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To determine the influence of media composition on Salmonella exclusion of Nurmi-type cultures, two and four types of cultures in the first and second trial, respectively, were prepared from the cecal contents of conventional laying hens, and Salmonella exclusion was assessed in newly hatched chicks. In the first trial, modified Viande Levure (VL) broth or nutrient broth (NB) were used to prepare Nurmi-type cultures (N-VL and N-NB), which were administered to the newly hatched chicks. Twenty-four hours later, the chicks were challenged with Salmonella enterica Typhimurium EF85-9 (ST). ST recoveries (log10 colony forming units/g of cecal contents) from the N-VL-, N-NB-, and control-treated groups 5 days after the challenge were 7.6±0.6, 0.9±1.9, and 7.7±0.4, respectively. The results suggested the influence of l-cysteine (Cys) present in the VL broth. Thus, we determined the effect of Cys in the second trial. We prepared two other cultures using VL broth without Cys (N-VL−Cys) and NB with Cys (NNB−Cys). ST recoveries from the cecal contents of the N-VL-, N-VL−Cys-, and control-treated groups were 6.3±0.9, 2.1±2.5, and 9.2±0.8, respectively. ST was not recovered from the N-NB- and N-NB−Cys-treated groups. To identify bacteria with Salmonella exclusion activity, we isolated 41 bacterial strains from the ceca of N-NB-treated chicks without Salmonella challenge. Most isolates were identified as Enterococcus faecalis or E. mundtii based on 16S rRNA gene sequencing, and only four cultures excluded Salmonella. Therefore, VL broth containing Cys was not always required for preparing Nurmi-type cultures. The use of media prepared with Cys at the lowest possible concentration or without Cys would promote to enhance Salmonella exclusion from Nurmi-type cultures.

Although several types of diet have been used in experimental steatohepatitis models, comparison of gut microbiota and immunological alterations in the gut among diets has not yet been performed. We attempted to clarify the difference in the gut environment between mice administrated several experimental diets. Male wild-type mice were fed a high-fat (HF) diet, a choline-deficient amino acid-defined (CDAA) diet, and a methionine-choline-deficient (MCD) diet for 8 weeks. We compared the severity of steatohepatitis, the composition of gut microbiota, and the intestinal expression of interleukin (IL)-17, an immune modulator. Steatohepatitis was most severe in the mice fed the CDAA diet, followed by the MCD diet, and the HF diet. Analysis of gut microbiota showed that the composition of the Firmicutes phylum differed markedly at order level between the mice fed the CDAA and HF diet. The CDAA diet increased the abundance of Clostridiales, while the HF diet increased that of lactate-producing bacteria. In addition, the CDAA diet decreased the abundance of lactate-producing bacteria and antiinflammatory bacterium Parabacteroides goldsteinii in the phylum Bacteroidetes. In CDAA-fed mice, IL-17 levels were increased in ileum as well as portal vein. In addition, the CDAA diet also elevated hepatic expression of chemokines, downstream targets of IL-17. The composition of gut microbiota and IL-17 expression varied considerably between mice administrated different experimental diets to induce steatohepatitis.

The formation of guaiacol, a potent phenolic off-odor compound in the Japanese sake brewing process, was investigated. Eight rice koji samples were analyzed, and one contained guaiacol and 4-vinylguaiacol (4-VG) at extraordinarily high levels: 374 and 2433 μg/kg dry mass koji, respectively. All samples contained ferulic and vanillic acids at concentrations of mg/kg dry mass koji. Guaiacol forming microorganisms were isolated from four rice koji samples. They were identified as Bacillus subtilis, B. amyloliquefaciens/subtilis, and Staphylococcus gallinarum using 16S rRNA gene sequence. These spoilage bacteria convert vanillic acid to guaiacol and ferulic acid to 4-VG. However, they convert very little ferulic acid or 4-VG to guaiacol. Nine strains of koji fungi tested produced vanillic acid at the mg/kg dry mass koji level after cultivation. These results indicated that spoilage bacteria form guaiacol from vanillic acid, which is a product of koji cultivation in the sake brewing process.

