The crystal structure of rat liver S-adenosyl-L-homocysteine hydrolase (AdoHcyase, EC 3.3.1.1) which catalyzes the reversible hydrolysis of S-adenosylhomocysteine (AdoHcy) has been determined at 2.8 A resolution. AdoHcyase from rat liver is a tetrameric enzyme with 431 amino acid residues in each identical subunit. The subunit is composed of the catalytic domain, the NAD+-binding domain, and the small C-terminal domain. Both catalytic and NAD+-binding domains are folded into an ellipsoid with a typical alpha/beta twisted open sheet structure. The C-terminal section is far from the main body of the subunit and extends into the opposite subunit. An NAD+ molecule binds to the consensus NAD+-binding cleft of the NAD+-binding domain. The peptide folding pattern of the catalytic domain is quite similar to the patterns observed in many methyltransferases. Although the crystal structure does not contain AdoHcy or its analogue, there is a well-formed AdoHcy-binding crevice in the catalytic domain. Without introducing any major structural changes, an AdoHcy molecule can be placed in the catalytic domain. In the structure described here, the catalytic and NAD+-binding domains are quite far apart from each other. Thus, the enzyme appears to have an "open" conformation in the absence of substrate. It is likely that binding of AdoHcy induces a large conformational change so as to place the ribose moiety of AdoHcy in close proximity to the nicotinamide moiety of NAD+. A catalytic mechanism of AdoHcyase has been proposed on the basis of this crystal structure. Glu155 acts as a proton acceptor from the O3'-H when the proton of C3'-H is abstracted by NAD+. His54 or Asp130 acts as a general acid-base catalyst, while Cys194 modulates the oxidation state of the bound NAD+. The polypeptide folding pattern of the catalytic domain suggests that AdoHcy molecules can travel freely to and from AdoHcyase and methyltransferases to properly regulate methyltransferase activities. We believe that the crystal structure described here can provide insight into the molecular architecture of this important regulatory enzyme.

Methyltransfer reactions are some of the most important reactions in biological systems. Glycine N-methyltransferase (GNMT) catalyzes the S-adenosyl-l-methionine- (SAM-) dependent methylation of glycine to form sarcosine. Unlike most SAM-dependent methyltransferases, GNMT has a relatively high value and is weakly inhibited by the product S-adenosyl-l-homocysteine (SAH). The major role of GNMT is believed to be the regulation of the cellular SAM/SAH ratio, which is thought to play a key role in SAM-dependent methyltransfer reactions. Crystal structures of GNMT complexed with SAM and acetate (a potent competitive inhibitor of Gly) and the R175K mutated enzyme complexed with SAM were determined at 2.8 and 3.0 A resolutions, respectively. With these crystal structures and the previously determined structures of substrate-free enzyme, a catalytic mechanism has been proposed. Structural changes occur in the transitions from the substrate-free to the binary complex and from the binary to the ternary complex. In the ternary complex stage, an alpha-helix in the N-terminus undergoes a major conformational change. As a result, the bound SAM is firmly connected to protein and a "Gly pocket" is created near the bound SAM. The second substrate Gly binds to Arg175 and is brought into the Gly pocket. Five hydrogen bonds connect the Gly in the proximity of the bound SAM and orient the lone pair orbital on the amino nitrogen (N) of Gly toward the donor methyl group (C(E)) of SAM. Thermal motion of the enzyme leads to a collision of the N and C(E) so that a S(N)2 methyltransfer reaction occurs. The proposed mechanism is supported by mutagenesis studies.

The fall webworm, Hyphantria cunea Drury (Lepidoptera: Arctiidae), was introduced from North America to Japan half a century ago. The critical photoperiod for diapause induction and its temperature dependence, as defined by the difference in the critical photoperiod between 20 and 25°C, were investigated in order to understand the mechanisms behind a shift from bi- to trivoltine life cycles. The critical photoperiod for diapause induction was shorter in the southern trivoltine populations than in the northern bivoltine populations, and this was more marked at 25°C than at 20°C. Although the critical photoperiod showed a positive correlation with the original latitude, the correlation was relatively low at both temperatures. Conversely, temperature dependence of the critical photoperiod for diapause induction correlated negatively with the original latitude. The trivoltine populations showed greater temperature sensitivity than the bivoltine populations. These results suggest that an increase in temperature sensitivity of the diapause response to photoperiods was involved in the shift to a trivoltine life cycle. The crossing experiments suggested that the photoperiodic control of diapause induction and its temperature dependence are under polygenic control without sex-linkage.

