suggests that the cysteine residue is not involved in ligation of the essential metal ion. Chemical modification studies using the reagent diethylpyrocarbonate have suggested that two histidines are involved in the ligation of the metal.

Studies on the interactions of glycerol dehydrogenase from Bacillus stearothermophilus with Zn2+ ions and NADH.

The interactions of the essential divalent cation, Zn2+, with the binary complex formed between glycerol dehydrogenase (glycerol:NAD+ 2-oxidoreductase, EC 1.1.1.6) and its coenzyme NADH have been examined by fluorescence spectroscopy. Both the metallo and non-metallo form of the enzyme bind the coenzyme NADH. The addition of Zn2+ ions to a solution of the binary complex formed between metal-depleted enzyme and NADH results in a rapid increase in fluorescence emission at 430 nm. This has been used to determine the on rate for Zn2+ to the enzyme/binary complex. A dissociation constant of 3.02 +/- 0.25.10(-9) M for the equilibrium between Zn2+ ions and the enzyme has been determined.

Spectroscopic studies of myo-inositol monophosphatase with a novel fluorescent substrate.

myo-Inositol monophosphatase catalyzes dephosphorylation of the synthetic substrate anthraniloyl-2'-AMP. Binding of this fluorescent substrate to Tb(III)-monophosphatase was monitored by luminescence spectroscopy. The anthraniloyl chromophore excited at 330 nm sensitizes the long lived luminescence of enzyme bound Tb(III) at 490, 545, 585 and 620 nm. Assuming a mechanism of radiationless energy transfer, the actual distance of separation between the donor anthraniloyl moiety and the acceptor Tb(III) was calculated to be R = 10 angstroms. The binding studies support the earlier observation of Bone et al. (Proc. Natl. Acad. Sci. USA 89 (1992) 10031-10035) that the substrate and the lanthanide Gd(III) interact with a common binding domain of the protein. The catalytic activity of the monophosphatase is completely dependent upon Mg(II) ions which elicit changes in the secondary structure of the protein as revealed by circular dichroism measurements. Binding of Mg(II) ions tend to stabilize the secondary structure of the phosphatase against guanidinium-HCl denaturation.

The production and characterisation of an immobilised chaperonin system.

Here we report a method of immobilising the chaperonins GroEL and GroES to a glass matrix. The immobilised chaperone system has been used to successfully refold target proteins denatured by guanidine hydrochloride and produce substantially higher levels of active protein than occur on dilution into aqueous solution alone. The chaperone system has been shown to refold proteins from each of the three categories of GroEL substrate. The refolding of the enzyme glycerol dehydrogenase from Bacillus stearothermophilus shows a two-fold increase in activity in the presence of immobilised GroEL compared to that in free solution. The lactate dehydrogenase from B. stearothermophilus also shows a two-fold higher yield of activity in the presence of the immobilised GroEL and ATP. The presence of immobilised GroEL in the absence of ATP arrests the refolding of LDH. The enzyme citrate synthetase from porcine heart demonstrates a three-fold increase in activity when refolded in the presence of immobilised GroEL, ATP and free GroES. Similar results are obtained in the presence of free GroEL, immobilised GroES and ATP. The matrix-bound chaperone can be removed from the refolding mixture by centrifugation, producing a reusable system that can be easily isolated and purified from the refolded substrate.

The identification of a lysine residue reactive to pyridoxal-5-phosphate in the glycerol dehydrogenase from the thermophile Bacillus stearothermophilus.

The glycerol dehydrogenase (GDH) from Bacillus stearothermophilus is inactivated by incubation with pyridoxal-5-phosphate (PALP). The complex formed between the two can be trapped by reduction with sodium borohydride to yield a protein with an absorbance band at 325 nm and a fluorescence emission band at 430 nm, typical of trapped pyridoxal-5-phosphate moieties. Total loss of catalytic activity of the enzyme is associated with the modification of approximately one equivalent of the reagent; the incorporation of the reagent and the loss of activity can be prevented by the additional presence of the oxidised or reduced coenzyme. Peptides derived from the labelled protein have been sequenced and have identified Lys-97 as the reactive residue. Site-directed mutagenesis had been used to replace Lys-97 by a His residue. This mutated enzyme has no catalytic activity and fluorescence spectroscopy studies suggest that it is unable to bind NADH.