To determine the influence of media composition on Salmonella exclusion of Nurmi-type cultures, two and four types of cultures in the first and second trial, respectively, were prepared from the cecal contents of conventional laying hens, and Salmonella exclusion was assessed in newly hatched chicks. In the first trial, modified Viande Levure (VL) broth or nutrient broth (NB) were used to prepare Nurmi-type cultures (N-VL and N-NB), which were administered to the newly hatched chicks. Twenty-four hours later, the chicks were challenged with Salmonella enterica Typhimurium EF85-9 (ST). ST recoveries (log10 colony forming units/g of cecal contents) from the N-VL-, N-NB-, and control-treated groups 5 days after the challenge were 7.6±0.6, 0.9±1.9, and 7.7±0.4, respectively. The results suggested the influence of l-cysteine (Cys) present in the VL broth. Thus, we determined the effect of Cys in the second trial. We prepared two other cultures using VL broth without Cys (N-VL-Cys) and NB with Cys (NNB-Cys). ST recoveries from the cecal contents of the N-VL-, N-VL-Cys-, and control-treated groups were 6.3±0.9, 2.1±2.5, and 9.2±0.8, respectively. ST was not recovered from the N-NB- and N-NB-Cys-treated groups. To identify bacteria with Salmonella exclusion activity, we isolated 41 bacterial strains from the ceca of N-NB-treated chicks without Salmonella challenge. Most isolates were identified as Enterococcus faecalis or E. mundtii based on 16S rRNA gene sequencing, and only four cultures excluded Salmonella. Therefore, VL broth containing Cys was not always required for preparing Nurmi-type cultures. The use of media prepared with Cys at the lowest possible concentration or without Cys would promote to enhance Salmonella exclusion from Nurmi-type cultures.

Aims: Allene oxide cyclase (AOC) (EC 5.3.99.6) is an important enzyme of jasmonates (JAs) biosynthesis. JAs are important signals that play a pivotal role in defense response of plants to environmental cues. Regulation JA biosynthesis is believed useful for elucidating the mechanism of plant defense system. Despite the high potential of AOC as a target for JA biosynthesis inhibitors, an efficient assay method suitable for screening AOC inhibitors is still not available. The aim of this work is to develop an efficient AOC assay method. Study Design: Using excess amount of purified recombinant allene oxide synthase (AOS) combined with 13(S)-hydroperxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid (13-HPOT), we established an efficient method to generate (12,13S)-epoxyoctadecatrienoic acid (EOT), the substrate of AOC. The AOS produced EOT was subsequently converted to (9S,13S)-12-oxo(10,15Z)-phytodienoic acid (OPDA) by using purified recombinant AOC in a real time manner and the amount of OPDA was determined by HPLC. Place and Duration of Study: All the experiments were conducted from October 2009 to March 2013 at Akita Prefectural University, Japan. Methodology: The recombinant AOS and AOC were expressed in E. coli. The target proteins were Original Research Article Keimei and Yoichiro; BBJ, 9(2): 1-7, 2015; Article no.BBJ.20089 2 purified using affinity chromatography, respectively. The unstable EOT was generated by using excess AOS combined with 13(S)-hydroperxy-9(Z), 11(E), 15(Z)-octadecatrienoic acid. The AOC synthesized OPDA was characterized by the comparison of HPLC retention time with the OPDA standard. AOC activity was calculated by determine the amount of OPDA in the assay system. Results: We found in the presence of 50 nmol of purified AOS together with 20 M 13-HPOT, the synthesis of OPDA was saturated when using 5 nmol of purified AOC in the enzyme reaction for 30 min. Our results indicated that the AOC activity can be determined by dual enzyme system. Conclusion: We established an efficient assay method for AOC which may be applied for screening of AOC inhibitors.

