No spare Tyr: Rational design of functional enzymes with a high number of turnovers is a challenge, especially those with a complex active site, such as respiratory oxidases. Introducing two His and ...one Tyr residues into myoglobin resulted in enzymes that reduce O sub(2) to H sub(2)O with more than 1000 turnovers (red line, see scheme) and minimal release of reactive oxygen species. The positioning of the Tyr residue is critical for activity.
A‐DNA conformation is favored by guanine‐rich sequences, such as (dG)
n
·(dC)
n
, or under low‐humidity conditions. Earlier A‐DNA crystal structures revealed some conformational variations which may ...be the result of sequence‐dependent effects and/or crystal packing forces. Here we report the high‐resolution crystal structure of d(AGGGGCCCCT) in two crystal forms (either in the P2
1
2
1
2
1
or the P6
1
22 space group) to gain insights into the conformation and dynamics of the (dG)
n
·(dC)
n
sequence. The P2
1
2
1
2
1
form has been analyzed using data to 1.1 Å resolution by the anisotropic temperature factor refinement procedure of the
shelx
97 program. Such analysis affords us with the detailed geometric, conformational and motional property of an A‐DNA structure. The backbone torsional angles fall in a narrow range, except for the α/γ angles which have two distinct combinations (
gauche
−
/
gauche
+
or
trans
/
trans
). An A‐DNA model of poly(dG)·poly(dC) has been constructed using the conformational parameters derived from the crystal structure of the P2
1
2
1
2
1
form. In the crystal structure of the P6
1
22 space group, the central eight base pairs of the decamer adopt A‐DNA conformation with the two terminal nucleotides flipped out to form base pairs with the neighboring nucleotides. Comparison of the A‐DNA structure of the same sequence from two different crystal forms, reinforced the conclusion that molecules crystallized in the same space group have a more similar conformation, whereas the same molecule crystallized in different space groups has different (local) conformations.
To clarify the correlation between alloy elements and growth pattern of the rust layers, the rusting evolution of the carbon and weathering steels was investigated by using the gold markers method. ...The corrosion experiments were carried out in a simulated environment containing chloride ions. The results indicate that Ni, Cu, Cr and Mo elements in the weathering steel suppress the rust crystallization and impede the rust layer growth to an internal development, and the suppression function benefits the formation of a protective rust layer. Moreover, c-FeOOH is mainly located in the outer rust layer, while b-FeOOH is mainly located in the inner rust layer, and the distribution of the rust compounds in the rust layer is closely related to the growth pattern of the rust layer.
Structural studies are essential to understand mechanisms of non‐sequence‐specific DNA binding used by chromosomal proteins. A non‐histone high‐mobility group (HMG) chromosomal protein from ...Drosophila melanogaster, HMG‐D, binds duplex DNA in a non‐sequence‐specific fashion. The DNA‐binding domain of HMG‐D has been co‐crystallized with a duplex DNA fragment in the primitive orthorhombic space group P212121, with unit‐cell dimensions a = 43.74, b = 53.80, c = 86.84 Å. Data have been collected to 2.20 Å at 99 K, with diffraction observed to at least 2.0 Å. Heavy‐atom derivative crystals have been obtained by co‐crystallization with oligonucleotides halogenated at major‐groove positions near the end of the DNA.
A conserved 2-His-1-Glu metal center, as found in natural non-heme iron-containing enzymes, was engineered into sperm whale myoglobin by replacing Leu29 and Phe43 with Glu and His, respectively (swMb ...L29E, F43H, H64, called Fe
B
Mb(-His)). A high resolution (1.65 Å) crystal structure of Cu(II)-CN
−
-Fe
B
Mb(-His) was determined, demonstrating that the unique 2-His-1-Glu metal center was successfully created within swMb. The Fe
B
Mb(-His) can bind Cu, Fe or Zn ions, with both Cu(I)-Fe
B
Mb(-His) and Fe(II)-Fe
B
Mb(-His) exhibiting nitric oxide reductase (NOR) activities. Cu dependent NOR activity was significantly higher than that of Fe in the same metal binding site. EPR studies showed that the reduction of NO to N
2
O catalyzed by these two enzymes resulted in different intermediates; a five-coordinate heme-NO species was observed for Cu(I)-Fe
B
Mb(-His) due to the cleavage of the proximal heme Fe-His bond, while Fe(II)-Fe
B
Mb(-His) remained six-coordinate. Therefore, both the metal ligand, Glu29, and the metal itself, Cu or Fe, play crucial roles in NOR activity. This study presents a novel protein model of NOR and provides insights into a newly discovered member of NOR family, gNOR.
Aedes aegypti kynurenine aminotransferase (AeKAT) is a multifunctional aminotransferase. It catalyzes the transamination of a number of amino acids and uses many biologically relevant α-keto acids as ...amino group acceptors. AeKAT also is a cysteine S-conjugate β-lyase. The most important function of AeKAT is the biosynthesis of kynurenic acid, a natural antagonist of NMDA and α7-nicotinic acetylcholine receptors. Here, we report the crystal structures of AeKAT in complex with its best amino acid substrates, glutamine and cysteine. Glutamine is found in both subunits of the biological dimer, and cysteine is found in one of the two subunits. Both substrates form external aldemines with pyridoxal 5-phosphate in the structures. This is the first instance in which one pyridoxal 5-phosphate enzyme has been crystallized with cysteine or glutamine forming external aldimine complexes, cysteinyl aldimine and glutaminyl aldimine. All the units with substrate are in the closed conformation form, and the unit without substrate is in the open form, which suggests that the binding of substrate induces the conformation change of AeKAT. By comparing the active site residues of the AeKAT−cysteine structure with those of the human KAT I−phenylalanine structure, we determined that Tyr286 in AeKAT is changed to Phe278 in human KAT I, which may explain why AeKAT transaminates hydrophilic amino acids more efficiently than human KAT I does.
