Akiko Kita

An archetypical extradiol-cleaving catecholic dioxygenase: the crystal structure of catechol 2,3-dioxygenase (metapyrocatechase) from Pseudomonas putida mt-2

BACKGROUND Catechol dioxygenases catalyze the ring cleavage of catechol and its derivatives in either an intradiol or extradiol manner. These enzymes have a key role in the degradation of aromatic molecules in the environment by soil bacteria. Catechol 2, 3-dioxygenase catalyzes the incorporation of dioxygen into catechol and the extradiol ring cleavage to form 2-hydroxymuconate semialdehyde. Catechol 2,3-dioxygenase (metapyrocatechase, MPC) from Pseudomonas putida mt-2 was the first extradiol dioxygenase to be obtained in a pure form and has been studied extensively. The lack of an MPC structure has hampered the understanding of the general mechanism of extradiol dioxygenases. RESULTS The three-dimensional structure of MPC has been determined at 2.8 A resolution by the multiple isomorphous replacement method. The enzyme is a homotetramer with each subunit folded into two similar domains. The structure of the MPC subunit resembles that of 2,3-dihydroxybiphenyl 1,2-dioxygenase, although there is low amino acid sequence identity between these enzymes. The active-site structure reveals a distorted tetrahedral Fe(II) site with three endogenous ligands (His153, His214 and Glu265), and an additional molecule that is most probably acetone. CONCLUSIONS The present structure of MPC, combined with those of two 2,3-dihydroxybiphenyl 1,2-dioxygenases, reveals a conserved core region of the active site comprising three Fe(II) ligands (His153, His214 and Glu265), one tyrosine (Tyr255) and two histidine (His199 and His246) residues. The results suggest that extradiol dioxygenases employ a common mechanism to recognize the catechol ring moiety of various substrates and to activate dioxygen. One of the conserved histidine residues (His199) seems to have important roles in the catalytic cycle.

Crystal structure of the [2Fe-2S] oxidative-stress sensor SoxR bound to DNA

The [2Fe-2S] transcription factor SoxR, a member of the MerR family, functions as a bacterial sensor of oxidative stress such as superoxide and nitric oxide. SoxR is activated by reversible one-electron oxidation of the [2Fe-2S] cluster and then enhances the production of various antioxidant proteins through the soxRS regulon. In the active state, SoxR and other MerR family proteins activate transcription from unique promoters, which have a long 19- or 20-bp spacer between the −35 and −10 operator elements, by untwisting the promoter DNA. Here, we show the crystal structures of SoxR and its complex with the target promoter in the oxidized (active) state. The structures reveal that the [2Fe-2S] cluster of SoxR is completely solvent-exposed and surrounded by an asymmetric environment stabilized by interaction with the other subunit. The asymmetrically charged environment of the [2Fe-2S] cluster probably causes redox-dependent conformational changes of SoxR and the target promoter. Compared with the promoter structures with the 19-bp spacer previously studied, the DNA structure is more sharply bent, by ≈1 bp, with the two central base pairs holding Watson–Crick base pairs. Comparison of the target promoter sequences of the MerR family indicates that the present DNA structure represents the activated conformation of the target promoter with a 20-bp spacer in the MerR family.

Crystal structure of the complex between calyculin A and the catalytic subunit of protein phosphatase 1.

The crystal structure of the catalytic subunit of the protein phosphatase 1 (PP1), PP1 gamma, in complex with a marine toxin, calyculin A, was determined at 2.0 A resolution. The metal binding site contains the phosphate group of calyculin A and forms a tight network via the hydrophilic interactions between PP1 and calyculin A. Calyculin A is located in two of the three grooves, namely, in the hydrophobic groove and the acidic groove on the molecular surface. This is the first observation to note that the inhibitor adopts not a pseudocyclic conformation but an extended conformation in order to form a complex with the protein. The amino acid terminus of calyculin A contributes, in a limited manner, to the binding to PP1 gamma, which is consistent with findings from the studies of dose-inhibition analysis.