The 2011 Tohoku tsunami had a serious impact, such as an increase in harmful substances and salinity over a large area. Herein, we evaluated transitions in bacterial communities in agricultural fields in the four years after the 2011 Tohoku tsunami. Bacterial communities were compared across four different types of soil&mdash, unflooded field (UF) soil, soil flooded for a short term (ST), soil flooded for the long term (LT), soil flooded long term and cultivated fields (LTC), and marine environmental materials (bay sediment, sea sand and sea water), using a polymerase chain reaction (PCR) and pyrosequencing of 16S ribosomal RNA genes. In the soil bacterial communities that were flooded by the 2011 Tohoku tsunami, these effects were not seen after 2013. Although the difference in bacterial communities between LT and UF became smaller during the four years, the bacterial communities in LT were different from those in UF in several ways, such as a higher tendency frequency of sulfur-oxidizing bacteria (SOB) and the presence of halotolerant SOB. Therefore, it is thought that the Tohoku tsunami affected the microbial communities in the soil for more than four years. Especially genus Halothiobacillus, which is Halotolerant SOB in flooded soils, was detected neither in unflooded soil nor in the marine environment. Therefore, it is thought that inundation by a tsunami produces a unique environment with bacterial communities to form in soil. Further, SOB structure, especially halotolerant, might serve as a good indicators of the impacts of inundation on bacterial communities in agricultural fields over the long term.

The effects of inundation caused by the 2011 Tohoku tsunami on soil bacterial communities in agricultural fields were evaluated. Bacterial communities were compared across three different types of soil, unflooded field (UF) soil, soil flooded for 2 weeks (short term (ST)), and soil flooded for 2 months (long term (LT)), using polymerase chain reaction-pyrosequencing of 16S rRNA genes. Acidobacteria were dominant in UF, with a relative abundance of approximately 35 %, and Proteobacteria dominated flooded soils (30-67 %). Hierarchical cluster analysis indicated that the community structure of soil bacteria in flooded soils (ST and LT) clearly differed from that in UF. Differences between LT and ST fields were rarely observed in terms of chemical properties and microbial community structure at the phylum level. However, sulfur-oxidizing bacteria (SOB) and nitrite-oxidizing bacteria (NOB) in LT tended to occur at high and low abundances, respectively. Halothiobacillus, a halotolerant SOB, was detected in all LT fields. Unexpectedly, a zeta-Proteobacteria, which had previously only been detected in marine environments, was detected in LT fields only. Our results demonstrate that the effects of the 2011 Tohoku tsunami on soil bacterial communities in agricultural fields may have lasted at least 1 year. Furthermore, SOB, NOB, and zeta-Proteobacteria may serve as indicators of the effects of seawater inundation on microorganisms.

We estimated the subsite structure of α-amylase II (TVA II) from Thermoactinomyces vulgaris R-47 expressed in Escherichia coli. TVA II has eight subsites, and the catalytic site is between the 5th and 6th subsite from the non-reducing end side. The subsite affinities, A-5, A-4, A-3, A-2, (A-1+A+1), A+2, and A+3, were calculated to be -0.35, 0.93, 0.55, 2.56, 1.18, 1.71, and 0.01 kcal mol(-1), respectively.

The preparation of both enantiomers of 8-[1-(2,4-dichlorophenyl)-2-imidazol-1-yl-ethoxy] octanoic acid heptyl ester (JM-8686), a potent inhibitor of allene oxide synthase, has been achieved using 2,4-dichlorophenacyl bromide as a starting material. The key step was the asymmetric reduction of 1-(2,4-dichlorophenyl)-2-imidazol-1-yl-ethanone with chiral BINAL-H. The products were purified by chiral high-performance liquid chromatography (HPLC) to afford pure ( R )-JM-8686 and ( S )-JM-8686. The inhibitory activities and binding affinities of these enantiomers toward allene oxide synthase were determined. We found that the inhibition potency of ( R )-JM-8686 is approximately 200 times greater than that of ( S )-JM-8686, with IC 50 values of approximately 5 ± 0.2 nM and 950 ± 18 nM, respectively. The dissociation constants of ( R )-JM-8686 and ( S )-JM-8686 with respect to the recombinant allene oxide synthase were approximately 1.4 ± 0.3 μM and 4.8 ± 0.6 μM, respectively.

