Existence of two D-alanine:D-alanine ligases in Escherichia coli: cloning and sequencing of the ddlA gene and purification and characterization of the DdlA and DdlB enzymes

Two distinct genes encoding D-alanine:D-alanine (D-Ala-D-Ala) ligase (ADP forming) activity in Escherichia coli have been cloned by complementation of E. coli strain ST640(lambda 112) deficient in D-Ala-D-Ala ligase activity with a lambda library of E. coli DNA. One of the two genes, designated as ddlB, is identical with the ddl gene already sequenced [Robinson, A.C., Kenan, D.L., Sweeney, J., & Donachie, W.D. (1986) J. Bacteriol. 167, 809-817]. We describe the subcloning and DNA sequencing of the other gene, designated as ddlA on the basis of similarities with the Salmonella typhimurium ddlA gene [Daub, E., Zawadzke, L.E., Botstein, D., & Walsh, C.T. (1988) Biochemistry 27, 3701-3708]. The predicted amino acid sequence of the E. coli DdlA enzyme shows 90% homology with the S. typhimurium DdlA sequence. The ddlB gene was subcloned by use of the polymerase chain reaction into an expression vector containing an optimized ribosome binding site, which expressed the DdlB enzyme to greater than 50% soluble cell protein. Both DdlA and DdlB enzymes were purified to greater than 90% homogeneity and characterized kinetically.     

Sequencing of the ddl gene and modeling of the mutated D-alanine:D-alanine ligase in glycopeptide-dependent strains of Enterococcus faecium

Glycopeptide dependence for growth in enterococci results from mutations in the ddl gene that inactivate the host D-Ala:D-Ala ligase. The strains require glycopeptides as inducers for synthesis of resistance proteins, which allows for the production of peptidoglycan precursors ending in D-Ala-D-Lac instead of D-Ala-D-Ala. The sequences of the ddl gene from nine glycopeptide-dependent Enterococcus faecium clinical isolates were determined. Each one had a mutation consisting either in a 5-bp insertion at position 41 leading to an early stop codon, an in-frame 6-bp deletion causing the loss of two residues (KDVA243-246 to KA), or single base-pair changes resulting in an amino acid substitution (E13 --> G, G99 --> R, V241 --> D, D295 --> G, P313 --> L). The potential consequences of the deletion and point mutations on the 3-D structure of the enzyme were evaluated by comparative molecular modeling of the E. faecium enzyme, using the X-ray structure of the homologous Escherichia coli D-Ala:D-Ala ligase DdlB as a template. All mutated residues were found either to interact directly with one of the substrates of the enzymatic reaction (E13 and D295) or to stabilize the position of critical residues in the active site. Maintenance of the 3-D structure in the vicinity of these mutations in the active site appears critical for D-Ala:D-Ala ligase activity.     

(1-Aminoethyl)boronic acid: a novel inhibitor for Bacillus stearothermophilus alanine racemase and Salmonella typhimurium D-alanine:D-alanine ligase (ADP-forming)

(1-Aminoethyl)boronic acid (Ala-B), an analogue of alanine in which a boronic acid group replaces the carboxyl group, has been synthesized and found to inhibit the first two enzymes, alanine racemase (from Bacillus stearothermophilus, EC 5.1.1.1) and D-alanine:D-alanine ligase (ADP-forming) (from Salmonella typhimurium, EC 6.3.2.4), of the D-alanine branch of bacterial peptidoglycan biosynthesis. In both cases, time-dependent, slow binding inhibition is observed due to the generation of long-lived, slowly dissociating complexes. Ala-B inhibits alanine racemase with a Ki of 20 mM and a kappa inact of 0.15-0.35 min-1. Time-dependent loss of activity is paralleled by conversion of the 420-nm chromophore of initial bound PLP aldimine to a 324-nm absorbing species. On dilution of Ala-B, racemase activity is regained with a t1/2 of ca. 1 h. The D-Ala-D-Ala ligase also shows progressive inhibition by Ala-B provided ATP (but not AMP-PNP or AMP-PCP) is present. The presence of D-alanine along with ATP also leads to Ala-B-induced inactivation. Kinetic analysis suggests Ala-B can compete with D-alanine at either of the two D-alanine binding sites, and on inactivation with Ala-B, labeled D-alanine, and labeled ATP, the inactive enzyme has stoichiometric amounts of D-alanine, ADP, Pi, and Ala-B bound. The half-life of inactive enzyme complexes varied from approximately 2 h (without D-alanine) to 4.5 days (with D-alanine). No D-Ala-D-Ala-B dipeptide was detected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of enterococci at the species level by sequencing of the genes for D-alanine:D-alanine ligases

