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.

amino acid ͉ arginine dehydrogenase ͉ racemase A lthough L-amino acids are the predominant amino acids in protein synthesis, D-amino acids serve as specialized components of many types of machineries in living organisms. In mammals, D-serine and D-aspartate are associated with cell aging and neural signaling (1, 2). In bacteria, some D-amino acids are essential ingredients of cell wall synthesis (3). Endogenous D-amino acids are produced by racemization from the prevalent L-amino acids through the action of racemases. Amino acid racemases are classified into 2 groups: pyridoxal 5Ј phosphate-dependent and phosphate-independent enzymes (4). Completely different reaction mechanisms have been proposed for these 2 groups of enzymes for the spatial rearrangement of ␣-hydrogen in the corresponding amino acids. Nevertheless, racemization of amino acids reported so far is catalyzed by a single enzyme.When provided in excess, some D-amino acids can be used as nutrients to support growth by bacteria. In most cases, D-amino acid oxidase or dehydrogenase catalyzes the oxidative deamination as the first step in catabolism. Pseudomonas aeruginosa, an opportunistic human pathogen with an enormous catabolic capacity, is capable of growing on D-arginine as the sole source of carbon and nitrogen (5). The presence of an inducible D-arginine dehydrogenase activity in this organism was initially reported by Haas and coworkers (6), and 2-ketoarginine derived from this reaction could be converged into the arginine transaminase (ATA) pathway (7,8), 1 of the 4 pathways for L-arginine catabolism in pseudomonads (Fig. 1). In fact, it has been proposed that L-arginine might be converted into D-arginine via racemization (6), reminiscent of L-alanine utilization through a catabolic alanine racemase and D-alanine dehydrogenase in Escherichia coli and many bacteria (9, 10). Existence of an arginine racemase in P. aeruginosa was supported by growth complementation of arginine auxotrophs with D-arginine (6). However, the activity of P. aeruginosa arginine racemase has never been demonstrated in vitro, presumably because of the instant decomposition of both L-and D-arginine in extracts.Under aerobic conditions, L-arginine is preferentially catabolized by the arginine succinyltransferase (AST) pathway, followed by the ATA pathway (7,11). Enzymes of the AST pathway are encoded by the aruCFGDBE operon (12), which is induced by exogenous L-arginine in the presence of a functional arginine regulator, ArgR (13). The ArgR protein belongs to the AraC family of transcriptional regulators. Depending on the location of its binding sites, ArgR serves as a repressor or activator of ArgR regulon in arginine and glutamate metabolism. Thus, when the AST pathway is absent or remains uninduced (e.g., in the argR mutant), the ATA pathway then takes charge as the auxiliary route of L-arginine utilization.

ELONGATED HYPOCOTYL 5 (HY5), a basic domain/leucine zipper (bZIP) transcription factor, acts as a master regulator of transcription to promote photomorphogenesis. At present, it's unclear whether HY5 uses additional mechanisms to inhibit hypocotyl elongation. Here, we demonstrate that HY5 enhances the activity of GSK3-like kinase BRASSINOSTEROID-INSENSITIVE 2 (BIN2), a key repressor of brassinosteroid signaling, to repress hypocotyl elongation. We show that HY5 physically interacts with and genetically acts through BIN2 to inhibit hypocotyl elongation. The interaction of HY5 with BIN2 enhances its kinase activity possibly by the promotion of BIN2 Tyr 200 autophosphorylation, and subsequently represses the accumulation of the transcription factor BRASSINAZOLE-RESISTANT 1 (BZR1). Leu 137 of HY5 is found to be important for the HY5-BIN2 interaction and HY5-mediated regulation of BIN2 activity, without affecting the transcriptional activity of HY5. HY5 levels increase with light intensity, which gradually enhances BIN2 activity. Thus, our work reveals an additional way in which HY5 promotes photomorphogenesis, and provides an insight into the regulation of GSK3 activity.

