Takamasa Tobimatsu

Characterization, Sequencing, and Expression of the Genes Encoding a Reactivating Factor for Glycerol-inactivated Adenosylcobalamin-dependent Diol Dehydratase

Diol dehydratase undergoes suicide inactivation by glycerol during catalysis involving irreversible cleavage of the Co-C bond of adenosylcobalamin. In permeabilized Klebsiella oxytoca and Klebsiella pneumoniae cells, the glycerol-inactivated holoenzyme or the enzyme-cyanocobalamin complex is rapidly activated by the exchange of the inactivated coenzyme or cyanocobalamin for free adenosylcobalamin in the presence of ATP and Mg2+ (Honda, S., Toraya, T., and Fukui, S. (1980) J. Bacteriol. 143, 1458–1465; Ushio, K., Honda, S., Toraya, T., and Fukui, S. (1982) J. Nutr. Sci. Vitaminol. 28, 225–236). Permeabilized Escherichia coli cells co-expressing the diol dehydratase genes with two open reading frames in the 3′-flanking region were capable of reactivating glycerol-inactivated diol dehydratase as well as activating the enzyme-cyanocobalamin complexin situ in the presence of free adenosylcobalamin, ATP, and Mg2+. These open reading frames, designated asddrA and ddrB genes, were identified as the genes of a putative reactivating factor for inactivated diol dehydratase. The genes encoded polypeptides consisting of 610 and 125 amino acid residues with predicted molecular weights of 64,266 and 13,620, respectively. Co-expression of the open reading frame in the 5′-flanking region was stimulatory but not obligatory for conferring the reactivating activity upon E. coli. Thus, the product of this gene was considered not an essential component of the reactivating factor.

Redesign of coenzyme B(12) dependent diol dehydratase to be resistant to the mechanism-based inactivation by glycerol and act on longer chain 1,2-diols

Coenzyme B12 dependent diol dehydratase undergoes mechanism‐based inactivation by glycerol, accompanying the irreversible cleavage of the coenzyme Co–C bond. Bachovchin et al. [Biochemistry16, 1082–1092 (1977)] reported that glycerol bound in the GS conformation, in which the pro‐S‐CH2OH group is oriented to the hydrogen‐abstracting site, primarily contributes to the inactivation reaction. To understand the mechanism of inactivation by glycerol, we analyzed the X‐ray structure of diol dehydratase complexed with cyanocobalamin and glycerol. Glycerol is bound to the active site preferentially in the same conformation as that of (S)‐1,2‐propanediol, i.e. in the GS conformation, with its 3‐OH group hydrogen bonded to Serα301, but not to nearby Glnα336. kinact of the Sα301A, Qα336A and Sα301A/Qα336A mutants with glycerol was much smaller than that of the wild‐type enzyme. kcat/kinact showed that the Sα301A and Qα336A mutants are substantially more resistant to glycerol inactivation than the wild‐type enzyme, suggesting that Serα301 and Glnα336 are directly or indirectly involved in the inactivation. The degree of preference for (S)‐1,2‐propanediol decreased on these mutations. The substrate activities towards longer chain 1,2‐diols significantly increased on the Sα301A/Qα336A double mutation, probably because these amino acid substitutions yield more space for accommodating a longer alkyl group on C3 of 1,2‐diols.

Specificities of reactivating factors for adenosylcobalamin-dependent diol dehydratase and glycerol dehydratase

Abstract. Adenosylcobalamin-dependent glycerol and diol dehydratases undergo inactivation by the physiological substrate glycerol during catalysis. In the permeabilized cells of Klebsiella pneumoniae, Klebsiella oxytoca, and recombinant Escherichia coli, glycerol-inactivated glycerol dehydratase and diol dehydratase are reactivated by their respective reactivating factors in the presence of ATP, Mg2+, and adenosylcobalamin. Both of the reactivating factors consist of two subunits. To examine the specificities of the reactivating factors, their genes or their hybrid genes were co-expressed with dehydratase genes in E. coli cells in various combinations. The reactivating factor of K. oxytoca for diol dehydratase efficiently cross-reactivated the inactivated glycerol dehydratase, whereas the reactivating factor of K. pneumoniae for glycerol dehydratase hardly cross-reactivated the inactivated diol dehydratase. Both of the two hybrid reactivating factors rapidly reactivated the inactivated glycerol dehydratase. In contrast, the hybrid reactivating factor containing the large subunit of the glycerol dehydratase reactivating factor hardly reactivated the inactivated diol dehydratase. These results indicate that the glycerol dehydratase reactivating factor is much more specific for the dehydratase partner than the diol dehydratase reactivating factor and that a large subunit of the reactivating factors principally determines the specificity for a dehydratase.