Crystal structure of stilbene synthase from Arachis hypogaea

Introduction. Stilbene synthase [STS; Enzyme Commission (EC) 2.3.1.95] and chalcone synthase (CHS; EC 2.3.1.74) are members of the type III polyketide synthases (PKSs) and plant-specific enzymes. 1 CHS is widely found in higher plants and plays a key role in the flavonoid biosynthesis by supplying chalcone to downstream enzymes. In contrast, a limited number of plants have STS essential for the synthesis of resveratrol utilized in the stilbenoid biosynthesis. 2 The members of CHS superfamily, including STS, produce linear polyketide intermediates by a common catalytic mechanism where coenzyme A (CoA)-linked starter molecules are iteratively condensed by acetyl units from malonyl-CoA. 3 STS and CHS share around 70% sequence identity without significant deletions and insertions; therefore, the enzymatic mechanism of STS has been considered to be very close to that of CHS. STS and CHS catalyze condensation reactions of pcoumaroyl-CoA and 3 acetyl units from malonyl-CoA, and produce a common linear tetraketide intermediate. In the following cyclization reaction, however, STS and CHS catalyze aldol and Claisen condensation of the tetraketide, resulting in 2 different final products, resveratrol and chalcone, respectively. The crystal structure and molecular mechanism of CHS from Medicago sativa (alfalfa) have recently been reported, 4 but the primary determinant of the cyclization reactions catalyzed by STS and CHS was not clear. More recently, the crystal structure of STS from Pinus silvestris (pine) was reported and provided a framework for understanding the specificity in the cyclization reaction. 5 This report describes the crystal structures of STS from Arachis hypogaea (peanut) in the absence and presence of its final product resveratrol at 2.4 A and 2.9 A, respectively. Detailed structural comparisons of STS from A. hypogaea (peanut STS) with STS from P. silvestris (pine STS) and CHS (alfalfa CHS) evidently revealed common differences between STS and CHS in the local conformation around the active site pocket.

Structure and Molecular Dynamics Simulation of Archaeal Prefoldin: The Molecular Mechanism for Binding and Recognition of Nonnative Substrate Proteins

Prefoldin (PFD) is a heterohexameric molecular chaperone complex in the eukaryotic cytosol and archaea with a jellyfish-like structure containing six long coiled-coil tentacles. PFDs capture protein folding intermediates or unfolded polypeptides and transfer them to group II chaperonins for facilitated folding. Although detailed studies on the mechanisms for interaction with unfolded proteins or cooperation with chaperonins of archaeal PFD have been performed, it is still unclear how PFD captures the unfolded protein. In this study, we determined the X-ray structure of Pyrococcus horikoshii OT3 PFD (PhPFD) at 3.0 A resolution and examined the molecular mechanism for binding and recognition of nonnative substrate proteins by molecular dynamics (MD) simulation and mutation analyses. PhPFD has a jellyfish-like structure with six long coiled-coil tentacles and a large central cavity. Each subunit has a hydrophobic groove at the distal region where an unfolded substrate protein is bound. During MD simulation at 330 K, each coiled coil was highly flexible, enabling it to widen its central cavity and capture various nonnative proteins. Docking MD simulation of PhPFD with unfolded insulin showed that the beta subunit is essentially involved in substrate binding and that the alpha subunit modulates the shape and width of the central cavity. Analyses of mutant PhPFDs with amino acid replacement of the hydrophobic residues of the beta subunit in the hydrophobic groove have shown that beta Ile107 has a critical role in forming the hydrophobic groove.