The Escherichia coliundecaprayl-pyrophosphate synthase (UPPs) structure has been solved using the single wavelength anomalous diffraction method. The putative substrate-binding site is located near ...the end of the βA-strand with Asp-26 playing a critical catalytic role. In both subunits, an elongated hydrophobic tunnel is found, surrounded by four β-strands (βA-βB-βD-βC) and two helices (α2 and α3) and lined at the bottom with large residues Ile-62, Leu-137, Val-105, and His-103. The product distributions formed by the use of the I62A, V105A, and H103A mutants are similar to those observed for wild-type UPPs. Catalysis by the L137A UPPs, on the other hand, results in predominantly the formation of the C70 polymer rather than the C55 polymer. Ala-69 and Ala-143 are located near the top of the tunnel. In contrast to the A143V reaction, the C30intermediate is formed to a greater extent and is longer lived in the process catalyzed by the A69L mutant. These findings suggest that the small side chain of Ala-69 is required for rapid elongation to the C55 product, whereas the large hydrophobic side chain of Leu-137 is required to limit the elongation to the C55product. The roles of residues located on a flexible loop were investigated. The S71A, N74A, or R77A mutants displayed 25–200-fold decrease in kcat values. W75A showed an 8-fold increase of the FPP Km value, and 22–33-fold increases in the IPP Km values were observed for E81A and S71A. The loop may function to bridge the interaction of IPP with FPP, needed to initiate the condensation reaction and serve as a hinge to control the substrate binding and product release.
The three-dimensional molecular structures of the complexes between an interesting antitumor drug, nogalamycin, and two DNA hexamers, dCGT(pS)ACG and dm5CGT(pS)Am5CG, were determined at high ...resolution by X-ray diffraction analyses. Two nogalamycins bind to the DNA double helix in a 2:1 ratio with the aglycon chromophore intercalated between the CpG steps at both ends of the helix. The nogalose and aminoglucose sugars lie in the minor and major grooves, respectively, of the distorted B-DNA double helix. The binding of nogalamycin to DNA requires that the base pairs in DNA open up transiently to allow the bulky sugars to go through. Specific hydrogen bonds are found in the complex between the drug and guanine bases. We suggest that nogalamycin may prefer GC sequences embedded in a stretch of AT sequences.
Aedes aegypti kynurenine aminotransferase (AeKAT) is a multifunctional aminotransferase. It catalyzes the transamination of a number of amino acids and uses many biologically relevant alpha-keto ...acids as amino group acceptors. AeKAT also is a cysteine S-conjugate beta2-lyase. The most important function of AeKAT is the biosynthesis of kynurenic acid, a natural antagonist of NMDA and alpha7-nicotinic acetylcholine receptors. Here, we report the crystal structures of AeKAT in complex with its best amino acid substrates, glutamine and cysteine. Glutamine is found in both subunits of the biological dimer, and cysteine is found in one of the two subunits. Both substrates form external aldemines with pyridoxal 5-phosphate in the structures. This is the first instance in which one pyridoxal 5-phosphate enzyme has been crystallized with cysteine or glutamine forming external aldimine complexes, cysteinyl aldimine and glutaminyl aldimine. All the units with substrate are in the closed conformation form, and the unit without substrate is in the open form, which suggests that the binding of substrate induces the conformation change of AeKAT. By comparing the active site residues of the AeKAT-cysteine structure with those of the human KAT I- phenylalanine structure, we determined that Tyr286 in AeKAT is changed to Phe278 in human KAT I, which may explain why AeKAT transaminates hydrophilic amino acids more efficiently than human KAT I does.
The Escherichia coli undecaprayl-pyrophosphate synthase (UPPs) structure has been solved using the single wavelength anomalous diffraction method. The putative substrate-binding site is located near ...the end of the betaA-strand with Asp-26 playing a critical catalytic role. In both subunits, an elongated hydrophobic tunnel is found, surrounded by four beta-strands (betaA-betaB-betaD-betaC) and two helices (alpha2 and alpha3) and lined at the bottom with large residues Ile-62, Leu-137, Val-105, and His-103. The product distributions formed by the use of the I62A, V105A, and H103A mutants are similar to those observed for wild-type UPPs. Catalysis by the L137A UPPs, on the other hand, results in predominantly the formation of the C(70) polymer rather than the C(55) polymer. Ala-69 and Ala-143 are located near the top of the tunnel. In contrast to the A143V reaction, the C(30) intermediate is formed to a greater extent and is longer lived in the process catalyzed by the A69L mutant. These findings suggest that the small side chain of Ala-69 is required for rapid elongation to the C(55) product, whereas the large hydrophobic side chain of Leu-137 is required to limit the elongation to the C(55) product. The roles of residues located on a flexible loop were investigated. The S71A, N74A, or R77A mutants displayed 25-200-fold decrease in k(cat) values. W75A showed an 8-fold increase of the FPP K(m) value, and 22-33-fold increases in the IPP K(m) values were observed for E81A and S71A. The loop may function to bridge the interaction of IPP with FPP, needed to initiate the condensation reaction and serve as a hinge to control the substrate binding and product release.
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