Isolation and characterisation of the glycerol dehydrogenase from Bacillus stearothermophilus.

A protocol for the rapid purification of the glycerol dehydrogenase (glycerol: NAD+ 2-oxidoreductase, EC 1.1.1.6) from the thermophile Bacillus stearothermophilus has been developed using a combination of chromatographic techniques including affinity chromatography on a Sepharose-immobilised triazine dye (Procion red, HE3B, ICI). Substrate specificity has been examined and Km values determined. The protein has been shown to have an oligomeric Mr of approx. 180,000 and consists of four identical subunits of Mr 42,000. Exposure to chelating agents (e.g., EDTA) leads to total loss of activity; the EDTA-inactivated enzyme can be reactivated by Zn2+ and requires 1 mol equivalent of zinc per subunit for full catalytic activity. Other divalent cations such as Cd2+ and Co2+ will reactivate the apo-enzyme but yields an enzyme of lower specific activity. The enzyme binds 1 equivalent of NADH per subunit and during catalysis transfers the 4-pro-R hydride from the nicotinamide ring of the reduced-coenzyme to the substrate. Glycerol increases the dissociation constant for the interaction between NADH and Zn-metallo-glycerol dehydrogenase (ZnGDH) but has no effect on the equilibrium between NADH and metal-depleted enzyme.

Localization of Cys-344 on the (Ca(2+)-Mg(2+)-ATPase of sarcoplasmic reticulum using resonance energy transfer.

4-Bromomethyl-6,7-dimethoxy-coumarin labels the (Ca(2+)-Mg(2+)-ATPase of skeletal muscle sarcoplasmic reticulum at Cys-344. Resonance energy transfer has been used to measure the distance between this site and Lys-515 labelled with fluorescein isothiocyanate as about 37 A. The height of Cys-344 above the phospholipid/water interface has been measured by resonance energy transfer for the ATPase reconstituted into bilayers containing fluorescein-labelled phosphatidylethanolamine; the height was found to be about 45 A. None of these distances was found to alter on changing pH, or on addition of Mg2+, Ca2+ or vanadate. Quenching of the fluorescence of the coumarin-labelled ATPase with KI suggested that the fluorophore is not fully exposed on the ATPase.

Bovine inositol monophosphatase. Studies on the binding interactions with magnesium, lithium and phosphate ions.

Rapid equilibrium dialysis has been used to show that recombinant bovine brain inositol monophosphatase binds one equivalent of Pi per subunit of enzyme. Pi is only bound in the presence of Mg2+ ions. The dissociation constant for the equilibrium is approximately 50 microM. This value of Kd is independent of the concentration of the Mg2+ ions and of the presence or absence of Li+ ions. Lithium ions which inhibit the enzyme uncompetitively are not able to support the binding of the Pi to the enzyme. The observation that Pi only binds in the presence of Mg2+ ions supports similar conclusions made in experiments which studied the protection of the enzyme from proteolytic degradation and chemical modification.

Bovine inositol monophosphatase: proteolysis and structural studies.

Bovine brain inositol monophosphatase is inactivated when trypsin catalyses the cleavage of a single peptide bond between Lys-36 and Ser-37. This proteolysis is closely followed by cleavage at two other sites in the protein between Lys-78 and Ser-79 and between Lys-156 and Ser-157 suggesting that all of these sites are exposed in the native conformation of the protein. All of these residues are predicted to lie at the ends of alpha helices. The most susceptible bond (Lys-36--Ser-37) is predicted to lie in a highly flexible region of the protein. Circular dichroism studies suggest that approximately 40% of the secondary structure of this protein is helical which is similar to that predicted by the algorithm of Garnier et al. [(1978) J. Mol. Biol. 120, 97-120].

Bovine inositol monophosphatase. The identification of a histidine residue reactive to diethylpyrocarbonate.