Survey of Catalytic Residues and Essential Roles of Glutamate-α170 and Aspartate-α335 in Coenzyme B12-dependent Diol Dehydratase

The importance of each active-site residue in adenosylcobalamin-dependent diol dehydratase of Klebsiella oxytoca was estimated using mutant enzymes in which one of the residues interacting with substrate and/or K+ was mutated to Ala or another amino acid residue. The Eα170A and Dα335A mutants were totally inactive, and the Hα143A mutant showed only a trace of activity, indicating that Glu-α170, Asp-α335, and His-α143 are catalytic residues. The Qα141A, Qα296A, and Sα362A mutants showed partial activity. It was suggested from kinetic parameters that Gln-α296 is important for substrate binding and Gln-α296 and Gln-α141 for preventing the enzyme from mechanism-based inactivation. The Eα221A, Eα170H, and Dα335A did not form the (αβγ)2 complex, suggesting that these mutations indirectly disrupt subunit contacts. Among other Glu-α170 and Asp-α335 mutants, Eα170D and Eα170Q were 2.2 ± 0.3% and 0.02% as active as the wild-type enzyme, respectively, whereas Dα335N was totally inactive. Kinetic analysis indicated that the presence and the position of a carboxyl group in the residue α170 are essential for catalysis as well as for the continuous progress of catalytic cycles. It was suggested that the roles of Glu-α170 and Asp-α335 are to participate in the binding of substrate and intermediates and keep them appropriately oriented and to function as a base in the dehydration of the 1,1-diol intermediate. In addition, Glu-α170 seems to stabilize the transition state for the hydroxyl group migration from C2 to C1 by accepting the proton of the spectator hydroxyl group on C1.

Heterologous high level expression, purification, and enzymological properties of recombinant rat cobalamin-dependent methionine synthase.

Rat methionine synthase was expressed chiefly as apoenzyme in recombinant baculovirus-infected insect cells (Yamada, K., Tobimatsu, T., and Toraya, T. (1998) Biosci. Biotech. Biochem. 62, 2155–2160). The apoenzyme produced was very unstable, and therefore, after complexation with methylcobalamin, the functional holoenzyme was purified to homogeneity. The specific activity and apparent K m values for substrates were in good agreement with those obtained with purified rat liver enzyme. The electronic spectrum of the purified recombinant enzyme resembled that of cob(II)alamin and changed to a methylcobalamin-like one upon incubation of the enzyme with titanium(III) andS-adenosylmethionine. The rate of oxidative inactivation of the enzyme in the absence of S-adenosylmethionine was slower with a stronger reducing agent like titanium(III). The nucleotide moiety, especially the phosphodiester group, was shown to play an important role in the binding of the coenzyme to apoprotein and thus for catalysis. Upon incubation with the apoenzyme in the absence of a reducing agent, cyano- and aquacobalamin were not effective or were effective only slightly in reconstituting holoenzyme. Ethyl- and propylcobalamin formed inactive complexes with apoenzyme, which were converted to holoenzyme by photolytic activation. Adenosylcobalamin was not able to form a complex with apoenzyme, which was convertible to holoenzyme by photoirradiation.

Crystallization and preliminary X-ray study of two crystal forms of Klebsiella oxytoca diol dehydratase–cyanocobalamin complex

Two crystal forms of Klebsiella oxytoca diol dehydratase complexed with cyanocobalamin have been obtained and preliminary crystallographic experiments have been performed. The crystals belong to two different space groups, depending on the crystallization conditions. One crystal (form I) belongs to space group P212121 with unit-cell parameters a = 76.2, b = 122.3, c = 209. 6 A, and diffracts to 2.2 A resolution using an X-ray beam from a synchrotron radiation source. The other crystal (form II) belongs to space group P21 with unit-cell parameters a = 75.4, b = 132.7, c = 298.8 A, beta = 91.9 degrees, and diffracts to 3.0 A resolution. For the purpose of structure determination, a heavy-atom derivative search was carried out and some mercuric derivatives were found to be promising. Structure analysis by the multiple isomorphous replacement method is now under way.

Low-solubility glycerol dehydratase, a chimeric enzyme of coenzyme B12-dependent glycerol and diol dehydratases

Coenzyme B12-dependent diol and glycerol dehydratases are isofunctional enzymes, which catalyze dehydration of 1, 2-diols to produce corresponding aldehydes. Although the two types of dehydratases have high sequence homology, glycerol dehydratase is a soluble cytosolic enzyme, whereas diol dehydratase is a low-solubility enzyme associated with carboxysome-like polyhedral organelles. Since both the N-terminal 20 and 16 amino acid residues of the β and γ subunits, respectively, are indispensable for the low solubility of diol dehydratase, we constructed glycerol dehydratase-based chimeric enzymes which carried N-terminal portions of the β and γ subunits of diol dehydratase in the corresponding subunits of glycerol dehydratase. Addition of the diol dehydratase-specific N-terminal 34 and 33 amino acid residues of the β and γ subunits, respectively, was not enough to lower the solubility of glycerol dehydratase. A chimeric enzyme which carries the low homology region (residues 35–60) of the diol dehydratase β subunit in addition to the diol dehydratase-specific extra-regions of β and γ subunits showed low solubility comparable to diol dehydratase, although its hydropathy plot does not show any prominent hydrophobic peaks in these regions. It was thus concluded that short N-terminal sequences are sufficient to change the solubility of the enzyme.