Mamoru Yamanishi

'Crystal lattice engineering,' an approach to engineer protein crystal contacts by creating intermolecular symmetry: crystallization and structure determination of a mutant human RNase 1 with a hydrophobic interface of leucines

A protein crystal lattice consists of surface contact regions, where the interactions of specific groups play a key role in stabilizing the regular arrangement of the protein molecules. In an attempt to control protein incorporation in a crystal lattice, a leucine zipper‐like hydrophobic interface (comprising four leucine residues) was introduced into a helical region (helix 2) of the human pancreatic ribonuclease 1 (RNase 1) that was predicted to form a suitable crystallization interface. Although crystallization of wild‐type RNase 1 has not yet been reported, the RNase 1 mutant having four leucines (4L‐RNase 1) was successfully crystallized under several different conditions. The crystal structures were subsequently determined by X‐ray crystallography by molecular replacement using the structure of bovine RNase A. The overall structure of 4L‐RNase 1 is quite similar to that of the bovine RNase A, and the introduced leucine residues formed the designed crystal interface. To characterize the role of the introduced leucine residues in crystallization of RNase 1 further, the number of leucines was reduced to three or two (3L‐ and 2L‐RNase 1, respectively). Both mutants crystallized and a similar hydrophobic interface as in 4L‐RNase 1 was observed. A related approach to engineer crystal contacts at helix 3 of RNase 1 (N4L‐RNase 1) was also evaluated. N4L‐RNase 1 also successfully crystallized and formed the expected hydrophobic packing interface. These results suggest that appropriate introduction of a leucine zipper‐like hydrophobic interface can promote intermolecular symmetry for more efficient protein crystallization in crystal lattice engineering efforts.

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.

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.

Purification and some properties of wild-type and N-terminal-truncated ethanolamine ammonia-lyase of Escherichia coli

The methods of homologous high-level expression and simple large-scale purification for coenzyme B(12)-dependent ethanolamine ammonia-lyase of Escherichia coli were developed. The eutB and eutC genes in the eut operon encoded the large and small subunits of the enzyme, respectively. The enzyme existed as the heterododecamer alpha(6)beta(6). Upon active-site titration with adeninylpentylcobalamin, a strong competitive inhibitor for coenzyme B(12), the binding of 1 mol of the inhibitor per mol of the alphabeta unit caused complete inhibition of enzyme, in consistent with its subunit structure. EPR spectra indicated the formation of substrate-derived radicals during catalysis and the binding of cobalamin in the base-on mode, i.e. with 5,6-dimethylbenzimidazole coordinating to the cobalt atom. The purified wild-type enzyme underwent aggregation and inactivation at high concentrations. Limited proteolysis with trypsin indicated that the N-terminal region is not essential for catalysis. His-tagged truncated enzymes were similar to the wild-type enzyme in catalytic properties, but more resistant to p-chloromercuribenzoate than the wild-type enzyme. A truncated enzyme was highly soluble even in the absence of detergent and resistant to aggregation and oxidative inactivation at high concentrations, indicating that a short N-terminal sequence is sufficient to change the solubility and stability of the enzyme.

Novel Pathway for Utilization of Cyclopropanecarboxylate by Rhodococcus rhodochrous

ABSTRACT A new strain isolated from soil utilizes cyclopropanecarboxylate as the sole source of carbon and energy and was identified as Rhodococcus rhodochrous (H. Nishihara, Y. Ochi, H. Nakano, M. Ando, and T. Toraya, J. Ferment. Bioeng. 80:400-402, 1995). A novel pathway for the utilization of cyclopropanecarboxylate, a highly strained compound, by this bacterium was investigated. Cyclopropanecarboxylate-dependent reduction of NAD+ in cell extracts of cyclopropanecarboxylate-grown cells was observed. When intermediates accumulated in vitro in the absence of NAD+ were trapped as hydroxamic acids by reaction with hydroxylamine, cyclopropanecarboxohydroxamic acid and 3-hydroxybutyrohydroxamic acid were formed. Cyclopropanecarboxyl-coenzyme A (CoA), 3-hydroxybutyryl-CoA, and crotonyl-CoA were oxidized with NAD+ in cell extracts, whereas methacrylyl-CoA and 3-hydroxyisobutyryl-CoA were not. When both CoA and ATP were added, organic acids corresponding to the former three CoA thioesters were also oxidized in vitro by NAD+, while methacrylate, 3-hydroxyisobutyrate, and 2-hydroxybutyrate were not. Therefore, it was concluded that cyclopropanecarboxylate undergoes oxidative degradation through cyclopropanecarboxyl-CoA and 3-hydroxybutyryl-CoA. The enzymes catalyzing formation and ring opening of cyclopropanecarboxyl-CoA were shown to be inducible, while other enzymes involved in the degradation were constitutive.

An electron paramagnetic resonance study on the mechanism-based inactivation of adenosylcobalamin-dependent diol dehydrase by glycerol and other substrates

Adenosylcobalamin-dependent diol dehydrase undergoes mechanism-based inactivation by glycerol or other substrates during catalysis. X-band electron paramagnetic resonance spectra of holoenzyme were measured at -130 degrees C after reaction with such substrates. After short time of incubation, broad signals assigned to low-spin Co(II) of cob(II)alamin and doublet signals assigned to an organic radical intermediate derived from each substrate were observed with 1,2-propanediol, 1,2-ethanediol, glycerol and meso-2,3-butanediol with the magnitude of their exchange interaction (J-value) decreasing in this order. A substrate with the smaller magnitude of exchange interaction between low-spin Co(II) and an organic radical intermediate seems to be an efficient mechanism-based inactivator. Since the magnitude of exchange interaction decreases with the distance between radical species in a radical pair, these results suggest that a stabilizing effect of holoenzyme on radical intermediates during reactions decreases with the distance between Co(II) and a radical.

Crystallization and preliminary X-ray analysis of molecular chaperone-like diol dehydratase-reactivating factor in ADP-bound and nucleotide-free forms.

Adenosylcobalamin (coenzyme B12) dependent diol dehydratase (EC 4.2.1.28) catalyzes the conversion of 1,2-diols and glycerol to the corresponding aldehydes. It undergoes mechanism-based inactivation by glycerol. The diol dehydratase-reactivating factor (DDR) reactivates the inactivated holoenzymes in the presence of adenosylcobalamin, ATP and Mg2+ by mediating the release of a damaged cofactor. This molecular chaperone-like factor was overexpressed in Escherichia coli, purified and crystallized in the ADP-bound and nucleotide-free forms by the sandwich-drop vapour-diffusion method. The crystals of the ADP-bound form belong to the orthorhombic system, with space group P2(1)2(1)2(1) and unit-cell parameters a = 83.26, b = 84.60, c = 280.09 A, and diffract to 2.0 A. In the absence of nucleotide, DDR crystals were orthorhombic, with space group P2(1)2(1)2(1) and unit-cell parameters a = 81.92, b = 85.37, c = 296.99 A and diffract to 3.0 A. Crystals of both forms were suitable for structural analysis.