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.

The mechanism of amine oxidation catalyzed by D-arginine dehydrogenase (DADH) has been investigated using steady-state and rapid reaction kinetics, with pH, substrate and solvent deuterium kinetic isotope effects (KIE) as mechanistic probes, and computational studies. Previous results showed that 85-90% of the flavin reduction reaction occurs in the mixing time of the stopped-flow spectrophotometer when arginine is the substrate, precluding a mechanistic investigation. Consequently, leucine, with slower kinetics, has been used here as the flavin-reducing substrate. Free energy calculations and the pH profile of the K(d) are consistent with the enzyme preferentially binding the zwitterionic form of the substrate. Isomerization of the Michaelis complex, yielding an enzyme-substrate complex competent for flavin reduction, is established due to an inverse hyperbolic dependence of k(cat)/K(m) on solvent viscosity. Amine deprotonation triggers the oxidation reaction, with cleavage of the substrate NH and CH bonds occurring in an asynchronous fashion, as suggested by the multiple deuterium KIE on the rate constant for flavin reduction (k(red)). A pK(a) of 9.6 signifies the ionization of a group that facilitates flavin reduction in the unprotonated form. The previously reported high-resolution crystal structures of the iminoarginine and iminohistidine complexes of DADH allow us to propose that Tyr(53), on a mobile loop covering the active site, may participate in substrate binding and facilitate flavin reduction.

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:The annotation of Ͼ4,900 genes for nitronate monooxygenase, which detoxifies a deadly toxin, is inaccurate due to the lack of functional data. Results: The protein PA4202 from Pseudomonas aeruginosa is characterized structurally and biochemically as the first bacterial nitronate monooxygenase. Conclusion: Four conserved motifs are identified in PA4202. Significance: New classes of enzymes are established based on the functional annotation of PA4202.

Objectives: To investigate the occupational and genetic risk factors inducing lumbar disc degeneration in a Chinese population, and to explore their synergistic interactions. Methods: A case-control study involving 178 low back pain patients with lumbar disc degeneration and 284 controls was carried out. Five types of work-related factors were investigated using questionnaires. Polymerase chain reaction and restriction fragments length polymorphism was used to detect the polymorphisms of MMP-3 (matrix metalloproteinase-3)(rs731236), VDR-Taq (vitamin D receptor-Taq) and VDR-Apa (vitamin D receptor-Apa)(rs35068180). Rothman's synergy index was used to measure the synergistic interactions between gene polymorphisms and occupational risk factors. Results: Family history of lumbar disc diseases, back injury history, whole-body vibration, bending/twisting, heavy physical workload, age, mutation alleles 5A of MMP-3 and A of VDR-Apa were significantly associated with lumbar disc degeneration (OR=12. 70, 11.79, 8.96, 5.46, 1.05, 1.96 and 1.70, respectively, p<0.05). Synergistic interactions existed between the mutation allele 5A of MMP-3 and wholebody vibration exposure, the mutation allele 5A of MMP-3 and bending/twisting, and the mutation allele A of VDR-Apa and bending/twisting (SI=13.27, 2.91, 2.35, respectively, p<0.05). Conclusions: The results of this study suggest that gene-occupation interaction might play a certain role in exaggerating lumbar disc degeneration. There is a possibility that subjects who carry mutation alleles 5A of MMP-3 and/or A of VDRApa are more vulnerable to lumbar disc degeneration when they are exposed to whole-body vibration and/or bending/twisting under ergonomic loads. It is estimated that 15% to 20% of adults have back pain during a single year, and 50% to 80% experience at least one episode of back pain during their lifetime 1) . Jin 2) reported that the incidence of low-back pain (LBP) was as high as 50% in an investigation of one population of Chinese workers. Lumbar disc degeneration is considered to be a primary cause of LBP 3,4) . The pathogenesis of lumbar disc degeneration includes radial fissures, rim tears in the annulus, and loss of water content in the nucleus pulpous and annulus. These changes are suspected of underlying many back pain symptoms.The etiology of lumbar disc degeneration is complex, and recent research on the causes of disc degeneration have shed light upon a better understanding of the risk factors contributing to disc degeneration. Magora 5) suggested that occupational factors contributing to the acceleration of spinal degeneration included heavy physical loads, manual materials handling consisting of lifting, bending and twisting, prolonged sitting, sustained non-neutral work postures, and vehicle driving. Videman 6) confirmed that occupational exposures are viewed as the primary source of the mechanical factors