The inositol monophosphatase from bovine brain is inactivated by the histidine-specific reagent diethylpyrocarbonate. Using 4 mM reagent at pH 6.5, the reaction results in the modification of 3 equivalents of histidine per polypeptide chain. The loss of activity occurs at the same rate as the slowest reacting of these residues. Site directed mutagenesis studies have been used to generate a mutated enzyme species bearing a His-217-->Gln replacement and have shown that it is the modification of histidine 217 which results in the inactivation of the enzyme.

Identification of a reversible structural transition in the metal-depleted glycerol dehydrogenase from Bacillus stearothermophilus.

Evidence is presented to demonstrate that the Zn2+ -depleted, inactive form of the glycerol dehydrogenase from Bacillus stearothermophilus exists in one of two possible conformations in equilibrium, the position of which is temperature sensitive. The conformation of the metal-depleted enzyme favoured by higher temperatures (20-40 degrees C) is able to bind Zn2+ and regain catalytic activity, whereas that favoured at lower temperatures (0-10 degrees C) is unable to bind metal ions and is thus inactive. This equilibrium is also pH dependent with a pK of 6.6. At pH 6.0, the equilibrium lies in favour of the form of the enzyme able to bind metal ions and exhibit activity.

Elution of human IgG from affinity columns containing immobilised variants of protein A.

Immobilised analogues of protein A have been used for affinity chromatographic separation of human IgG. Truncation of the C-terminal region of an engineered IgG-binding domain based upon the B domain from protein A, in combination with site-directed mutagenesis, has led to the generation of a number of proteins with a decreased affinity for IgG. The elution of human IgG from these proteins when immobilised onto a solid support occurs over the pH range 3.2-5.0 with 0.5 M acetate buffer. These proteins may be effective alternatives to standard protein A columns when milder elution conditions are required.

A study of the interactions between an IgG-binding domain based on the B domain of staphylococcal protein A and rabbit IgG.

The nonantigenic interaction between a recombinant immunoglobulin G (IgG)-binding protein based on the B domain of Protein A from Staphylococcus aureus (termed SpA1) and the Fc fragment of rabbit IgG has been investigated. The contribution to binding of four putative hydrogen bond contacts between SpA1 and IgG-Fc were examined by the individual substitution of the residues in SpA1 involved in these interactions by others unable to form hydrogen bonds. It was found that the most important of the hydrogen bonds involved Tyr 18 which, when replaced by Phe, resulted in a twofold decrease in IgG-binding affinity. The residues of SpA1 proposed to make close, mainly hydrophobic, contacts with Fc were replaced by residues with potential electrostatic charge to establish the importance of the hydrophobic interaction in the complex. The IgG-binding affinities of the mutant proteins were compared to the wild-type protein by a competitive enzyme-linked immunosorbent assay. The replacement of individual hydrophobic residues by His generated a number of novel IgG-binding proteins with reduced binding affinity at pH 5.0 but which maintained strong binding affinities at pH 8.0. The elution profile of human IgG1-Fc (Fc fragment of human IgG1) from a column made from an immobilized two-domain mutant protein shows that the complex dissociates at a higher pH relative to that of the non-mutated protein thus offering favorable elution characteristics.

Bovine inositol monophosphatase: development of a continuous fluorescence assay of enzyme activity.

This paper describes a continuous assay for the enzyme inositol monophosphatase which has been developed using a new substrate, the fluorescent compound 4-methylumbelliferyl phosphate. The hydrolysis of the phosphate group from this compound can be readily detected by a resultant large red shift in the emission spectrum from 390-450 nm. The kinetic constants for the enzyme using this new substrate are described.

Glutamate dehydrogenase from wild type Neurospora crassa; inactivation by photo-oxidation.

The NADP dependent glutamate dehydrogenase from wild type Neurospora crassa is inactivated by exposure to light in the presence of the dye, Methylene Blue. Photo-oxidation appears to disturb the conformational equilibrium which controls the activity of this enzyme. Data obtained suggests that the modified group is the same as that reactive to the histidine reagent, diethylpyrocarbonate.