A PCR assay based on the use of degenerate oligodeoxyribonucleotides allowed characterization of a fragment internal to the ddl genes encoding D-alanine:D-alanine ligases in Enterococcus columbae, E. durans, E. malodoratus, E. mundtii, E. raffinosus, E. seriolicida, E. solitarius, and E. sulfureus. Phylogenetic analysis of the sequence of the amplification products and of those already obtained from E. aviuni, E. casseliflavus, E. cecorum, E. dispar, E. faecalis, E. faecium, E. flavescens, E. gallinarurm, E. hirae, E. pseudoavium, and E. saccharolvticus yielded an evolutionary tree with a topology similar to that based on 16S rRNA sequences. Partial sequencing of the ddl gene can therefore be used for genotypic identification of Enterococcus spp.     

Characterization of Chlamydia MurC-Ddl, a fusion protein exhibiting D-alanyl-D-alanine ligase activity involved in peptidoglycan synthesis and D-cycloserine sensitivity

Recent characterization of chlamydial genes encoding functional peptidoglycan (PG)-synthesis proteins suggests that the Chlamydiaceae possess the ability to synthesize PG yet biochemical evidence for the synthesis of PG has yet to be demonstrated. The presence of D-amino acids in PG is a hallmark of bacteria. Chlamydiaceae do not appear to encode amino acid racemases however, a D-alanyl-D-alanine (D-Ala-D-Ala) ligase homologue (Ddl) is encoded in the genome. Thus, we undertook a genetics-based approach to demonstrate and characterize the D-Ala-D-Ala ligase activity of chlamydial Ddl, a protein encoded as a fusion with MurC. The full-length murC-ddl fusion gene from Chlamydia trachomatis serovar L2 was cloned and placed under the control of the arabinose-inducible ara promoter and transformed into a D-Ala-D-Ala ligase auxotroph of Escherichia coli possessing deletions of both the ddlA and ddlB genes. Viability of the E. coliDeltaddlADeltaddlB mutant in the absence of exogenous D-Ala-D-Ala dipeptide became dependent on the expression of the chlamydial murC-ddl thus demonstrating functional ligase activity. Domain mapping of the full-length fusion protein and site-directed mutagenesis of the MurC domain revealed that the structure of the full fusion protein but not MurC enzymatic activity was required for ligase activity in vivo. Recombinant MurC-Ddl exhibited substrate specificity for D-Ala. Chlamydia growth is inhibited by D-cycloserine (DCS) and in vitro analysis provided evidence for the chlamydial MurC-Ddl as the target for DCS sensitivity. In vivo sensitivity to DCS could be reversed by addition of exogenous D-Ala and D-Ala-D-Ala. Together, these findings further support our hypothesis that PG is synthesized by members of the Chlamydiaceae family and suggest that D-amino acids, specifically D-Ala, are present in chlamydial PG.     

