Yoshitaka Nakajima

Crystal Structure of Aminopeptidase N (Proteobacteria Alanyl Aminopeptidase) from Escherichia coli and Conformational Change of Methionine 260 Involved in Substrate Recognition

Aminopeptidase N from Escherichia coli is a broad specificity zinc exopeptidase belonging to aminopeptidase clan MA, family M1. The structures of the ligand-free form and the enzyme-bestatin complex were determined at 1.5- and 1.6-Å resolution, respectively. The enzyme is composed of four domains: an N-terminal β-domain (Met1-Asp193), a catalytic domain (Phe194-Gly444), a middle β-domain (Thr445-Trp546), and a C-terminal α-domain (Ser547-Ala870). The structure of the catalytic domain exhibits similarity to thermolysin, and a metal-binding motif (HEXXHX18E) is found in the domain. The zinc ion is coordinated by His297, His301, Glu320, and a water molecule. The groove on the catalytic domain that contains the active site is covered by the C-terminal α-domain, and a large cavity is formed inside the protein. However, there exists a small hole at the center of the C-terminal α-domain. The N terminus of bestatin is recognized by Glu121 and Glu264, which are located in the N-terminal and catalytic domains, respectively. Glu298 and Tyr381, located near the zinc ion, are considered to be involved in peptide cleavage. A difference revealed between the ligand-free form and the enzyme-bestatin complex indicated that Met260 functions as a cushion to accept substrates with different N-terminal residue sizes, resulting in the broad substrate specificity of this enzyme.

Unusual Disordered Crystal Structure of a Racemate Exhibiting a Novel Enantiomeric Resolution: Preferential Enrichment

Simple recrystallization of racemic (±)-NC leads to preferential enrichment of one enantiomer in the mother liquor, which allows the efficient resolution of the two enantiomers. In the unique disordered crystal structure of the racemate, the two enantiomers form centrosymmetric dimers as the major component.

Structure of aminopeptidase N from Escherichia coli complexed with the transition-state analogue aminophosphinic inhibitor PL250

Aminopeptidase N (APN; EC 3.4.11.2) purified from Escherichia coli has been crystallized with the optically pure aminophosphinic inhibitor PL250, H3N+‐CH(CH3)‐P(O)(OH)‐CH2‐CH(CH2Ph)‐CONH‐CH(CH2Ph)CO2 −, which mimics the transition state of the hydrolysis reaction. PL250 inhibits APN with a K i of 1.5–2.2 nM and its three‐dimensional structure in complex with E. coli APN showed its interaction with the S1, S′1 and S′2 subsites of the catalytic site. In this structure, the Zn ion was shown to be pentacoordinated by His297, His301 and Glu320 of APN and the two O atoms of the phosphinic moiety of PL250. One of these O atoms is also involved in a hydrogen bond to Tyr381, supporting the proposed role of this amino acid in the stabilization of the transition state of the enzymatic process. The strength of the phosphinic zinc binding and the occupancy of the S′2 subsite account for the 100‐fold increase in affinity of PL250 compared with the dipeptide‐derived inhibitor bestatin (K i = 4.1 × 10−6  M). Accordingly, the removal of the C‐terminal phenylalanine of PL250 resulted in a large decrease in affinity (K i = 2.17 × 10−7  M). Furthermore, it was observed that the C‐terminal carboxyl group of the inhibitor makes no direct interactions with the amino acids of the APN active site. Interestingly, PL250 exhibits the same inhibitory potency for E. coli APN and for mammalian enzymes, suggesting that the structure of the complex could be used as a template for the rational design of various human APN inhibitors needed to study the role of this aminopeptidase in various pathologies.

