Bacterial resistance to ␤-lactam antibiotics is a serious problem limiting current clinical therapy. The most common form of resistance is the production of ␤-lactamases that inactivate ␤-lactam antibiotics. Toho-1 is an extended-spectrum ␤-lactamase that has acquired efficient activity not only to penicillins but also to cephalosporins including the expanded-spectrum cephalosporins that were developed to be stable in former ␤-lactamases. We present the acyl-intermediate structures of Toho-1 in complex with cefotaxime (expanded-spectrum cephalosporin), cephalothin (non-expanded-spectrum cephalosporin), and benzylpenicillin at 1.8-, 2.0-, and 2.1-Å resolutions, respectively. These structures reveal distinct features that can explain the ability of Toho-1 to hydrolyze expanded-spectrum cephalosporins. First, the ⍀-loop of Toho-1 is displaced to avoid the steric contacts with the bulky side chain of cefotaxime. Second, the conserved residues Asn 104 and Asp 240 form unique interactions with the bulky side chain of cefotaxime to fix it tightly. Finally, the unique interaction between the conserved Ser 237 and cephalosporins probably helps to bring the ␤-lactam carbonyl group to the suitable position in the oxyanion hole, thus increasing the cephalosporinase activity.

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The crystallographic structure of the class A beta-lactamase Toho-1, an extended-spectrum beta-lactamase with potent activity against expanded-spectrum cephems, has been determined at 1.65 A resolution. The result reveals that the Lys73 side chain can adopt two alternative conformations. The predominant conformation of Lys73 is different from that observed in the E166A mutant, indicating that removal of the Glu166 side chain changes the conformation of the Lys73 side chain and thus the interaction between Lys73 and Glu166. The Lys73 side chain would play an important role in proton relay, switching its conformation from one to the other depending on the circumstances. The electron density map also implies possible rotation of Ser237. Comparison of the Toho-1 structure with the structure of other class A beta-lactamases shows that the hydroxyl group of Ser237 is likely to rotate through interaction with the carboxyl group of the substrate. Another peculiarity is the existence of three sulfate ions positioned in or near the substrate-binding cavity. One of these sulfate ions is tightly bound to the active center, while the other two are held by a region of positive charge formed by two arginine residues, Arg274 and Arg276. This positively charged region is speculated to represent a pseudo-binding site of the beta-lactam antibiotics, presumably catching the methoxyimino group of the third-generation cephems prior to proper binding in the substrate-binding cleft for hydrolysis. This high-resolution structure, together with detailed kinetic analysis of Toho-1, provides a new hypothesis for the catalytic mechanism and substrate specificity of Toho-1.

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Neoculin (NCL), a protein with sweetness approximately 500-fold that of sugar, can be utilized as a nonglycemic sweetener. It also has taste-modifying activity to convert sourness to sweetness. NCL is a heterodimer composed of an N-glycosylated acidic subunit (NAS) and a basic subunit (NBS), which are conjugated by disulfide bonds. For the production of recombinant NCL (rNCL) by Aspergillus oryzae, ␣-amylase with a KEX2 cleavage site, -K-R-, was fused upstream of each of NAS and NBS and the resulting fusion proteins were simultaneously expressed. For accurate and efficient cleavage of the fusion construct by KEX2-like protease, a triglycine motif was inserted after the KEX2 cleavage site. As NBS showed lower production efficiency than did NAS, a larger amount of the NBS expression plasmid than of NAS expression plasmid was introduced during cotransformation, resulting in successful production of rNCL in the culture medium. Moreover, to obtain a higher production yield of rNCL, the active form of hacA cDNA encoding a transcription factor that induces an unfolded protein response was cloned and expressed constitutively. This resulted in a 1.5-fold increase in the level of rNCL production (2.0 mg/liter). rNCL was purified by chromatography, and its NAS was found to be N-glycosylated as expected. The original sweetness and taste-modifying activity of rNCL were comparable to those of native NCL when confirmed by calcium imaging with human embryonic kidney cells expressing the human sweet taste receptor and by sensory tests.Neoculin (NCL) is a sweet protein present in the fruits of Curculigo latifolia, a tropical plant that is native to West Malaysia. In our previous study, we characterized NCL as a heterodimeric glycoprotein composed of an N-glycosylated acidic subunit (NAS) of ca. 13 kDa and a basic subunit (NBS) of ca. 11 kDa conjugated by disulfide bonds (25). NCL per se is approximately 500 times sweeter than sugar by weight (11,33).