The MvaT and MvaU proteins belonging to the H-NS family were identified as DNA-binding proteins that interact with the regulatory region of the aotJQMOP-argR operon for arginine uptake and regulation. Recombinant MvaT and MvaU proteins were purified, and binding of these purified proteins to the aotJ regulatory region was demonstrated using electromobility shift assays. Polyclonal antibodies against purified MvaT and MvaU were prepared and employed in supershift assays to support these observations. Knockout mutations resulting in a single lesion in mvaT or mvaU, as well as knockout mutations resulting in double lesions, were constructed using biparental conjugation, and the absence of MvaT and MvaU in the resulting mutants was confirmed by immunoblot analysis. Using measurements of the ␤-galactosidase activities from aotJ::lacZ fusions in the mutants and the parental strain, it was found that MvaT and MvaU serve as repressors in control of aotJ expression. The effects of MvaT and MvaU on pyocyanin synthesis and CupA fimbrial expression in these mutants were also analyzed. Pyocyanin synthesis was induced in the single mutants but was completely abolished in the double mutant, suggesting that there is a complicated regulatory scheme in which MvaT and MvaU are essential elements. In comparison, MvaT had a more profound role than MvaU as a repressor of cupA expression; however, a combination of MvaT depletion and MvaU depletion had a strong synergistic effect on cupA. Moreover, prophage Pf4 integrated into the chromosome of Pseudomonas aeruginosa PAO1 was activated in an mvaT mvaU double mutant but not in a single mutant. These results were supported by purification and nucleotide sequencing of replicative-form DNA and by the release of phage particles in plaque assays. In summary, the mvaT mvaU double mutant was viable, and depletion of MvaT and MvaU had serious effects on a variety of physiological functions in P. aeruginosa.The MvaT and MvaU proteins of Pseudomonas aeruginosa belong to the H-NS family of small DNA-binding proteins. MvaT was initially identified in P. mevalonii as a positive regulator for mevalonate catabolism (25). Subsequently, MvaT homologues have been identified in other pseudomonads based on structural and functional similarities; five homologues have been identified in P. putida, three homologues have been identified in P. fluorescens, four homologues have been identified in P. syringae, and two homologues have been identified in P. aeruginosa. In P. putida, the TurA protein represses the Pu promoter of the TOL plasmid in a temperature-dependent manner (24). In P. fluorescens, the MvaT and MvaV proteins regulate the expression of two biocontrol exoproducts, 2,4-diacetyl phloroglucinol and pyoluteorin (1). In P. aeruginosa, MvaT is involved in quorum-sensing responses and biofilm formation. Inactivation of mvaT resulted in increased production of PA-IL lectin and the toxic exoproduct pyocyanin, reduced biofilm formation, increased drug resistance, and reduced swarming motility (5, 33). In ad...

(14) and to trigger biofilm disassembly (12). Therefore, it is important to understand how bacterial cells regulate D-amino acid homeostasis.In living organisms, biosynthesis of free-form D-amino acids is catalyzed by racemases, with L-enantiomers as substrates. Peptidyl D-amino acids occur either by taking free D-amino acid as a substrate in the cell wall synthesis or by L-to-D epimerization, as in the nonribosomal peptide synthesis in microorganisms (4). In higher eukaryotic organisms, this process is infrequently catalyzed by enzyme-driven posttranslational isomerization (11). Amino acid racemization also occurs at an accelerated rate with physical and/or chemical treatments. For example, racemization of L-lysine at elevated temperatures has great potential as a commercial process of D-lysine production (20).The biochemistry of D-amino acid catabolism has not been intensively studied in comparison to those of L-amino acids. In general, D-amino acids are metabolized either directly or after conversion into the L-enantiomers. Pseudomonas aeruginosa is able to utilize many D-amino acids as nutrients and hence serves as an excellent model organism to explore novel pathways and enzymes for D-amino acid metabolism. A new type of D-to-L arginine racemization by coupled catabolic and anabolic dehydrogenases encoded by the dauBA operon was recently reported by our group (15-16). Furthermore, the molecular structure of DauA, a flavin adenine dinucleotide (FAD)-dependent D-amino acid dehydrogenase, has been determined (8).In contrast, L-alanine catabolism in Escherichia coli and other Gram-negative bacteria is mediated by DadX-dependent L-to-D racemization followed by DadA-dependent oxidative deamination of D-alanine (Fig. 1). An early report by Wasserman and coworkers established the presence of two alanine racemases, the importance of L-to-D racemization for L-Ala catabolism, and the physical proximity and coregulation of two genes encoding D-alanine dehydrogenase and catabolic alanine racemase in Salmonella enterica serovar Typhimurium (21). The same gene organization was later found in E. coli (17). The dadAX operon and its regulation by the leucine-responsive regulator Lrp and carbon catabolite repression in enteric bacteria have been characterized (23-24). DadA of E. coli has