Crystal Structures of N-Acetylglucosamine-phosphate Mutase, a Member of the α-d-Phosphohexomutase Superfamily, and Its Substrate and Product Complexes

N-Acetylglucosamine-phosphate mutase (AGM1) is an essential enzyme in the synthetic process of UDP-N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is a UDP sugar that serves as a biosynthetic precursor of glycoproteins, mucopolysaccharides, and the cell wall of bacteria. Thus, a specific inhibitor of AGM1 from pathogenetic fungi could be a new candidate for an antifungal reagent that inhibits cell wall synthesis. AGM1 catalyzes the conversion of N-acetylglucosamine 6-phosphate (GlcNAc-6-P) into N-acetylglucosamine 1-phosphate (GlcNAc-1-P). This enzyme is a member of the α-d-phosphohexomutase superfamily, which catalyzes the intramolecular phosphoryl transfer of sugar substrates. Here we report the crystal structures of AGM1 from Candida albicans for the first time, both in the apoform and in the complex forms with the substrate and the product, and discuss its catalytic mechanism. The structure of AGM1 consists of four domains, of which three domains have essentially the same fold. The overall structure is similar to those of phosphohexomutases; however, there are two additional β-strands in domain 4, and a circular permutation occurs in domain 1. The catalytic cleft is formed by four loops from each domain. The N-acetyl group of the substrate is recognized by Val-370 and Asn-389 in domain 3, from which the substrate specificity arises. By comparing the substrate and product complexes, it is suggested that the substrate rotates about 180° on the axis linking C-4 and the midpoint of the C-5—O-5 bond in the reaction.

Crystal Structure of Uridine-diphospho-N-acetylglucosamine Pyrophosphorylase from Candida albicans and Catalytic Reaction Mechanism

Uridine-diphospho-N-acetylglucosamine (UDP-GlcNAc) is a precursor of the bacterial and fungal cell wall. It is also used in a component of N-linked glycosylation and the glycosylphosphoinositol anchor of eukaryotic proteins. It is synthesized from N-acetylglucosamine-1-phosphate (GlcNAc-1-P) and uridine-5′-triphosphate (UTP) by UDP-GlcNAc pyrophosphorylase (UAP). This is an SN2 reaction; the non-esterified oxygen atom of the GlcNAc-1-P phosphate group attacks the α-phosphate group of UTP. We determined crystal structures of UAP from Candida albicans (CaUAP1) without any ligands and also complexed with its substrate or with its product. The series of structures in different forms shows the induced fit movements of CaUAP1. Three loops approaching the ligand molecule close the active site when ligand is bound. In addition, Lys-421, instead of the metal ion in prokaryotic UAPs, is coordinated by both phosphate groups of UDP-Glc-NAc and acts as a cofactor. However, a magnesium ion enhances the enzymatic activity of CaUAP1, and thus we propose that the magnesium ion increases the affinity between UTP and the enzyme by coordinating to the α- and γ-phosphate group of UTP.

Crystal Structure of the C2 Domain of Class II Phosphatidylinositide 3-Kinase C2α

Phosphatidylinositide (PtdIns) 3-kinase catalyzes the addition of a phosphate group to the 3′-position of phosphatidyl inositol. Accumulated evidence shows that PtdIns 3-kinase can provide a critical signal for cell proliferation, cell survival, membrane trafficking, glucose transport, and membrane ruffling. Mammalian PtdIns 3-kinases are divided into three classes based on structure and substrate specificity. A unique characteristic of class II PtdIns 3-kinases is the presence of both a phox homolog domain and a C2 domain at the C terminus. The biological function of the C2 domain of the class II PtdIns 3-kinases remains to be determined. We have determined the crystal structure of the mCPK-C2 domain, which is the first three-dimensional structural model of a C2 domain of class II PtdIns 3-kinases. Structural studies reveal that the mCPK-C2 domain has a typical anti-parallel β-sandwich fold. Scrutiny of the surface of this C2 domain has identified three small, shallow sulfate-binding sites. On the basis of the structural features of these sulfate-binding sites, we have studied the lipid binding properties of the mCPK-C2 domain by site-directed mutagenesis. Our results show that this C2 domain binds specifically to PtdIns(3,4)P2 and PtdIns(4,5)P2 and that three lysine residues at SBS I site, Lys-1420, Lys-1432, and Lys-1434, are responsible for the phospholipid binding affinity.