Molecular basis for vancomycin resistance in Enterococcus faecium BM4147: biosynthesis of a depsipeptide peptidoglycan precursor by vancomycin resistance proteins VanH and VanA

Vancomycin resistance in Enterococcus faecium BM4147 is mediated by vancomycin resistance proteins VanA and VanH. VanA is a D-alanine:D-alanine ligase of altered substrate specificity [Bugg, T. D. H., Dutka-Malen, S., Arthur, M., Courvalin, P., & Walsh, C. T. (1991) Biochemistry 30, 2017-2021], while the sequence of VanH is related to those of alpha-keto acid dehydrogenases [Arthur, M., Molinas, C., Dutka-Malen, S., & Courvalin, P. (1991) Gene (submitted)]. We report purification of VanH to homogeneity, characterization as a D-specific alpha-keto acid dehydrogenase, and comparison with D-lactate dehydrogenases from Leuconostoc mesenteroides and Lactobacillus leichmanii. VanA was found to catalyze ester bond formation between D-alanine and the D-hydroxy acid products of VanH, the best substrate being D-2-hydroxybutyrate (Km = 0.60 mM). The VanA product D-alanyl-D-2-hydroxybutyrate could then be incorporated into the UDPMurNAc-pentapeptide peptidoglycan precursor by D-Ala-D-Ala adding enzyme from Escherichia coli or by crude extract from E. faecium BM4147. The vancomycin binding constant of a synthetic modified peptidoglycan analogue N-acetyl-D-alanyl-D-2-hydroxybutyrate (Kd greater than 73 mM) was greater than 1000-fold higher than the binding constant for N-acetyl-D-alanyl-D-alanine (Kd = 54 microM), partly due to the disruption of a hydrogen bond in the vancomycin-target complex, thus providing a molecular rationale for high-level vancomycin resistance.     

Genetic analysis of peptidoglycan biosynthesis in mycobacteria: characterization of a ddlA mutant of Mycobacterium smegmatis

A temperature-sensitive mutant of Mycobacterium smegmatis was characterized that contains a mutation in ddlA, the gene encoding D-alanine:D-alanine ligase. Enzymatic assays using recombinant proteins and D-cycloserine susceptibility indicate that the A365V mutation in the SMEG35 DdlA protein causes a reduction in enzymatic activity in vitro and in vivo.     

Enzymatic characterization and crystal structure analysis of the D-alanine-D-alanine ligase from Helicobacter pylori

D-Alanine-D-alanine ligase is the second enzyme in the D-Ala branch of bacterial cell wall peptidoglycan assembly, and recognized as an attractive antimicrobial target. In this work, the D-Ala-D-Ala ligase of Helicobacter pylori strain SS1 (HpDdl) was kinetically and structurally characterized. The determined apparent K(m) of ATP (0.87 microM), the K(m1) (1.89 mM) and K(m2) of D-Ala (627 mM), and the k(cat) (115 min(-1)) at pH 8.0 indicated its relatively weak binding affinity and poor catalytic activity against the substrate D-Ala in vitro. However, by complementary assay of expressing HpDdl in Escherichia coli Delta ddl mutant, HpDdl was confirmed to be capable of D-Ala-D-Ala ligating in vivo. Through sequence alignment with other members of the D-Ala-D-X ligase superfamily, HpDdl keeps two conservatively substituted residues (Ile16 and Leu241) and two nonconserved residues (Leu308 and Tyr311) broadly located in the active region of the enzyme. Kinetic analyses against the corresponding HpDdl mutants (I16V, L241Y, L241F, L308T, and Y311S) suggested that these residues, especially Leu308 and Tyr311, might partly contribute to the unique catalytic properties of the enzyme. This was fairly proved by the crystal structure of HpDdl, which revealed that there is a 3(10)-helix (including residues from Gly306 to Leu312) near the D-Ala binding region in the C-terminal domain, where HpDdl has two sequence deletions compared with other homologs. Such 3(10)-helix may participate in D-Ala binding and conformational change of the enzyme. Our present work hopefully provides useful information for understanding the D-Ala-D-Ala ligase of Helicobacter pylori.     

