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The overexpression of fdm, which encodes the formaldehyde dismutase from Pseudomonas putida F61, resulted in the formation of inclusion bodies made up of aggregated enzyme, leaving little activity in the soluble fraction of the transformant cells. On the other hand, coexpression of groESL along with fdm facilitated in vivo solubilization of the enzyme protein in its active form. When coexpressed with groESL, formaldehyde dismutase purified from E. coli had the same crystalline form (i.e., a regular octahedron) as the native enzyme, and like the native enzyme, it bound 1 mol of NAD(H) and 2 mol of zinc in each subunit.

Aminodeoxychorismate lyase is a pyridoxal 5'-phosphate-dependent enzyme that converts 4-aminodeoxychorismate to pyruvate and p-aminobenzoate, a precursor of folic acid in bacteria. The enzyme exhibits significant sequence similarity to two aminotransferases, D-amino acid aminotransferase and branched-chain L-amino acid aminotransferase. In the present study, we have found that aminodeoxychorismate lyase catalyzes the transamination between D-alanine and pyridoxal phosphate to produce pyruvate and pyridoxamine phosphate. L-Alanine and other D- and L-amino acids tested were inert as substrates of transamination. The pro-R hydrogen of C4' of pyridoxamine phosphate was stereospecifically abstracted during the reverse half transamination from pyridoxamine phosphate to pyruvate. Aminodeoxychorismate lyase is identical to D-amino acid aminotransferase and branched-chain L-amino acid aminotransferase in the stereospecificity of the hydrogen abstraction, and differs from all other pyridoxal enzymes that catalyze pro-S hydrogen transfer. Aminodeoxychorismate lyase is the first example of a lyase that catalyzes pro-R-specific hydrogen abstraction. The result is consistent with recent X-ray crystallographic findings showing that the topological relationships between the cofactor and the catalytic residue for hydrogen abstraction are conserved among aminodeoxychorismate lyase, D-amino acid aminotransferase and branched-chain L-amino acid aminotransferase [Nakai, T., Mizutani, H., Miyahara, I., Hirotsu, K., Takeda, S., Jhee, K.-H., Yoshimura, T., and Esaki, N. (2000) J. Biochem. 128, 29-38].

beta-Glucosidase from Cellvibrio gilvus was successfully overproduced in soluble form in Escherichia coli, with the coexpression of GroEL/ES. Without the GroEL/ES protein, the beta-glucosidase overexpressed in E. coli constituted a huge amount (80%) of the total cellular protein, but was localized in the insoluble fraction, and little activity was detected in the soluble fraction. Coexpression of the E. coli GroEL/ES had a drastic impact on the proper folding of the beta-glucosidase; 20% of the overexpressed enzyme was recovered in the soluble fraction in active form. In addition, the synergistic effect of GroEL/ES and the low induction temperature led to 70% solubilization of the total expressed target protein and more than a 20-fold increase in activity. Similar effects of GroEL/ES were also observed on the overexpressed beta-glucosidase from Agrobacterium tumefaciens.

A trypsin-like serine protease from Streptomyces erythraeus (abbreviated as SET) has been crystallized at pH 7, which is within its active pH range. The crystal structure of SET has been solved by molecular replacement using the atomic model of Streptomyces griseus trypsin (SGT), which is 37% homologous with SET, and refined to the crystallographic R factor of 0.199 for 15,878 reflections with Fo/sigma(F) > 3 between 7 and 1.9 A resolution. The final model of SET contains 1,619 protein atoms and 97 water molecules. No Ca2+ ion is present in SET apparently because (i) the two carboxylate groups from two Glu residues, which bind a Ca2+ ion in bovine trypsin (BT) or SGT, have disappeared; and (ii) a guanidino group from an Arg residue is unfavorably present in the potential binding region. There is an unusual type II beta-turn in which the third residue is Asp instead of Gly. This Asp residue is the only non-Gly residue significantly outside the allowed regions in the Ramachandran map. The three-dimensional structure of SET is essentially the same as those of other trypsins of mammalian origin. The 211 C alpha atoms of SET exhibit an r.m.s. deviation of 1.16 A with equivalent atoms of BT, and the 208 equivalent C alpha atoms between SET and SGT exhibit an r.m.s. deviation of 1.09 A. The large deviations in C alpha positions between SET and BT or between SET and SGT are mainly observed in the first domain. The conformations of the side-chains of the catalytic triad are mutually similar to each other in these three proteases. Each of the chi 1 torsion angles of the three residues is distributed within +/- 5 degrees from each corresponding mean value. The hydrogen bond distances related to the side-chains in the triad coincide fairly well, though the relative disposition of the side-chains differs by 0.1-0.6 A among SET, BT, and SGT. The hydrogen bond network concerned with Ser(195), Asp(189), and water molecules in the substrate binding pocket differs from that in BT or SGT.

The unfolding and refolding characteristics of Escherichia coli tryptophanase (tryptophan indole-lyase) [EC 4.1.99.1] in guanidine hydrochloride were studied. Tryptophanase unfolded by first dissociating its coenzyme, pyridoxal 5'-phosphate, from the active site. This dissociation caused a significant destabilization of structure, and global unfolding of the protein followed. During this global unfolding step, an intermediate was formed which had a strong tendency to aggregate irreversibly, as detected by light scattering experiments. Tryptophanase was unable to refold quantitatively after unfolding in 4 M guanidine hydrochloride. The low refolding yield was due to non-specific aggregation which occurs during refolding. Various conditions which limited this aggregation were probed, and it was found that by initiating the refolding reaction at low temperature, the aggregation of tryptophanase folding intermediates during the reaction could be avoided to a certain extent, and the refolding yield improved.