Burkholderiales bacterium GJ-E10, isolated from the Tamagawa River in Akita Prefecture, Japan, is an unclassified, iron-oxidizing chemolithoautotrophic bacterium. Its single circular genome, consisting of 3,276,549 bp, was sequenced by using three types of next-generation sequencers and the sequences were then confirmed by PCR-based Sanger sequencing.

An α-amylase II (TVA II) produced by Thermoactinomyces vulgaris R-47 exhibits wide substrate specificity for starch, pullulan, cyclodextrins and isopanose. Asp465 and Arg469 are located at subsite (-2) and are observed among many α-amylase family enzymes. Here, we have investigated the relationship between the substrate specificity of TVA II and the substrate binding by Asp465 and Arg469 using site-directed mutagenesis and X-ray crystallographic analyses. The five mutated TVA IIs (D465N, D465E, D465Q, R469L and R469K) showed lower specific activities for all substrates tested here than the wild-type TVA II. In particular, the mutation of Arg469 significantly decreased the hydrolyzing activities compared to the mutation of Asp465. While the Km values for α-cyclodextrin were increased for all the mutants, large decreases of the kcat values were observed only for R469L and R469K. The side chain of Arg469 is close to that of Asp421, which is one of three catalytic residues, and the positive charge of Arg469 seems to affect the catalytic mechanism via Asp421. These findings suggest that Asp465 and Arg469 are important residues for the substrate binding and the catalytic reaction. Furthermore, the binding of the α-maltosyl unit by Asp465 and Arg469 is commonly observed among TVA II and other α-amylase family enzymes, which is thought to be the basic mechanism of substrate binding.

Membrane type 1 matrix metalloproteinase (MT1-MMP) is a type I transmembrane MMP shown to play a critical role in normal development and in malignant processes. Emerging evidence indicates that MT1-MMP is regulated by a process of ectodomain shedding. Active MT1-MMP undergoes autocatalytic processing on the cell surface, leading to the formation of an inactive 44-kDa fragment and release of the entire catalytic domain. Analysis of the released MT1-MMP forms in various cell types revealed a complex pattern of shedding involving two major fragments of 50 and 18 kDa and two minor species of 56 and 31-35 kDa. Protease inhibitor studies and a catalytically inactive MT1-MMP mutant revealed both autocatalytic (18 kDa) and non-autocatalytic (56, 50, and 31-35 kDa) shedding mechanisms. Purification and sequencing of the 18-kDa fragment indicated that it extends from Tyr(112) to Ala(255). Structural and sequencing data indicate that shedding of the 18-kDa fragment is initiated at the Gly(284)-Gly(285) site, followed by cleavage between the conserved Ala(255) and Ile(256) residues near the conserved methionine turn, a structural feature of the catalytic domain of all MMPs. Consistently, a recombinant 18-kDa fragment had no catalytic activity and did not bind TIMP-2. Thus, autocatalytic shedding evolved as a specific mechanism to terminate MT1-MMP activity on the cell surface by disrupting enzyme integrity at a vital structural site. In contrast, functional data suggest that the non-autocatalytic shedding generates soluble active MT1-MMP species capable of binding TIMP-2. These studies suggest that ectodomain shedding regulates the pericellular and extracellular activities of MT1-MMP through a delicate balance of active and inactive enzyme-soluble fragments.

