Structural basis of substrate recognition by the substrate binding protein (SBP) of a hydrazide transporter, obtained from Microbacterium hydrocarbonoxydans.

Microbacterium hydrocarbonoxydans was isolated, using hydrazide compounds as its sole carbon source. The key enzyme that metabolizes these compounds was identified as hydrazidase, and the operon containing the gene coding for the enzyme, was revealed by genome sequencing. The operon also contained genes coding for an ATP-binding cassette transporter (ABC transporter), which was expected to transport the hydrazide compounds. Substrate binding protein (SBP), a component subunit of the transporter, plays an important role in recognizing the correct substrates for transport. Therefore, to elucidate the mechanism of recognition of the unnatural hydrazide compounds, we determined the crystal structures of the SBP, obtained from M. hydrocarbonoxydans (Mh-SBP), complexed with and without the hydrazide compound, at 2.2 Å and 1.75 Å resolutions, respectively. The overall structures of Mh-SBP were similar to those of the SBP in oligopeptide transporters such as OppA. On comparison, the liganded and unliganded structures of Mh-SBP showed an open - close conformation change. Interestingly, the binding mode of the compound to Mh-SBP was almost identical to that of the compound to hydrazidase, suggesting that the ABC transporter served transporting these compounds. Furthermore, based on the hydrazide complex structure, paraben, the other putative substrate of the protein, was successfully used with Mh-SBP to obtain the paraben complex structure.

Structural basis of the substrate recognition of hydrazidase isolated from Microbacterium sp. strain HM58-2, which catalyzes acylhydrazide compounds as its sole carbon source.

Hydrazidase was an enzyme that remained unidentified for a half century. However, recently, it was purified, and its encoding gene was cloned. Microbacterium sp. strain HM58-2 grows with acylhydrazides as its sole carbon source; it produces hydrazidase and degrades acylhydrazides to acetate and hydrazides. The bacterial hydrazidase belongs to the amidase signature enzyme family and contains a Ser-cisSer-Lys catalytic motif. The condensation of hydrazine and carbonic acid produces various hydrazides, some of which are raw materials for synthesizing pharmaceuticals and other useful chemicals. Although natural hydrazide compounds have been identified, the metabolic systems for hydrazides are not fully understood. Here, we report the crystal structure of hydrazidase from Microbacterium sp. strain HM58-2. The active site was revealed to consist of a Ser-cisSer-Lys catalytic triad, in which Ser179 forms a covalent bond with a carbonyl carbon of the substrate. 4-Hydroxybenzoic acid hydrazide bound to the S179A mutant, showing an oxyanion hole composed of the three backbone amide groups. Furthermore, H336 in the non-conserved region in the amidase family may define the substrate specificity, which was confirmed by mutation analysis. A wild-type apoenzyme structure revealed an unidentified molecule covalently bound to S179, representing a tetrahedral intermediate.

Conformational divergence in the HA-33/HA-17 trimer of serotype C and D botulinum toxin complex.

Clostridium botulinum produces a large toxin complex (L-TC) comprising botulinum neurotoxin associated with auxiliary nontoxic proteins. A complex of 33- and 17-kDa hemagglutinins (an HA-33/HA-17 trimer) enhances L-TC transport across the intestinal epithelial cell layer via binding HA-33 to a sugar on the cell surface. At least two subtypes of serotype C/D HA-33 exhibit differing preferences for the sugars sialic acid and galactose. Here, we compared the three-dimensional structures of the galactose-binding HA-33 and HA-33/HA-17 trimers produced by the C-Yoichi strain. Comparisons of serotype C/D HA-33 sequences reveal a variable region with relatively low sequence similarity across the C. botulinum strains; the variability of this region may influence the manner of sugar-recognition by HA-33. Crystal structures of sialic acid- and galactose-binding HA-33 are broadly similar in appearance. However, small-angle X-ray scattering revealed distinct solution structures for HA-33/HA-17 trimers. A structural change in the C-terminal variable region of HA-33 might cause a dramatic shift in the conformation and sugar-recognition mode of HA-33/HA-17 trimer.

Designed abscisic acid analogs as antagonists of PYL-PP2C receptor interactions.

