bThe enzymes LinB UT and LinB MI (LinB from Sphingobium japonicum UT26 and Sphingobium sp. MI1205, respectively) catalyze the hydrolytic dechlorination of ␤-hexachlorocyclohexane (␤-HCH) and yield different products, 2,3,4,5,6-pentachlorocyclohexanol (PCHL) and 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL), respectively, despite their 98% identity in amino acid sequence. To reveal the structural basis of their different enzymatic properties, we performed site-directed mutagenesis and X-ray crystallographic studies of LinB MI and its seven point mutants. The mutation analysis revealed that the seven amino acid residues uniquely found in LinB MI were categorized into three groups based on the efficiency of the first-step (from ␤-HCH to PCHL) and second-step (from PCHL to TCDL) conversions. Crystal structure analyses of wild-type LinB MI and its seven point mutants indicated how each mutated residue contributed to the first-and second-step conversions by LinB MI . The dynamics simulation analyses of wild-type LinB MI and LinB UT revealed that the entrance of the substrate access tunnel of LinB UT was more flexible than that of LinB MI , which could lead to the different efficiencies of dehalogenation activity between these dehalogenases.H exachlorocyclohexane (HCH) is a six-chlorine-substituted cyclohexane. One of its isomers, the ␥ isomer, has insecticidal properties and has been widely used as an insecticide around the world (1). Although the use of ␥-HCH has been prohibited in most countries due to its toxicity and long persistence, the largescale production, widespread use, and dumping of the other noninsecticidal isomers (␣-, ␤-, and ␦-HCHs) in past decades still continue to create problems with HCH contamination in soil and groundwater (2). ␤-HCH in particular is a persistent and problematic isomer of HCH.Several ␤-HCH-degrading bacteria whose ␤-HCH-degrading enzymes can be utilized for bioremediation have been identified (3-5). LinB MI and LinB UT are haloalkane dehalogenases isolated from Sphingobium sp. MI1205 and Sphingobium japonicum UT26, respectively, that can cleave the carbon-halogen bond in ␤-HCH. Haloalkane dehalogenases belong to the ␣/␤-hydrolase family, and their catalytic mechanism consists of the following steps: (i) substrate binding, (ii) cleavage of the carbon-halogen bond in the substrate and formation of an intermediate covalently bound to the nucleophile, (iii) hydrolysis of the alkyl-enzyme intermediate, and (iv) release of halide ion and alcohol (6). LinB MI and LinB UT share 98% sequence identity, with only 7 different amino acid residues (at positions 81, 112, 134, 135, 138, 247, and 253) out of 296 residues, but these enzymes exhibit different enzymatic properties (Fig. 1). LinB MI catalyzes the two-step dehalogenation and converts ␤-HCH to 2,3,4,5,6-pentachlorocyclohexanol (PCHL) and further to 2,3,5,6-tetrachlorocyclohexane-1,4-diol (TCDL) (7) in the manner of LinB2 from Sphingomonas sp. BHC-A (8) and LinB from Sphingobium indicum B90A (9), whereas LinB UT catalyzes only the firs...

Calmodulin (CaM) is a Ca(2+)-binding protein that regulates a number of fundamental cellular activities. Nicotiana tabacum CaM (NtCaM) comprises 13 genes classified into three types, among which gene expression and target enzyme activation differ. We performed Fourier-transform infrared spectroscopy to compare the secondary and coordination structures of Mg(2+) and Ca(2+) among NtCaM1, NtCaM3, and NtCaM13 as representatives of the three types of NtCaMs. Data suggested that NtCaM13 has a different secondary structure due to the weak β-strand bands and the weak 1661 cm(-1) band. Coordination structures of Mg(2+) of NtCaM3 and NtCaM13 were similar but different from that of NtCaM1, while the Ca(2+)-binding manner was similar among the three CaMs. The amplitude differences of the band at 1554-1550 cm(-1) obtained by second-derivative spectra indicated that the intensity change of the band of NtCaM13 was smaller in response to [Ca(2+)] increases under low [Ca(2+)] conditions than were those of NtCaM1 and NtCaM3, while the intensity reached the same level under high [Ca(2+)]. Therefore, NtCaM13 has a characteristic secondary structure and specific Mg(2+)-binding manner and needs higher [Ca(2+)] for bidentate Ca(2+) coordination of 12th Glu in EF-hand motifs. The Ca(2+)-binding mechanisms of the EF-hand motifs of the three CaMs are similar; however, the cation-dependent conformational change in NtCaM13 is unique among the three NtCaMs.

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