Matrix metalloproteinases (MMPs) are important hydrolytic enzymes with profound physiological and pathological functions in living organisms. MMPs are produced in their inactive zymogenic forms, which are subsequently proteolytically activated in an elaborate set of events. The propeptide in the zymogen blocks the active site, with a cysteine side-chain thiolate from this propeptide achieving coordination with the catalytically important zinc ion in the active site. Molecular dynamics simulations, ab initio calculations, and wet chemistry experiments presented herein argue for the critical importance of a protonation event at the coordinated thiolate as a prerequisite for the departure of the propeptide from the active site. Furthermore, a catalytically important glutamate is shown to coordinate transiently to the active-site zinc ion to "mask" the positive potential of the zinc ion and lower the energy barrier for dissociation of the protonated cysteine side chain from the zinc ion. In addition, a subtle conformational change by the propeptide is needed in the course of zymogen activation. These elaborate processes take place in concert in the activation process of MMPs, and the insight into these processes presented herein sheds light on a highly regulated physiological process with profound consequences for eukaryotic organisms. Proper interactions of eukaryotic cells with the extracellular matrix (ECM) are important for the normal functions within a tissue. ECM-cell interactions are regulated by a family of at least 27 zinc-dependent endopeptidases known as matrix met- alloproteinases (MMPs), the functions of which have profound consequences in organ development, tissue remodeling, em- briogenesis, wound healing, and angiogenesis. 1 Because of the critical functions of these enzymes in many physiological processes, their activities are strictly regulated. 2 Each is produced as an inactive zymogen form (pro-MMP), which requires activation for generation of the catalytically competent enzyme. The process of zymogen activation appears to be complicated. 3,4 A second level of control of MMP activity is exerted by inhibition of the active enzymes by a group of four specific proteins known as the tissue inhibitors of metalloproteinases ("TIMPs"). When these elaborate regulatory processes fail, the

The tissue inhibitors of metalloproteinases (TIMPs) are specific inhibitors of MMP enzymatic activity. However, TIMP-2 can promote the activation of pro-MMP-2 by MT1-MMP. This process is mediated by the formation of a complex between MT1-MMP, TIMP-2, and pro-MMP-2. Binding of TIMP-2 to active MT1-MMP also inhibits the autocatalytic turnover of MT1-MMP on the cell surface. Thus, under certain conditions, TIMP-2 is a positive regulator of MMP activity. TIMP-4, a close homologue of TIMP-2 also binds to pro-MMP-2 and can potentially participate in pro-MMP-2 activation. We coexpressed MT1-MMP with TIMP-4 and investigated its ability to support pro-MMP-2 activation. TIMP-4, unlike TIMP-2, does not promote pro-MMP-2 activation by MT1-MMP. However, TIMP-4 binds to MT1-MMP inhibiting its autocatalytic processing. When coexpressed with TIMP-2, TIMP-4 competitively reduced pro-MMP-2 activation by MT1-MMP. A balance between TIMP-2 and TIMP-4 may be a critical factor in determining the degradative potential of cells in normal and pathological conditions.

The structures of Thermoactinomyces vulgaris R-47 alpha-amylase II mutant (d325nTVA II) complexed with substrate analogues, methyl beta-cyclodextrin (m beta-CD) and maltohexaose (G6), were solved by X-ray diffraction at 3.2 A and 3.3 A resolution, respectively. In d325nTVA II-m beta-CD complex, the orientation and binding-position of beta-CD in TVA II were identical to those in cyclodextin glucanotransferase (CGTase). The active site residues were essentialy conserved, while there are no residues corresponding to Tyr89, Phe183, and His233 of CGTase in TVA II. In d325nTVA II-G6 complex, the electron density maps of two glucosyl units at the non-reducing end were disordered and invisible. The four glucosyl units of G6 were bound to TVA II as in CGTase, while the others were not stacked and were probably flexible. The residues of TVA II corresponding to Tyr89, Lys232, and His233 of CGTase were completely lacking. These results suggest that the lack of the residues related to alpha-glucan and CD-stacking causes the functional distinctions between CGTase and TVA II.