ATP-dependent inactivation and slow binding inhibition of Salmonella typhimurium D-alanine:D-alanine ligase (ADP) by (aminoalkyl)phosphinate and aminophosphonate analogues of D-alanine

In Salmonella typhimurium, D-alanine:D-alanine ligase (ADP) (EC 6.3.2.4) is the second enzyme in the three enzyme D-alanine branch pathway of peptidoglycan biosynthesis. The interaction of this enzyme with a possible transition-state analogue, the (aminoalkyl)phosphinate D-3-[(1-aminoethyl)phosphinyl]-2-heptylpropionic acid [Parsons et al. (1987) Abstracts of Papers, 193rd National Meeting of the American Chemical Society, Denver, CO, MEDI 63, American Chemical Society, Washington, DC], has been studied. This compound is a potent active site directed inhibitor and is competitive with D-alanine (Ki = 1.2 microM); it exhibits time-dependent inhibition in the presence of ATP. Kinetic analysis revealed a rapid onset of steady-state inhibition (kon = 1.35 X 10(4) M-1 s-1) followed by slow dissociation of inhibitory complex(es) with a half-life of 8.2 h. The inhibitory complex was shown to consist of E...I...ATP in equilibrium with E...I, Pi, and ADP. Similar time-dependent inhibition was also observed with D-(1-aminoethyl)phosphonic acid (D-Ala-P) (Ki = 0.5 mM; kon = 27 M-1 s-1; t1/2 for regain = 1.73 min) but not with D-(1-aminoethyl)phosphinic acid, which behaved as a simple competitive inhibitor (Ki = 0.4 mM). The mechanism of inhibition is discussed in the light of the precedents of glutamine synthase inhibition by methionine sulfoximine and phosphinothricin.     

Sequence of active site peptides from the penicillin-sensitive D-alanine carboxypeptidase of Bacillus subtilis. Mechanism of penicillin action and sequence homology to beta-lactamases

It has been proposed that penicillin and other beta-lactam antibiotics are substrate analogs which inactivate certain essential enzymes of bacterial cell wall biosynthesis by acylating a catalytic site amino acid residue (Tipper, D.J., and Strominger, J.L. (1965) Proc. Natl. Acad. Sci. U.S.A. 54, 1133-1141). A key prediction of this hypothesis, that the penicilloyl moiety and an acyl moiety derived from substrate both bind to the same active site residue, has been examined. D-Alanine carboxypeptidase, a penicillin-sensitive membrane enzyme, was purified from Bacillus subtilis and labeled covalently at the antibiotic binding site with [14C]penicillin G or with the cephalosporin [14C]cefoxitin. Alternatively, an acyl moiety derived from the depsipeptide substrate [14C]diacetyl L-Lys-D-Ala-D-lactate was trapped at the catalytic site in near-stoichiometric amounts by rapid denaturation of an acyl-enzyme intermediate. Radiolabeled peptides were purified from a pepsin digest of each of the 14C-labeled D-alanine carboxypeptidases and their amino acid sequences determined. Antibiotic- and substrate-labeled peptic peptides had the same sequence: Tyr-Ser-Lys-Asn-Ala-Asp-Lys-Arg-Leu-Pro-Ile-Ala-Ser-Met. Acyl moieties derived from antibiotic and from substrate were shown to be bound covalently in ester linkage to the identical amino acid residue, a serine at the penultimate position of the peptic peptide. These studies establish that beta-lactam antibiotics are indeed active site-directed acylating agents. Additional amino acid sequence data were obtained by isolating and sequencing [14C]penicilloyl peptides after digestion of [14C]penicilloyl D-alanine carboxypeptidase with either trypsin or cyanogen bromide and by NH2-terminal sequencing of the uncleaved protein. The sequence of the NH2-terminal 64 amino acids was thus determined and the active site serine then identified as residue 36. A computer search for homologous proteins indicated significant sequence homology between the active site of D-alanine carboxypeptidase and the NH2-terminal portion of beta-lactamases. Maximum homology was obtained when the active site serine of D-alanine carboxypeptidase was aligned correctly with a serine likely to be involved in beta-lactamase catalysis. These findings provide strong evidence that penicillin-sensitive D-alanine carboxypeptidases and penicillin-inactivating beta-lactamases are related evolutionarily.     