Porphyromonas gingivalis is a major pathogen associated with adult periodontitis. We cloned and sequenced the gene (dpp) coding for dipeptidyl aminopeptidase IV (DPPIV) from P. gingivalis W83, based on the amino acid sequences of peptide fragments derived from purified DPPIV. An Escherichia coli strain overproducing P. gingivalis DPPIV was constructed. The enzymatic properties of recombinant DPPIV purified from the overproducer were similar to those of DPPIV isolated from P. gingivalis. The three amino acid residues Ser, Asp, and His, which are thought to form a catalytic triad in the C-terminal catalytic domain of eukaryotic DPPIV, are conserved in P. gingivalis DPPIV. When each of the corresponding residues of the enzyme was substituted with Ala by site-directed mutagenesis, DPPIV activity significantly decreased, suggesting that these three residues of P. gingivalis DPPIV are involved in the catalytic reaction. DPPIV-deficient mutants of P. gingivalis were constructed and subjected to animal experiments. Mice injected with the wild-type strain developed abscesses to a greater extent and died more frequently than those challenged with mutant strains. Mice injected with the mutants exhibited faster recovery from the infection, as assessed by weight gain and the rate of lesion healing. This decreased virulence of mutants compared with the parent strain suggests that DPPIV is a potential virulence factor of P. gingivalis and may play important roles in the pathogenesis of adult periodontitis induced by the organism.

The effects of global warming on the life cycle and life-history traits of the fall webworm, Hyphantria cunea (Drury) (Lepidoptera: Arctiidae), were investigated in Fukui, Japan. Our previous studies showed that the Fukui population had a predominantly bivoltine life cycle, but recently the life cycle has changed. In the present study, the life-history traits of individuals collected in 2002 in Fukui were clarified and compared to those in the previous studies. The lower threshold temperature for development and the thermal constant for one generation were 10.6 ° C and 724.4 degree days, respectively. Although these developmental parameters were not investigated in the previous studies, the difference of the developmental period between the present and previous results was negligible in the larval and pupal stages at 20 ° C. The critical photoperiod for diapause induction was 14 h 29 min at 20 ° C and 14 h 10 min at 25 ° C. The critical photoperiod at 25 ° C was shortened to 14 min from that of the previous studies for individuals collected in 1995. The incidence of pupal diapause in the second generation was investigated in individuals that were collected as fourth and fifth instars in the field. Some portion of individuals averted diapause even if they were kept under a short photoperiod of L14:D10 at 25 ° C after collection. These results, together with climate data and field observations in Fukui, suggest that at least a part of the population has three generations per year at present. This shift of the life cycle occurred within 7 years and is probably related to global warming in recent years.

The fall webworm, Hyphantria cunea (Drury) (Lepidoptera: Arctiidae), invaded Japan from North America about 60 years ago. Immediately after its invasion -and for the first three decades -its life cycle was bivoltine, two generations per year throughout its entire distribution in Japan. Thereafter, its life cycle shifted to trivoltine in the southwestern areas of Japan. In the present study we examined the life-history traits proposed to be implicated in this event with the aim of clarifying the mechanism of this life-cycle shift. The critical photoperiod for diapause induction, as defined by the photoperiod at which 50% of individuals enter diapause, was shorter in the trivoltine populations than in their bivoltine counterparts. The temperature sensitivity of the photoperiodic response, as defined by the difference in the critical photoperiod between 20 and 25°C, was greater in the trivoltine populations than in the bivoltine ones. The geographic variation in larval and pupal periods was positively correlated to the latitude of the original localities of the populations. The change in the number of larval instars would be one of the main factors accounting for the regional differences in the developmental period. These results suggest that some life-history traits of H. cunea have changed following its invasion of Japan as an adaptive response to local climates.

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