Crystal structure of decameric peroxiredoxin (AhpC) from Amphibacillus xylanus.

Introduction. Peroxiredoxins (Prxs) act as antioxidants to protect organisms against reactive oxygen species. In Escherichia coli, Prx is responsible for the reduction of endogenously generated hydrogen peroxides. In mammalian cells, the enzyme is essential for tumor suppression. Prxs are currently divided into three classes: typical 2-Cys, atypical 2-Cys, and 1-Cys Prxs. All Prxs contain one conserved cysteine in the N-terminal region (peroxidatic cysteine, Cys47 in Amphibacillus xylanus AhpC), which is the site of oxidation. 2-Cys Prx contains an additional conserved cysteine near the C-terminal region (resolving cysteine, Cys166). The oxidized peroxidatic cysteine rapidly forms an intermolecular disulfide bond with the resolving cysteine of the other subunit, which is subsequently reduced with electrons supplied by reducing agents such as thioredoxin. To date, the crystal structures of seven eukaryotic and bacterial Prxs have been determined (homodecamer or homodimer). A. xylanus, isolated from alkaline compost, lacks a respiratory system and the heme proteins catalase and peroxidase, and has the same growth rate and cell yield under strictly anaerobic and aerobic conditions. A typical 2-Cys Prx from this facultatively anaerobic bacterium (A. xylanus AhpC) plays an essential role in reducing oxygen species coupled with the flavoprotein, A. xylanus NADH oxidase. Here we report the crystal structure of A. xylanus AhpC in its oxidized and decameric form.

Crystal structure of TTHA1657 (AT-rich DNA-binding protein; p25) from Thermus thermophilus HB8 at 2.16 Å resolution

Introduction. The p25 protein from Thermus aquaticus (Taq) was initially identified as a protein that bound preferentially to AT-rich DNA. The purified protein was characterized as a homodimer. In vitro, this protein displays nonsequence specific dsDNA-binding activity. Database searches revealed more than 30 gram-positive bacteria displaying a sequential similarity (30% identity) to p25. However, the precise roles of these proteins were unclear. Recently, Rex from Streptomyces coelicolor, one of the sequence homologues of Taq p25 (45% identity), was characterized as a novel redox sensing repressor. The DNA-binding site of Rex has been localized to an 8-bp inverted repeat recognition element called a ROP, which is located upstream of several respiratory genes, including the cydABCD and rex-hemACD operons. It has also been shown that the DNA-binding activity of Rex is regulated by the NADH/NADþ redox poise. NADþ competes with NADH for Rex binding, and a raised NADH/ NADþ ratio results in inhibition of the DNA binding of Rex. The sequence analysis has shown that Rex family proteins have a pyridine dinucleotide-binding domain, the Rossmann fold. However, NAD(H) binding to p25 has not been studied. Most recently, the X-ray structure of Taq p25 (Thermus aquaticus Rex; T-Rex) bound to NADH has been determined at 2.9 Å resolution, revealing a structural feature with two main domains organized as a domain-swapped homodimer and demonstrating that TRex exhibits functional characteristics similar to those of its homologue, Streptomyces coelicolor Rex. The Cterminal domain containing one NADH molecule belongs to the Rossmann fold family, whose members are commonly observed among the dinucleotide-dependent enzymes, whereas the N-terminal domain has a fold related to the winged helix DNA-binding motif. Here, we report the 2.16 Å resolution X-ray structure of Thermus thermophilus p25 encoded by the TTHA1657 gene, which is bound to NADH. Though Thermus thermophilus p25 is sequentially identical to Thermus aquaticus p25 and the two have very similar overall structures, several structural differences, including two newly found well-ordered sulfate anions in the N-terminal DNA-binding domain, were found in the present higher-resolution structure. These provide a structural insight into the DNA recognition by the Rex protein.