The crystal structure of Thermoactinomyces vulgaris R-47 alpha-Amylase II (TVAII) has been determined by multiple isomorphous replacement at 2.6 A resolution. TVAII was crystallized in an orthorhombic system with the space group P212121 and the cell dimensions a=118.5 A, b=119.5 A, c=114.5 A. There are two molecules in an asymmetric unit, related by the non-crystallographic 2-fold symmetry. Diffraction data were collected at 113 K and the cell dimensions reduced to a=114.6 A, b=117.9 A, c=114.2 A, and the model was refined against 7.0-2.6 A resolution data giving an R-factor of 0.204 (Rfree=0.272). The final model consists of 1170 amino acid residues (two molecules) and 478 water molecules with good chemical geometry. TVAII has three domains, A, B, and C, like other alpha-amylases. Domain A with a (beta/alpha)8 barrel structure and domain C with a beta-sandwich structure are very similar to those found in other alpha-amylases. Additionally, TVAII has an extra domain N composed of 121 amino acid residues at the N-terminal site, which has a beta-barrel-like structure consisting of seven antiparallel beta-strands. Domain N is one of the driving forces in the formation of the dimer structure of TVAII, but its role in the enzyme activity is still not clear. TVAII does not have the Ca2+ binding site that connects domains A and B in other alpha-amylases, rather the NZ atom of Lys299 of TVAII serves as the connector between these domains. TVAII can hydrolyze cyclodextrins and pullulan as well as starch. Based on a structural comparison with the complex between a mutant cyclodextrin glucanotransferase and a beta-cyclodextrin derivative, Phe286 located at domain B is considered the residue most likely to recognize the hydrophobic cavity of cyclodextrins. The active-site cleft of TVAII is wider and shallower than that of other alpha-amylases, and seems to be suitable for the binding of pullulan which is expected not to adopt the helical structure of amylose.

Thermoactinomyces vulgaris R-47 produces two alpha-amylases, TVA I, an extracellular enzyme, and TVA II, an intracellular enzyme. Both enzymes hydrolyze pullulan to produce panose, and also hydrolyze cyclodextrins. We cloned and sequenced the TVA I gene. The TVA I gene consisted of 1833 base pairs, and the deduced primary structure was composed of 611 amino-acid residues, including an N-terminal signal sequence consisting of 29 amino-acid residues. The similarity between the amino-acid sequence of mature TVA I with those of other pullulan/cyclodextrin-hydrolyzing enzymes, such as TVA II and Bacillus stearothermophilus neopullulanase, was only 30%, although that of TVA II with neopullulanase was 48%. TVA II prefers specific small oligosaccharides and alpha- and beta-cyclodextrins. Whereas kcat/Km values of TVA I for pullulan were larger than that of TVA II, and TVA II could not hydrolyze starch completely. TVA II was inhibited by maltose, the hydrolysate of starch, which seems to be the reason for inefficient hydrolysis of starch. These kinetic properties indicate that TVA I and TVA II have differential physiological roles in sugar metabolism extracellularly and intracellularly, respectively.

The transition of the bacterial community structure and predominant bacteria in the ceca of chicks from hatching to 2 wk of age was investigated using denaturing gradient gel electrophoresis with the 16S ribosomal RNA gene, followed by phylogenetic analysis. The results demonstrated that most of the cecal bacterial flora from hatching to a few days old consisted of Escherichia coli (sequence similarity: 100%), and the floral diversity was still low 2 wk posthatch. These findings will help contribute to the development of a novel competitive exclusion product.