The plant stress hormone abscisic acid (ABA) is critical for several abiotic stress responses. ABA signaling is normally repressed by group-A protein phosphatases 2C (PP2Cs), but stress-induced ABA binds Arabidopsis PYR/PYL/RCAR (PYL) receptors, which then bind and inhibit PP2Cs. X-ray structures of several receptor-ABA complexes revealed a tunnel above ABA's 3' ring CH that opens at the PP2C binding interface. Here, ABA analogs with sufficiently long 3' alkyl chains were predicted to traverse this tunnel and block PYL-PP2C interactions. To test this, a series of 3'-alkylsulfanyl ABAs were synthesized with different alkyl chain lengths. Physiological, biochemical and structural analyses revealed that a six-carbon alkyl substitution produced a potent ABA antagonist that was sufficiently active to block multiple stress-induced ABA responses in vivo. This study provides a new approach for the design of ABA analogs, and the results validated structure-based design for this target class.

Vialinin A is a ubiquitin-specific peptidase inhibitor.

Vialinin A, a small compound isolated from the Chinese mushroom Thelephora vialis, exhibits more effective anti-inflammatory activity than the widely used immunosuppressive drug tacrolimus (FK506). Here, we show that ubiquitin-specific peptidase 5/isopeptidase T (USP5/IsoT) is a target molecule of vialinin A, identified by using a beads-probe method. Vialinin A inhibited the peptidase activity of USP5/IsoT and also inhibited the enzymatic activities of USP4 among deubiquitinating enzymes tested. Although USPs are a member of thiol protease family, vialinin A exhibited no inhibitions for other thiol proteases, such as calpain and cathepsin.

Structural basis of the conformational changes in Microbacterium hydrocarbonoxydans IclR transcription factor homolog due to ligand binding.

Microbacterium hydrocarbonoxydans has been isolated using an unnatural acylhydrazide compound as the sole carbon source. The compound is hydrolyzed by bacterial hydrazidase, and the gene expression of the enzyme is considered to be controlled by a transcription factor of the Isocitrate lyase Regulator (IclR) family, belonging to the one-component signaling systems. Recently, we reported the crystal structure of an unliganded IclR homolog from M. hydrocarbonoxydans, named putative 4-hydroxybenzoate response regulator (pHbrR), which has a unique homotetramer conformation. In this study, we report the crystal structure of pHbrR complexed with 4-hydroxybenzoic acid, the catalytic product of hydrazidase, at 2.0 Å resolution. pHbrR forms a homodimer with multimeric rearrangement in the unliganded state. Gel filtration column chromatography results suggested dimer-tetramer rearrangement. We observed conformational change in the loop region covering the ligand-binding site, and domain rearrangements in the monomer. This study reports the first liganded IclR family protein structure that demonstrates large structural rearrangements between liganded and unliganded proteins, which may represent a general model for IclRs.

Structure-Based Chemical Design of Abscisic Acid Antagonists That Block PYL-PP2C Receptor Interactions.

In Arabidopsis, signaling of the stress hormone abscisic acid (ABA) is mediated by PYR/PYL/RCAR receptors (PYLs), which bind to and inhibit group-A protein phosphatases 2C (PP2Cs), the negative regulators of ABA. X-ray structures of several PYL-ABA and PYL-ABA-PP2C complexes have revealed that a conserved tryptophan in PP2Cs is inserted into a small tunnel adjacent to the C4' of ABA in the PYL-ABA complex and plays a crucial role in the formation and stabilization of the PYL-ABA-PP2C complex. Here, 4'-modified ABA analogues were designed to prevent the insertion of the tryptophan into the tunnel adjacent to the C4' of ABA in these complexes. These analogues were predicted to block PYL-PP2C receptor interactions and thus block ABA signaling. To test this, 4'- O-phenylpropynyl ABA analogues were synthesized as novel PYL antagonists (PANs). Structural, thermodynamic, biochemical, and physiological studies demonstrated that PANs completely abolished ABA-induced PYL-PP2C interactions in vitro and suppressed stress-induced ABA responses in vivo more strongly than did 3'-hexylsulfanyl-ABA (AS6), a PYL antagonist we developed previously. The PANs and AS6 antagonized the effects of ABA to different degrees in different plants, suggesting that these PANs can function as chemical scalpels to dissect the complicated regulatory mechanism of ABA signaling in plants.