The guanidine-HCl (Gu-HCl)-induced denaturation of a tetrameric enzyme, aspartase from Escherichia coli has been studied by size-exclusion chromatography, and circular dichroism and fluorescence spectroscopies. The size-exclusion analysis showed that in the presence of 0.4 M Gu-HCl, the enzyme has a dimeric structure with 45% of the native activity. The fluorescence and CD studies showed that only a small change occurred in the secondary and tertiary structures in 0.4 M Gu-HCl. In the range of 0.4 to 1 M Gu-HCl, decrease in the activity was observed as the secondary and tertiary structures were disrupted, whereas the dimeric enzyme did not dissociate into inactive monomer until 1 M Gu-HCl. When the enzyme was denatured in less than 1 M Gu-HCl, more than 90% of the original activity was recovered from the renaturation reaction, indicating that the dissociation process from tetramer to dimer is reversible. In contrast, the renaturation yield was 43% when the enzyme was diluted from more than 1 M Gu-HCl, indicating that the process of dissociation into monomer is not reversible. Thus, we identified an active dimeric form as a denaturation intermediate in this study, although the intermediates (including dimer) that were detected in renaturation experiments at low temperature were inactive, as reported previously [Imaishi, H., Yumoto, N., & Tokushige, M. (1989) Physiol. Chem. Phys. Med. NMR 21, 221-228].

The biodegradative threonine deaminase from Escherichia coli is activated allosterically by AMP. To identify the residues interacting with the phosphate group of AMP at the binding site, we used the affinity labeling reagent, adenosine diphosphopyridoxal (AP2-PL). In the absence of AMP, the enzyme formed the Schiff base with AP2-PL and Scatchard plot analysis showed a biphasic pattern, the respective Kd values for the high- and low-affinity binding phases being 20 and 110 microM. The former value is comparable to the Kd value of the enzyme for AMP. In the presence of AMP, the Schiff base formation was greatly reduced. Although the maximal activating effect of adenosine diphosphopyridoxine, a non-reactive derivative of AP2-PL, was about 13% of that of AMP, the half-saturation concentration was almost the same. These findings suggest that AP2-PL specifically labeled the lysyl residue(s) at the AMP-binding site of the enzyme. To identify the labeled residue(s), we reduced the modified enzyme with sodium borohydride, then cleaved it with cyanogen bromide and Achromobacter lyticus protease I. Reverse-phase HPLC was used to isolate two labeled peptides from the digest. Their amino acid compositions and sequences showed that Lys-111 and Lys-113 were labeled. We conclude that these two lysyl residues are located around the phosphate group of AMP at the allosteric regulation site of the enzyme.

The tyrosine residues of lambda cro repressor were partially nitrated with tetranitromethane under mild conditions. After digestion by Achromobacter protease I, the extent of nitration was determined by HPLC and amino acid analysis. Tyr 26 was most easily nitrated and Tyr 51 followed it. Tyr 10 was resistant to nitration. By comparison of the proton magnetic resonance spectrum of the partially nitrated cro protein with the above result, the aromatic proton resonances of the tyrosine side chains could be assigned to individual tyrosine residues. The extent of nitration is parallel to the accessibility to a flavin dye as measured by photo CIDNP (chemically induced dynamic nuclear polarization).

In order to understand the conformations and stabilities of the immunoglobulin domains, a derivative of the type-lambda constant fragment in which the peptide bond Arg190-Ser191 is cleaved and a derivative of the type-kappa constant fragment in which the peptide bond Arg142-Glu143 is cleaved were prepared by limited proteolysis with the proteinase from mouse submaxillary gland, endoproteinase Arg-C [EC 3.4.21.40]. The cleaved peptide bond of each derivative is located in the loop formed by the intrachain disulfide bond and the two peptides formed are linked by the disulfide bond. The two nicked CL fragments did not assume any ordered conformation. On the other hand, a derivative in which the intrachain disulfide bond is reduced had a conformation very similar to that of the intact CL fragment, although the stability was considerably low. Since the entropy of the nicked fragment should be nearly the same as that of the reduced CL fragment in the unfolded state, a destabilizing effect of cleavage of the polypeptide bond in the folded state in addition to the entropic effect in the unfolded state seems necessary to account for the conformations of the nicked CL fragments.

1H and 13C NMR titrations were performed on PMS-St. trypsin derived by PMSF modification of Ser 195 in Streptomyces erythraeus trypsin, which is devoid of auto-catalytic degradation activity at pH approximately 8. NMR titration of the imidazole C2 proton showed that His 57 had the pKa value of 6.9 in both native St. trypsin and PMS-St. trypsin, suggesting that the adjacent hydroxyl group of Ser 195 had no effect or a very weak effect on the acid-base properties of the imidazole ring in the catalytic triad. A small change in the chemical shift of the isotopically enriched methylene carbon in the PMS moiety of [methylene-13C]PMS-St. trypsin was observed between pH 6 and 8. The titration curve had an inflexion at pH 6.9 and the mode of transition was apparently sigmoidal, although a Hill coefficient of more than unity was suggested. It is thus likely that His 57 is responsible for this transition. Based on these results, the role of His 57 in the catalytic triad of the active site in serine protease is discussed.