Cooperative Effect of Two Surface Amino Acid Mutations (Q252L and E170K) in Glucose Dehydrogenase from Bacillus megaterium IWG3 on Stabilization of Its Oligomeric State

A thermostable glucose dehydrogenase (GlcDH) mutant of Bacillus megaterium IWG3 harboring the Q252L substitution (Y. Makino, S. Negoro, I. Urabe, and H. Okada, J. Biol. Chem. 264:6381-6385, 1989) is stable at pH values above 9, but only in the presence of 2 M NaCl. Another GlcDH mutant exhibiting increased stability at an alkaline pH in the absence of NaCl has been isolated previously (S.-H. Baik, T. Ide, H. Yoshida, O. Kagami, and S. Harayama, Appl. Microbiol. Biotechnol. 61:329-335, 2003). This mutant had two amino acid substitutions, Q252L and E170K. In the present study, we characterized three GlcDH mutants harboring the substitutions Q252L, E170K, and Q252L/E170K under low-salt conditions. The GlcDH mutant harboring two substitutions, Q252L/E170K, was stable, but mutants harboring a single substitution, either Q252L or E170K, were unstable at an alkaline pH. Gel filtration chromatography analyses demonstrated that the oligomeric state of the Q252/E170K enzyme was stable, while the tetramers of the enzymes harboring a single substitution (Q252L or E170K) dissociated into dimers at an alkaline pH. These results indicated that the Q252L and E170K substitutions synergistically strengthened the interaction at the dimer-dimer interface. The crystal structure of the E170K/Q252L mutant, determined at 2.0-Å resolution, showed that residues 170 and 252 are located in a hydrophobic cavity at the subunit-subunit interface. We concluded that these residues in the wild-type enzyme have thermodynamically unfavorable effects, while the Q252L and E170K substitutions increase the subunit-subunit interactions by stabilizing the hydrophobic cavity.     

Stability-increasing mutants of glucose dehydrogenase from Bacillus megaterium IWG3

A glucose dehydrogenase gene was isolated from Bacillus megaterium IWG3, and its nucleotide sequence was identified. The amino acid sequence of the enzyme deduced from the nucleotide sequence is very similar to the protein sequence of the enzyme from B. megaterium M1286 reported by Jany et al. (Jany, K.-D., Ulmer, W., Froschle, M., and Pfleiderer, G. (1984) FEBS Lett. 165, 6-10). The isolated gene was mutagenized with hydrazine, formic acid, or sodium nitrite, and 12 clones (H35, H39, F18, F20, F191, F192, N1, N13, N14, N28, N71, and N72) containing mutant genes for thermostable glucose dehydrogenase were obtained. The nucleotide sequences of the 12 genes show that they include 8 kinds of mutants having the following amino acid substitutions: H35 and H39, Glu-96 to Gly; F18 and F191, Glu-96 to Ala; F20, Gln-252 to Leu; F192, Gln-252 to Leu and Ala-258 to Gly; N1, Glu-96 to Lys and Val-183 to Ile; N13 and N14, Glu-96 to Lys, Val-112 to Ala, Glu-133 to Lys, and Tyr-217 to His; N28, Glu-96 to Lys, Asp-108 to Asn, Pro-194 to Gln, and Glu-210 to Lys; and N71 and N72, Tyr-253 to Cys. These mutant enzymes have higher stability at 60 degrees C than the wild-type enzyme. The results of this study indicate that the tetrameric structure of glucose dehydrogenase is stabilized by several kinds of mutation, and at least one of the following amino acid substitutions stabilizes the enzyme: Glu-96 to Gly, Glu-96 to Ala, Gln-252 to Leu, and Tyr-253 to Cys.     

Crystal structure of glucose dehydrogenase from Bacillus megaterium IWG3 at 1.7 A resolution

The crystal structure of glucose dehydrogenase (GlcDH) from Bacillus megaterium IWG3 has been determined to an R-factor of 17.9% at 1.7 A resolution. The enzyme consists of four identical subunits, which are similar to those of other short-chain reductases/dehydrogenases (SDRs) in their overall folding and subunit architecture, although cofactor binding sites and subunit interactions differ. Whereas a pair of basic residues is well conserved among NADP(+)-preferring SDRs, only Arg39 was found around the adenine ribose moiety of GlcDH. This suggests that one basic amino acid is enough to determine the coenzyme specificity. The four subunits are interrelated by three mutually perpendicular diad axes (P, Q, and R). While subunit interactions through the P-axis for GlcDH are not so different from those of the other SDRs, those through the Q-axis differ significantly. GlcDH was found to have weaker hydrophobic interactions in the Q-interface. Moreover, GlcDH lacks the salt bridge that stabilizes the subunit interaction in the Q-interface in the other SDRs. Hydrogen bonds between Q-axis related subunits are also less common than in the other SDRs. The GlcDH tetramer dissociates into inactive monomers at pH 9.0, which can be attributed mainly to the weakness of the Q-axis interface.     

