The molecular structure of the D-alanine:D-alanine ligase of the ddlB gene of Escherichia coli, co-crystallized with an S,R-methylphosphinate and adenosine triphosphate, was determined by x-ray diffraction to a resolution of 2.3 angstroms. A catalytic mechanism for the ligation of two D-alanine substrates is proposed in which a helix dipole and a hydrogen-bonded triad of tyrosine, serine, and glutamic acid assist binding and deprotonation steps. From sequence comparison, it is proposed that a different triad exists in a recently discovered D-alanine:D-lactate ligase (VanA) present in vancomycin-resistant enterococci. A molecular mechanism for the altered specificity of VanA is suggested.

The crystallographic structure of the D-alanine:D-alanine ligase of the ddlB gene of Escherichia coli complexed with a D-Ala-D-alpha-hydroxybutyrate phosphonate and the structure of the Y216F mutant ligase complexed with a D-Ala-D-Ala phosphinate have been determined to 2.2 and 1.9 A resolution, respectively, and refined to R factors of 0.156 and 0.158. In each complex the inhibitor has reacted with ATP to produce ADP and a tight-binding phosphorylated transition state intermediate. Comparison of these two structures with the known crystal structure of the phosphinate intermediate of the wild-type ligase shows no major conformational changes, but B factors indicate differences in mobility of loops covering the binding site. The weaker inhibition of the Y216F mutant by both inhibitors is thought to be due in part to the loss of an interloop hydrogen bond. A similar mechanism may account for poor inhibition of VanA, the homologous D-Ala:D-lactate ligase produced by vancomycin-resistant enterococci.

Examination of x-ray crystallographic structures shows the tertiary structure of D-alanine:D-alanine ligase (EC 6.3.2.4), a bacterial cell wall synthesizing enzyme, is similar to that of glutathione synthetase (EC 6.3.2.3) despite low sequence homology. Both Escherichia coli enzymes, which convert ATP to ADP during ligation to produce peptide products, are made of three domains, each folded around a 4-to 6-stranded ,B-sheet core. Sandwiched between the 8-sheets of the C-terminal and central domains of each enzyme is a nonclassical ATP-binding site that contains a common set of spatially equivalent amino acids. In recently have x-ray structures of various members of this class become available. The Escherichia coli glutamine synthetase, where H2NR' = NH3, is a homododecamer of 50-kDa subunits that is highly regulated by intersubunit interactions (1). For a long time, it was the sole representative whose structure was known (6). More recently the structure of GSHase from E.coli, a homotetramer of 36-kDa subunits, was solved to 2.0-A resolution (7). Crystal soaking with ATP and the analog y-Glu-L-aminobutyrate revealed approximate locations of nucleotide and dipeptide binding. Details of the binding interactions were not published, however, because two loops near the binding sites were disordered.We recently reported the x-ray structure of DD-ligase of the ddlB gene of E. coli complexed with ADP and a phosphoryScheme I lated phosphinate analog of tetrahedral adduct 2 (8). A dramatic similarity was noted in the three-dimensional structures of the DD-ligase and GSHase, in spite of only marginal sequence homology. The crystallographic structure of the DD-ligase complex may, therefore, provide significant structural details and mechanistic insights not available from the disordered structure of the homologous GSHase complex. Thus, the two crystal structures suggest the enzymes possess a signature fold and catalytic site that may prove characteristic of a family of ADP-forming peptide synthetases. METHODS Primary Sequence Comparison. For the protein data base search of primary amino acid sequences, the BLASTP program (9) was used. Sequence alignments were done with the CLUSTAL program (10) using a Dayhoff weighting scheme with PAM250 (11).Tertiary Structures. The crystallographic structure of the ternary complex of E. coli DD-ligase with ADP and a phosphorylphosphinate, refined at 2.3-A resolution to an R factor of 0.171 (8), is deposited in the Protein Data Bank, Chemistry Department, Brookhaven National Laboratory, entry 2DLN. The enzyme has 306 residues, all of which are visible in the electron density map. DD-Ligase is dimeric across the diad of space group P21212.The crystallographic structure of E. coli GSHase, refined at 2.0-A resolution to an R factor of 0.186 (7), was taken from the Protein Data Bank, entry 1GLT. The enzyme has 315 amino acid residues but atomic coordinates of only 296 residues are available because two regions, Gly-164 to to are invisible in the electron density map. Coordi-

BackgroundGram-negative bacteria of the genus Serratia are potential producers of many useful secondary metabolites, such as prodigiosin and serrawettins, which have potential applications in environmental bioremediation or in the pharmaceutical industry. Several Serratia strains produce prodigiosin and serrawettin W1 as the main bioactive compounds, and the biosynthetic pathways are co-regulated by quorum sensing (QS). In contrast, the Serratia strain, which can simultaneously produce prodigiosin and serrawettin W2, has not been reported. This study focused on analyzing the genomic sequence of Serratia sp. strain YD25T isolated from rhizosphere soil under continuously planted burley tobacco collected from Yongding, Fujian province, China, which is unique in producing both prodigiosin and serrawettin W2.ResultsA hybrid polyketide synthases (PKS)-non-ribosomal peptide synthetases (NRPS) gene cluster putatively involved in biosynthesis of antimicrobial serrawettin W2 was identified in the genome of YD25T, and its biosynthesis pathway was proposed. We found potent antimicrobial activity of serrawettin W2 purified from YD25T against various pathogenic bacteria and fungi as well as antitumor activity against Hela cells. Subsequently, comparative genomic analyses were performed among a total of 133 Serratia species. The prodigiosin biosynthesis gene cluster in YD25T belongs to the type I pig cluster, which is the main form of pig-encoding genes existing in most of the pigmented Serratia species. In addition, a complete autoinducer-2 (AI-2) system (including luxS, lsrBACDEF, lsrGK, and lsrR) as a conserved bacterial operator is found in the genome of Serratia sp. strain YD25T. Phylogenetic analysis based on concatenated Lsr and LuxS proteins revealed that YD25T formed an independent branch and was clearly distant from the strains that solely produce either prodigiosin or serrawettin W2. The Fe (III) ion reduction assay confirmed that strain YD25T could produce an AI-2 signal molecule. Phylogenetic analysis using the genomic sequence of YD25T combined with phylogenetic and phenotypic analyses support this strain as a member of a novel and previously uncharacterized Serratia species.ConclusionGenomic sequence and metabolite analysis of Serratia surfactantfaciens YD25T indicate that this strain can be further explored for the production of useful metabolites. Unveiling the genomic sequence of S. surfactantfaciens YD25T benefits the usage of this unique strain as a model system for studying the biosynthesis regulation of both prodigiosin and serrawettin W2 by the QS system.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3171-7) contains supplementary material, which is available to authorized users.