The objective of this study was to investigate the genetic basis of high level aminoglycoside resistance in Acinetobacter baumannii clinical isolates from Beijing, China. 173 A. baumannii clinical isolates from hospitals in Beijing from 2006 to 2009 were first subjected to high level aminoglycoside resistance (HLAR, MIC to gentamicin and amikacin>512 µg/mL) phenotype selection by broth microdilution method. The strains were then subjected to genetic basis analysis by PCR detection of the aminoglycoside modifying enzyme genes (aac(3)-I, aac(3)-IIc, aac(6′)-Ib, aac(6′)-II, aph(4)-Ia, aph(3′)-I, aph(3′)-IIb, aph(3′)-IIIa, aph(3′)-VIa, aph(2″)-Ib, aph(2″)-Ic, aph(2″)-Id, ant(2″)-Ia, ant(3″)-I and ant(4′)-Ia) and the 16S rRNA methylase genes (armA, rmtB and rmtC). Correlation analysis between the presence of aminoglycoside resistance gene and HLAR phenotype were performed by SPSS. Totally 102 (58.96%) HLAR isolates were selected. The HLAR rates for year 2006, 2007, 2008 and 2009 were 52.63%, 65.22%, 51.11% and 70.83%, respectively. Five modifying enzyme genes (aac(3)-I, detection rate of 65.69%; aac(6′)-Ib, detection rate of 45.10%; aph(3′)-I, detection rate of 47.06%; aph(3′)-IIb, detection rate of 0.98%; ant(3″)-I, detection rate of 95.10%) and one methylase gene (armA, detection rate of 98.04%) were detected in the 102 A. baumannii with aac(3)-I+aac(6′)-Ib+ant(3″)-I+armA (detection rate of 25.49%), aac(3)-I+aph(3′)-I+ant(3″)-I+armA (detection rate of 21.57%) and ant(3″)-I+armA (detection rate of 12.75%) being the most prevalent gene profiles. The values of chi-square tests showed correlation of armA, ant(3″)-I, aac(3)-I, aph(3′)-I and aac(6′)-Ib with HLAR. armA had significant correlation (contingency coefficient 0.685) and good contingency with HLAR (kappa 0.940). The high rates of HLAR may cause a serious problem for combination therapy of aminoglycoside with β-lactams against A. baumannii infections. As armA was reported to be able to cause high level aminoglycoside resistance to most of the clinical important aminoglycosides (gentamicin, amikacin, tobramycin, etc), the function of aminoglycoside modifying enzyme gene(s) in A. baumannii carrying armA deserves further investigation.

A unique D-to-L racemization of arginine by coupled arginine dehydrogenases DauA and DauB encoded by the dauBAR operon has been recently reported as a prerequisite for D-arginine utilization as the sole source of carbon and nitrogen through L-arginine catabolic pathways in P. aeruginosa. In this study, enzymic properties of the catabolic FAD-dependent D-amino acid dehydrogenase DauA and the physiological functions of the dauBAR operon were further characterized with other D-amino acids. These results establish DauA as a D-amino acid dehydrogenase of broad substrate specificity, with D-Arg and D-Lys as the two most effective substrates, based on the kinetic parameters. In addition, expression of dauBAR is specifically induced by exogenous D-Arg and D-Lys, and mutations in the dauBAR operon affect utilization of these two amino acids alone. The function of DauR as a repressor in the control of the dauBAR operon was demonstrated by dauB promoter activity measurements in vivo and mobility shift assays with purified His-tagged protein in vitro. The potential effect of 2-ketoarginine (2-KA) derived from D-Arg deamination by DauA as a signal molecule in dauBAR induction was first revealed by mutation analysis and further supported by its in vitro effect on alleviation of DauR-DNA interactions. Through sequence analysis, putative DauR operators were identified and confirmed by mutation analysis. Induction of the dauBAR operon to the maximal level was found to require the L-arginine-responsive regulator ArgR, as supported by the loss of inductive effect by LArg on dauBAR expression in the argR mutant and binding of purified ArgR to the dauB regulatory region in vitro. In summary, this study establishes that optimal induction of the dauBAR operon requires relief of DauR repression by 2-KA and activation of ArgR by L-Arg as a result of D-Arg racemization by the encoded DauA and DauB.

The results validated the potent efficacy of nemonoxacin in vivo. The higher efficacy of nemonoxacin than of levofloxacin towards infections caused by Gram-positive cocci (especially MRSA, levofloxacin-resistant MRSC, PRSP and VRE) warrants investigation of its clinical use.