Crystal structure of an IclR homologue from Microbacterium sp. strain HM58-2.

The bacterial transcription factor IclR (isocitrate lyase regulator) is a member of a one-component signal transduction system, which shares the common motif of a helix-turn-helix (HTH)-type DNA-binding domain (DBD) connected to a substrate-binding domain (SBD). Here, the crystal structure of an IclR homologue (Mi-IclR) from Microbacterium sp. strain HM58-2, which catabolizes acylhydrazide as the sole carbon source, is reported. Mi-IclR is expected to regulate an operon responsible for acylhydrazide degradation as an initial step. Native single-wavelength anomalous diffraction (SAD) experiments were performed in combination with molecular replacement. CRANK2 from the CCP4 suite successfully phased and modelled the complete structure of a homotetramer composed of 1000 residues in an asymmetric unit, and the model was refined to 2.1 Šresolution. The overall structure of Mi-IclR shared the same domain combination as other known IclR structures, but the relative geometry between the DBD and SBD differs. Accordingly, the geometry of the Mi-IclR tetramer was unique: the putative substrate-binding site in each subunit is accessible from the outside of the tetramer, as opposed to buried inside as in the previously known IclR structures. These differences in the domain geometry may contribute to the transcriptional regulation of IclRs.

Draft Genome Sequence of Microbacterium sp. Strain HM58-2, Which Hydrolyzes Acylhydrazides.

We report the draft genome sequence of Microbacterium sp. strain HM58-2, which produces hydrazidase, an enzyme hydrolyzing acylhydrazides. The estimated genome size is 3.9 Mb. Genome sequence information of this strain will help to identify an assimilating mechanism of nonnatural compounds in this strain and to develop ecological applications.

Crystallization and preliminary X-ray analysis of a novel haemagglutinin component of the toxin complex of serotype C Clostridium botulinum.

The botulinum toxin complex, the causative agent of botulism, passes through the intestinal wall via sugar-chain-dependent cell binding of a haemagglutinin of 33 kDa molecular weight (HA-33). The amino-acid sequence of the C-terminal half of HA-33 of the serotype C strain Yoichi (C-Yoichi) shares only 46% identity with those of the major serotype C strains. Additionally, C-Yoichi HA-33 exhibits a unique sugar-binding specificity. In the present work, C-Yoichi HA-33 was expressed in Escherichia coli and crystallized. Diffraction data were collected at a resolution of 2.2 Å. The crystals belonged to space group R3. The complete detailed protein structure will yield insight into how the unique HA-33 protein recognizes sugar moieties.

Moraxella catarrhalis bacteraemia associated with prosthetic vascular graft infection.

Moraxella catarrhalis, formerly called Branhamella catarrhalis, 'Neisseria catarrhalis' or 'Micrococcus catarrhalis', is a Gram-negative, aerobic diplococcus frequently found as a colonizer of the upper respiratory tract. Over the last 20-30 years, this bacterium has emerged as a genuine pathogen, and is now considered an important cause of otitis media in children and an aetiological agent in pneumonia in adults with chronic obstructive pulmonary disease. However, bacteraemia due to M. catarrhalis has rarely been reported. Presented here is a case of M. catarrhalis bacteraemia associated with prosthetic vascular graft infection along with a review of the relevant literature.

Development of a novel PCR method to comprehensively analyze salivary bacterial flora and its application to patients with odontogenic infections.

OBJECTIVE: The objective of this study was to develop a novel polymerase chain reaction (PCR) method to comprehensively analyze salivary bacterial flora. STUDY DESIGN: The bacterial flora in the saliva of 10 healthy persons and 11 patients with odontogenic infections were examined using a DNA extraction method with a high level of cell destruction efficiency and a novel universal primer set to amplify approximately 580 bp of the 16S rDNA sequence. RESULTS: Streptococcus (54.5%), Neisseria (14.7%), Actinomyces (8.4%), Gemella (4.1%), Granulicatella (3.8%), and Prevotella (1.4%) were dominant in a total of 1655 clones examined from the saliva of the healthy subjects. The dominant genera differed among the patients with odontogenic infections (a total of 823 clones) and were entirely different from those of the healthy subjects. CONCLUSION: This novel comprehensive salivary bacterial flora analysis method may be a useful supportive method to identify causative agents of odontogenic infections.