DADH catalyzes the flavin-dependent oxidative deamination of d-amino acids to the corresponding α-keto acids and ammonia. Here we report the first X-ray crystal structures of DADH at 1.06 Å resolution and its complexes with iminoarginine (DADH(red)/iminoarginine) and iminohistidine (DADH(red)/iminohistidine) at 1.30 Å resolution. The DADH crystal structure comprises an unliganded conformation and a product-bound conformation, which is almost identical to the DADH(red)/iminoarginine crystal structure. The active site of DADH was partially occupied with iminoarginine product (30% occupancy) that interacts with Tyr53 in the minor conformation of a surface loop. This flexible loop forms an "active site lid", similar to those seen in other enzymes, and may play an essential role in substrate recognition. The guanidinium side chain of iminoarginine forms a hydrogen bond interaction with the hydroxyl of Thr50 and an ionic interaction with Glu87. In the structure of DADH in complex with iminohistidine, two alternate conformations were observed for iminohistidine where the imidazole groups formed hydrogen bond interactions with the side chains of His48 and Thr50 and either Glu87 or Gln336. The different interactions and very distinct binding modes observed for iminoarginine and iminohistidine are consistent with the 1000-fold difference in k(cat)/K(m) values for d-arginine and d-histidine. Comparison of the kinetic data for the activity of DADH on different d-amino acids and the crystal structures in complex with iminoarginine and iminohistidine establishes that this enzyme is characterized by relatively broad substrate specificity, being able to oxidize positively charged and large hydrophobic d-amino acids bound within a flask-like cavity.

D-arginine dehydrogenase from Pseudomonas aeruginosa catalyzes the oxidation of D-arginine to iminoarginine, which is hydrolyzed in solution to ketoarginine and ammonia. In the present study, we have genetically engineered an untagged form of the enzyme that was purified to high levels and characterized in its kinetic properties. The enzyme is a true dehydrogenase that does not react with molecular oxygen. Steady-state kinetic studies with D-arginine or D-histidine as substrate and PMS as the electron acceptor established a ping-pong bi-bi kinetic mechanism. With the fast substrate D-arginine a dead-end complex of the reduced enzyme and the substrate occurs at high concentrations of D-arginine yielding substrate inhibition, while the overall turnover is partially limited by the release of the iminoarginine product. With the slow substrate D-histidine the initial Michaelis complex undergoes an isomerization involving multiple conformations that are not all equally catalytically competent for the subsequent oxidation reaction, while the overall turnover is at least partially limited by flavin reduction. The kinetic data are interpreted in view of the high-resolution crystal structures of the iminoarginine--and iminohistidine--enzyme complexes.

Background: With the improvement of bacterial detection, the theory of the sterile female upper reproductive tract has been frequently challenged in recent years. However, thus far, no researchers have used ovaries as study targets. Methods: Six women who were diagnosed with ovarian cancer were included in the cancer group, and ten women who were diagnosed with a noncancerous ovarian condition (including three patients with uterine myoma and seven patients with uterine adenomyosis) were included in the control group. Immunohistochemistry staining using an antibacterial lipopolysaccharide (LPS) antibody was used to confirm the presence of bacteria in the ovarian tissues. In addition, 16S rRNA sequencing was used to compare the differences in the bacteria between ovarian cancer tissues and noncancerous ovarian tissues. BugBase and Phylogenetic Investigation of Communities by Reconstruction of Unobserved States (PICRUSt) were used to predict the functional composition of the bacteria. Results: Bacterial LPS was present in ovarian cancer tissue and noncancerous ovarian tissue, which implied the presence of bacteria in ovarian tissue. When compared to the noncancerous ovarian bacteria at the phylum level, the cancerous ovarian bacteria were composed of increased Aquificae and Planctomycetes and decreased Crenarchaeota. When predicting metagenomes, gene functions associated with the potentially pathogenic and the oxidative stress-tolerant phenotype were enriched in the ovaries of the cancer group. Forty-six significantly different KEGG pathways existed in the ovarian bacteria of the cancer group compared to that of the control group. Conclusions: Different bacteria compositions were present in cancerous and noncancerous ovarian tissues.

Caspase-3, 6 and 7 cleave many proteins at specific sites to induce apoptosis. Their recognition of the P5 position in substrates has been investigated by kinetics, modeling and crystallography. Caspase-3 and -6 recognize P5 in pentapeptides as shown by enzyme activity data and interactions observed in the crystal structure of caspase-3/LDESD and in a model for caspase-6. In caspase-3 the P5 main-chain was anchored by interactions with Ser209 in loop-3 and the P5 Leu side-chain interacted with Phe250 and Phe252 in loop-4 consistent with 50% increased hydrolysis of LDEVD relative to DEVD. Caspase-6 formed similar interactions and showed a preference for polar P5 in QDEVD likely due to interactions with polar Lys265 and hydrophobic Phe263 in loop-4. Caspase-7 exhibited no preference for P5 residue in agreement with the absence of P5 interactions in the caspase-7/LDESD crystal structure. Initiator caspase-8, with Pro in the P5-anchoring position and no loop-4, had only 20% activity on tested pentapeptides relative to DEVD. Therefore, caspases-3 and -6 bind P5 using critical loop-3 anchoring Ser/Thr and loop-4 side-chain interactions, while caspase-7 and -8 lack P5-binding residues.

Caspase-3 recognition of various P4 residues in its numerous protein substrates was investigated by crystallography, kinetics, and calculations on model complexes. Asp is the most frequent P4 residue in peptide substrates, although a wide variety of P4 residues are found in the cellular proteins cleaved by caspase-3. The binding of peptidic inhibitors with hydrophobic P4 residues, or no P4 residue, is illustrated by crystal structures of caspase-3 complexes with Ac-IEPD-Cho, Ac-WEHD-Cho, Ac-YVAD-Cho, and Boc-D(OMe)-Fmk at resolutions of 1.9-2.6 A. The P4 residues formed favorable hydrophobic interactions in two separate hydrophobic regions of the binding site. The side chains of P4 Ile and Tyr form hydrophobic interactions with caspase-3 residues Trp206 and Trp214 within a non-polar pocket of the S4 subsite, while P4 Trp interacts with Phe250 and Phe252 that can also form the S5 subsite. These interactions of hydrophobic P4 residues are distinct from those for polar P4 Asp, which indicates the adaptability of caspase-3 for binding diverse P4 residues. The predicted trends in peptide binding from molecular models had high correlation with experimental values for peptide inhibitors. Analysis of structural models for the binding of 20 different amino acids at P4 in the aldehyde peptide Ac-XEVD-Cho suggested that the majority of hydrophilic P4 residues interact with Phe250, while hydrophobic residues interact with Trp206, Phe250, and Trp214. Overall, the S4 pocket of caspase-3 exhibits flexible adaptation for different residues and the new structures and models, especially for hydrophobic P4 residues, will be helpful for the design of caspase-3 based drugs.