Expression, Purification, Crystallization and Preliminary X-ray Studies of Histamine Dehydroganase from Nocardioides simplex

This is the publisher's version, also available electronically from http://scripts.iucr.org/cgi-bin/paper?S1744309108023336.Histamine dehydrogenase (HADH) from Nocardioides simplex catalyzes the oxidative deamination of histamine to produce imidazole acetaldehyde and an ammonium ion. HADH is functionally related to trimethylamine dehydrogenase (TMADH), but HADH has strict substrate specificity towards histamine. HADH is a homodimer, with each 76 kDa subunit containing two redox cofactors: a [4Fe-4S] cluster and an unusual covalently bound flavin mononucleotide, 6-S-cysteinyl-FMN. In order to understand the substrate specificity of HADH, it was sought to determine its structure by X-ray crystallography. This enzyme has been expressed recombinantly in Escherichia coli and successfully crystallized in two forms. Diffraction data were collected to 2.7 Å resolution at the SSRL synchrotron with 99.7% completeness. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 101.14, b = 107.03, c = 153.35 Å

Hangekobokuto (Banxia-houpo-tang), a Kampo Medicine that Treats Functional Dyspepsia

Although abdominal bloating is one of the most bothersome symptoms experienced by patients with functional dyspepsia (FD), therapeutic drugs to relieve abdominal bloating have not been established. We investigated the Kampo (Chinese herbal) medicine, Hangekobokuto (Banxia-houpo-tang, HKT) for patients with FD from the standpoint of bowel gas retention. The bowel gas volume calculated from a plain abdominal radiogram (gas volume score, GVS) in FD patients was significantly higher than that in healthy subjects. Two week administration of HKT in the FD patients showed a significant decrease of GVS. Furthermore, gastrointestinal symptoms, especially symptoms of abdominal pain, indigestion and constipation, all of which are closely related to abdominal bloating, improved significantly in FD patients after the administration of HKT. These results suggest that HKT improves abdominal bloating accompanied by the reduction of bowel gas in FD patients

Artificial Self-Sufficient P450 in Reversed Micelles

Cytochrome P450s are heme-containing monooxygenases that require electron transfer proteins for their catalytic activities. They prefer hydrophobic compounds as substrates and it is, therefore, desirable to perform their reactions in non-aqueous media. Reversed micelles can stably encapsulate proteins in nano-scaled water pools in organic solvents. However, in the reversed micellar system, when multiple proteins are involved in a reaction they can be separated into different micelles and it is then difficult to transfer electrons between proteins. We show here that an artificial self-sufficient cytochrome P450, which is an enzymatically crosslinked fusion protein composed of P450 and electron transfer proteins, showed micelle-size dependent catalytic activity in a reversed micellar system. Furthermore, the presence of thermostable alcohol dehydrogenase promoted the P450-catalyzed reaction due to cofactor regeneration

Fine Tuning of Spatial Arrangement of Enzymes in a PCNA-Mediated Multienzyme Complex Using a Rigid Poly-L-Proline Linker

<div><p>Inspired by natural multienzyme complexes, many types of artificial multienzyme complexes have recently been constructed. We previously constructed a self-assembled complex of a bacterial cytochrome P450 and its ferredoxin and ferredoxin reductase partners using heterotrimerization of proliferating cell nuclear antigen (PCNA) from <i>Sulfolobus solfataricus</i>. In this study, we inserted different peptide linkers between ferredoxin and the PCNA subunit, and examined the effect on activity of the self-assembled multienzyme complex. Although the activity was affected by the lengths of both the rigid poly-L-proline-rich linkers and the flexible Gly₄-Ser repeating linkers, the poly-L-proline-rich linkers provided the greatest activity enhancement. The optimized poly-L-proline-rich linker enhanced the activity 1.9-fold compared with the GGGGSLVPRGSGGGGS linker used in the previously reported complex, while the Gly₄-Ser repeating linkers, (G₄S)_n (n = 1–6), did not yield higher activity than the maximum activity by the optimized poly-L-proline linker. Both the rigidity/flexibility and length of the peptide linker were found to be important for enhancing the overall activity of the multienzyme complex.</p> </div

細菌由来シトクロムP450の触媒活性に必須な電子伝達システムの探索

科学研究費助成事業 研究成果報告書：基盤研究(C)2018-2021課題番号 : 18K0484

Models for binding between PdX and P450cam in PUPPET.

<p>(A) A docking model of P450cam and PdX. The docking program GRAMM-X [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075114#B42" target="_blank">42</a>] was used to generate the model from crystal structures of P450cam (PDB: 1DZ4) and PdX (PDB: 1XLP) according to a previous report [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075114#B40" target="_blank">40</a>]. (B, C) Spatial arrangement of P450cam and the PCNA ring when the PdX-binding site of P450cam faces (B) in the same direction as or (C) in a perpendicular direction to the PCNA ring. The distance between the C-termini of PCNA2 and PCNA3 was estimated from the crystal structure of the PCNA heterotrimer (PDB: 2NTI).</p

P450cam oxidation activities of the PUPPET linker variants.

<p>The activities of (A) PUPPET-P_n (n = 1–5) and (B) PUPPET-G_n (n = 1–6) were evaluated in reaction mixtures containing 18 nM enzyme. The activity of the previously reported PUPPET, containing a GGGGSLVPRGSGGGGS linker (indicated as “O”), and the activity of an equimolar mixture of PdR, PdX and P450cam (indicated as “R”), were also evaluated under the same reaction conditions. Error bars represent the standard error of three replicates; *P < 0.05, ***P < 0.001 (Student’s t-test).</p

UV–Vis spectra of PCNA2-G ₄S-PdX and PUPPET-G₁.

<p>(A) UV–Vis spectrum of PCNA2-G ₄S-PdX. The peaks at 314, 412, and 460 nm, and the broad band in the long wavelength region, are specific to [2Fe-2S] cluster-containing proteins. UV–Vis spectra of PCNA2-(G₄S)_n-PdX (n = 2–6) and PCNA2-G ₄S(P₅)_nG₄S-PdX (n = 1–5) are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075114#pone.0075114.s001" target="_blank">Figure S1</a>. (B) UV–Vis spectra of PUPPET-G₁ (solid line), and a linear combination of the individual component protein spectra (broken line). PUPPET-G_n (n = 2–6) and PUPPET-P_n (n = 1–5) are shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0075114#pone.0075114.s002" target="_blank">Figure S2</a>.</p

SDS-PAGE analyses of PCNA2-PdX fusion proteins and PUPPETs.

<p>(A) PCNA2-PdX fusion proteins: lane 1–5, PCNA2-G ₄S(P₅)_nG₄S-PdX (n = 1–5); lane 6–11, PCNA2-(G₄S)_n-PdX (n = 1–6) (B) PUPPETs: lane 1–5, PUPPET-P_n (n = 1–5); lane 6–11, PUPPET-G_n (n = 1–6). P1-PdR, P2-PdX and P3-P450 are PCNA1-PdR, PCNA2-PdX and PCNA3-P450cam, respectively.</p