Amino acid sequence of the K-peptide generated by limited proteolysis of glucose dehydrogenase from Bacillus megaterium by proteinase K1

Proteolysis of glucose dehydrogenase from Bacillus megaterium with proteinase K apparently generated two fragments. The small fragment, designated as K-peptide, was sequenced and its covalent structure was determined as Ser-Ser-Glu-Ala-Ser-Tyr-Val-Thr-Gly-Ile-Thr-Leu-Phe-Ala-Asp-Gly-Gly-Met-Thr- Gln-Tyr-Pro-Ser-Phe-Glu-Ala-Gly-Arg-Gly. The sequence analysis showed that the K-peptide consists of two identical fragments, one of which is lacking one serine residue at the amino-terminus.     

Stereospecific synthesis of (R)-2-hydroxy carboxylic acids using recombinant E. coli BL21 overexpressing YiaE from Escherichia coli K12 and glucose dehydrogenase from Bacillus subtilis

The yiaE gene from Escherichia coli K12 was functionally expressed in E. coli BL21 using an IPTG inducible pET expression system (2.1 U/mg), and YiaE was purified to a specific activity of 18 U/mg. The purified enzyme catalyzes reduction of various aromatic and aliphatic 2-oxo carboxylic acids to the corresponding (R)-2-hydoxy carboxylic acids using NADPH. For practical applications, the problem of NADPH recycle was effectively solved by using recombinant E. coli overexpressing YiaE and glucose dehydrogenase from Bacillus subtilis in the same cell. The recombinant E. coli was used to prepare (R)-phenyllactic acid and (R)-2-hydroxy-4-phenylbutanoic acid from the corresponding 2-oxo carboxylic acids (98% ee) while the alpha-carbonyl group of 2,4-dioxo-4-phenylbutyric acid was reduced regio- and stereospecifically to give (R)-2-hydroxy-4-oxo-4-phenylbutyric acid (97% ee) in quantitative yields. The cells could be recycled for 3 days at room temperature in 100 mM phosphate buffer (pH 7.0) without loss of activity, which reduced to 70% after 1 week.     

Insecticidal properties of 3-aminopropyl(methyl)phosphinic acid and its effect on K+-evoked release of acetylcholine from cockroach synaptosomes

3-Aminopropyl(methyl)phosphinic acid (APMP), a potent agonist of mammalian GABA_B receptors, caused prostration in houseflies (Musca domestica L.) on injection into their thoraces, with an ED₅₀ value of 0.42 μg/fly. The 48-h LD₅₀ values of APMP were estimated to be 0.45 and 5.6 μg/fly in the presence and absence of piperonyl butoxide, a mixed-function oxidase inhibitor, respectively. Analogues of APMP, bearing a longer or shorter side chain by a CH₂ unit, or a phenyl group in the place of the methyl group, were without effects. In perfusion assays with synaptosomes prepared from the thoracic/abdominal nerve cords of cockroaches (Periplaneta americana L.), 1 mM APMP reduced K⁺-evoked acetylcholine release to 10.4% of the control. These findings indicate that the physiologically important site of action of APMP, which might be implicated in neurotransmitter release, is present in insect neurons.     

The local environment at the cytoplasmic end of TM6 of the mu opioid receptor differs from those of rhodopsin and monoamine receptors: introduction of an ionic lock between the cytoplasmic ends of helices 3 and 6 by a L6.30(275)E mutation inactivates the mu opioid receptor and reduces the constitutive activity of its T6.34(279)K mutant

Activation of rhodopsin and monoamine G protein-coupled receptors (GPCRs) has been proposed to involve in part the disruption of a conserved E6.30-R3.50 ionic interaction between transmembrane segments (TMs) 3 and 6. However, this interaction does not occur in the opioid receptors, which have L275 at 6.30. On the basis of our findings that mutations of T6.34(279) to K and D produced, respectively, a constitutively active and an inactive form of the mu opioid receptor, we previously suggested that the functional role of the 6.30(275) residue could be assumed by T6.34(279), but the interplay between residues at positions 6.30 and 6.34 remained unresolved. In this study, we examined the effects of introducing an E in position 6.30(275) of the wild type (WT) and of the T6.34(279) mutants of the mu opioid receptor to compare the participation of the 6.30 locus in molecular events during activation in this receptor with its role in other GPCRs. The L6.30(275)E and the L6.30(275)E/T6.34(279)D mutants displayed no constitutive activity and could not be activated by the agonist DAMGO or morphine. The L6.30(275)E/T6.34(279)K mutant had some constitutive activity, but much less than the T6.34(279)K mutant, and could be activated by both agonists. The rank order of affinity for the agonist DAMGO is as follows: T6.34(279)K > WT congruent with L6.30(275)E/T6.34(279)K > L6.30(275)E congruent with T6.34(279)D > L6.30(275)E/T6.34(279)D; however, all constructs have a similar affinity for the antagonist [(3)H]diprenorphine. These data are interpreted in the context of interactions with the conserved R3.50(165) in TM3. When L6.30(275) is mutated to E, the favorable E6.30(275)-R3.50(165) interaction stabilizes an inactive state, as in rhodopsin, and hence reduces the activities of T6.34(279) mutants. Thus, the mu opioid receptor is shown to be different from rhodopsin and monoamine GPCRs, of which the WTs with native E6.30 can be activated, and the 6.34D or 6.34K mutants display enhanced constitutive activities. Our molecular modeling results suggest that some specific differences in local geometry at the cytoplasmic ends of TM5 and TM6 may account in part for the observed differences in the molecular mechanisms of receptor activation.