Transglycosylation reactions are useful for preserving a specific sugar structure during the synthesis of branched oligosaccharides. We have previously reported a panosyl unit transglycosylation reaction by pullulan-hydrolyzing amylase II (TVA II) cloned from Thermoactinomyces vulgaris R-47 (Tonozuka et al., Carbohydr. Res., 1994, 261, 157-162). The acceptor specificity of the TVA II transglycosylation reaction was investigated using pullulan as the donor and sugar alcohols as the acceptor. TVA II transferred the α-panosyl unit to the C-1 hydroxyl group of meso-erythritol, C-1 and C-2 of xylitol, and C-1 and C-6 of d-sorbitol. TVA II differentiated between the sugar alcohols' hydroxyl groups to produce five novel non-reducing branched oligosaccharides, 1-O-α-panosylerythritol, 1-O-α-panosylxylitol, 2-O-α-panosylxylitol, 1-O-α-panosylsorbitol, and 6-O-α-panosylsorbitol. The Trp(356)→Ala mutant showed similar transglycosylation reactions; however, panose production by the mutant was 4.0-4.5-fold higher than that of the wild type. This suggests that Trp(356) is important for recognizing both water and the acceptor molecules in the transglycosylation and the hydrolysis reaction.

Membrane type 1 matrix metalloproteinase (MT1-MMP) is a type I transmembrane MMP shown to play a critical role in normal development and in malignant processes. Emerging evidence indicates that MT1-MMP is regulated by a process of ectodomain shedding. Active MT1-MMP undergoes autocatalytic processing on the cell surface, leading to the formation of an inactive 44-kDa fragment and release of the entire catalytic domain. Analysis of the released MT1-MMP forms in various cell types revealed a complex pattern of shedding involving two major fragments of 50 and 18 kDa and two minor species of 56 and 31-35 kDa. Protease inhibitor studies and a catalytically inactive MT1-MMP mutant revealed both autocatalytic (18 kDa) and non-autocatalytic (56, 50, and 31-35 kDa) shedding mechanisms. Purification and sequencing of the 18-kDa fragment indicated that it extends from Tyr(112) to Ala(255). Structural and sequencing data indicate that shedding of the 18-kDa fragment is initiated at the Gly(284)-Gly(285) site, followed by cleavage between the conserved Ala(255) and Ile(256) residues near the conserved methionine turn, a structural feature of the catalytic domain of all MMPs. Consistently, a recombinant 18-kDa fragment had no catalytic activity and did not bind TIMP-2. Thus, autocatalytic shedding evolved as a specific mechanism to terminate MT1-MMP activity on the cell surface by disrupting enzyme integrity at a vital structural site. In contrast, functional data suggest that the non-autocatalytic shedding generates soluble active MT1-MMP species capable of binding TIMP-2. These studies suggest that ectodomain shedding regulates the pericellular and extracellular activities of MT1-MMP through a delicate balance of active and inactive enzyme-soluble fragments.

Phe286 located in the center of the active site of α-amylase 2 from Thermoactinomyces vulgaris R-47 (TVAII) plays an important role in the substrate recognition for cyclomaltooligosaccharides (cyclodextrins). The X-ray structures of mutant TVAIIs with the replacement of Phe286 by Ala (F286A) and Tyr (F286Y) were determined at 3.2 A resolution. Their structures have no significant differences from that of the wild-type enzyme. The kinetic analyses of Phe286-replaced variants showed that the variants with non-aromatic residues, Ala (F286A) and Leu (F286L), have lower enzymatic activities than those with aromatic residues, Tyr (F286Y) and Trp (F286W), and the replacement of Phe286 affects enzymatic activities for CDs more than those for starch.

To confirm that the catalytic residues (Asp325, Glu354, and Asp421) are necessary for the hydrolysis of starch, pullulan, and cyclodextrins, we constructed TVA II mutated by site-directed mutagenesis. The mutated enzymes (D325N, E354Q, and D421N) had markedly reduced levels of activity, less than 0.006% of the wild type, indicating that these three residues are the catalytic sites for these substrates. Even E354D had reduced levels of activity, less than 0.05% of wild type. These four mutated enzymes retained a trace of activity. From the result of hydrolysis patterns for maltohexaose, in particular, D421N, unlike D325N and E354Q, catalyzed transglycosylation rather than hydrolysis. The results suggest that Asp421 could function to capture water molecules.