Dipeptidyl Aminopeptidase IV from Stenotrophomonas maltophilia Exhibits Activity against a Substrate Containing a 4-Hydroxyproline Residue

The crystal structure of dipeptidyl aminopeptidase IV from Stenotrophomonas maltophilia was determined at 2.8-A resolution by the multiple isomorphous replacement method, using platinum and selenomethionine derivatives. The crystals belong to space group P4(3)2(1)2, with unit cell parameters a = b = 105.9 A and c = 161.9 A. Dipeptidyl aminopeptidase IV is a homodimer, and the subunit structure is composed of two domains, namely, N-terminal beta-propeller and C-terminal catalytic domains. At the active site, a hydrophobic pocket to accommodate a proline residue of the substrate is conserved as well as those of mammalian enzymes. Stenotrophomonas dipeptidyl aminopeptidase IV exhibited activity toward a substrate containing a 4-hydroxyproline residue at the second position from the N terminus. In the Stenotrophomonas enzyme, one of the residues composing the hydrophobic pocket at the active site is changed to Asn611 from the corresponding residue of Tyr631 in the porcine enzyme, which showed very low activity against the substrate containing 4-hydroxyproline. The N611Y mutant enzyme was generated by site-directed mutagenesis. The activity of this mutant enzyme toward a substrate containing 4-hydroxyproline decreased to 30.6% of that of the wild-type enzyme. Accordingly, it was considered that Asn611 would be one of the major factors involved in the recognition of substrates containing 4-hydroxyproline.

Comparison of Crystal Structures of New Racemic Chiral Compounds Showing and Not Showing the Phenomenon of Preferential Enrichment

Abstract The crystal structures of (±)-[2-[4-(3-ethoxy-2-hydroxypropoxy)phenylcarbamoyl]ethyl] trimethylammonium p-chlorobenzenesulfonate [(±)-NCMe3] and its terminal methoxy derivative, (±)-NCMe3-OMe, are compared. The former racemate exhibited the phenomenon of Preferential Enrichment, whereas the latter failed to do so. Crystal data, (±)-NCMe3: CuKα radiation, space group P 1, Z = 2, a = 9.848(5), b = 14.823(3), c = 9.147(1) Å, α = 97.81(1), β = 92.68(3), γ = 105.92(2)°, D calc = 1.355 g/cm3, R = 0.056 for 3213 observed reflections; (±)-NCMe3-OMe: CuKα radiation, space group P 1, Z = 2, a = 11.350(1), b = 14.568(2), c = 8.2981(4) Å, α = 94.346(7), β = 112.376(5), γ = 78.622(9)°, D calc = 1.343 g/cm3, R = 0.069 for 1519 observed reflections.

Preferential Enrichment and Crystal Structure

Abstract Comparison of three different stable crystal structures of (±)-[2-[4-(3-ethoxy-2-hydroxypropoxy)phenylcarbamoyl]ethyl]dimethylammonium p-nitrobenzenesulfonate [(±)-NNMe2], and its p-chlorobenzene-sulfonate [(±)-NCMe2] and p-toluenesulfonate [(±)-NTMe2] derivatives is described. The first two racemates exhibited the phenomenon of the Preferential Enrichment, but the last one failed to do. It is clear that the nature of the para substituent on the benzenesulfonate group greatly affects the crystal structure and thereby governs the occurrence of the Preferential Enrichment; i.e., substitution by an electron-withdrawing group leads to the formantion of an ordered racemic compound crystal or a fairly ordered racemic mixed crystal of the two enantiomers, while the presence of an electron-donating group results in the formation of a highly disordered racemic mixed crystal.

Ungewöhnliche fehlgeordnete Kristallstruktur eines Racemats als neues Phänomen bei der Racematspaltung: bevorzugte Anreicherung

Durch einfaches Umkristallisieren des Racemats (±)-NC findet eine bevorzugte Anreicherung eines Enantiomers in der Mutterlauge statt, was eine effiziente Racematspaltung ermoglicht. Das Racemat weist eine einzigartige fehlgeordnete Kristallstruktur auf, in der hauptsachlich zentrosymmetrische cyclische Dimere beider Enantiomere vorliegen.

Crystallization and preliminary X-ray characterization of rat liver acyl-CoA oxidase

A recombinant form of the flavoenzyme acyl-CoA oxidase from rat liver has been crystallized by the hanging-drop vapour-diffusion technique using PEG 20 000 as a precipitating agent. The crystals grew as yellow prisms, with unit-cell parameters a = 71.05, b = 87.29, c = 213.05 A, alpha = beta = gamma = 90 degrees. The crystals exhibit the symmetry of space group P2(1)2(1)2(1) and are most likely to contain a dimer in the asymmetric unit, with a V(M) value of 2.21 A(3) Da(-1). The crystals diffract to a resolution of 2.5 A at beamline BL6A of the Photon Factory. Two heavy-atom derivatives have been identified.