Temperature-sensitive mutant of Escherichia coli K-12 with an impaired D-alanine:D-alanine ligase

The temperature-sensitive Escherichia coli mutant strain ST-640 lyses at the restrictive temperature except when an osmotic stabilizer or a high concentration of d-alanine is present. The presence of dl-alanyl-dl-alanine does not prevent lysis. The rate of murein synthesis, followed in a wall medium, is decreased at both 30 and 42 C. d-Alanyl-d-alanine and uridine diphosphate-N-acetyl-muramyl (UDP-MurNAc)-pentapeptide are synthesized in decreased amounts, accompanied by accumulation of UDP-MurNAc-tripeptide at 42 C but not at 30 C. Uridine nucleotide precursors leak into the medium, especially out of the mutant cells. This leakage is prevented when NaCl is present. The d-alanine: d-alanine ligase (ADP) (EC 6.3.2.4) of the mutant strain, assayed in crude extracts, is temperature sensitive. The impaired ligase is relatively resistant to d-cycloserine and other inhibitors of the enzyme. Combined genetic and enzymatic results show that the low ligase activity is due to a mutation in the ddl gene, the structural gene for d-alanine: d-alanine ligase.     

Sequence of the vanC gene of Enterococcus gallinarum BM4174 encoding a D-alanine:D-alanine ligase-related protein necessary for vancomycin resistance.

The amplification product obtained with DNA from vancomycin-resistant (VmR) Enterococcus gallinarum BM4174 and a pair of degenerate oligodeoxyribonucleotides that correspond to conserved amino acid (aa) motifs in Escherichia coli D-alanine (D-Ala):D-Ala ligases and in En. faecium VmR protein (VanA) was used as a probe to clone the vanC gene of that strain. The vanC product, with a calculated Mr of 37,504, exhibits 29 to 38% aa identity with VanA and E. coli ligases. Insertional inactivation of vanC led to Vm sensitivity of BM4174 suggesting that the gene may encode a D-Ala:D-Ala ligase of altered specificity.     

Molecular, genetic, and topological characterization of O-antigen chain length regulation in Shigella flexneri.

The rfb region of Shigella flexneri encodes the proteins required to synthesize the O-antigen component of its cell surface lipopolysaccharides (LPS). We have previously reported that a region adjacent to rfb was involved in regulating the length distribution of the O-antigen polysaccharide chains (D. F. Macpherson et al., Mol. Microbiol. 5:1491-1499, 1991). The gene responsible has been identified in Escherichia coli O75 (called rol [R. A. Batchelor et al., J. Bacteriol. 173:5699-5704, 1991]) and in E. coli O111 and Salmonella enterica serovar typhimurium strain LT2 (called cld [D. A. Bastin et al., Mol. Microbiol. 5:2223-2231, 1991]). Through a combination of subcloning, deletion, and transposon insertion analysis, we have identified a gene adjacent to the S. flexneri rfb region which encodes a protein of 36 kDa responsible for the length distribution of O-antigen chains in LPS as seen on silver-stained sodium dodecyl sulfate-polyacrylamide gels. DNA sequence analysis identified an open reading frame (ORF) corresponding to the rol gene. The corresponding protein was almost identical in sequence to the Rol protein of E. coli O75 and was highly homologous to the functionally identical Cld proteins of E. coli O111 and S. enterica serovar typhimurium LT2. These proteins, together with ORF o349 adjacent to rfe, had almost identical hydropathy plots which predict membrane-spanning segments at the amino- and carboxy-terminal ends and a hydrophilic central region. We isolated a number of TnphoA insertions which inactivated the rol gene, and the fusion end points were determined. The PhoA+ Rol::PhoA fusion proteins had PhoA fused within the large hydrophilic central domain of Rol. These proteins were located in the whole-membrane fraction, and extraction with Triton X-100 indicated a cytoplasmic membrane location. This finding was supported by sucrose density gradient fractionation of the whole-cell membranes and of E. coli maxicells expressing L-[35S]methionine-labelled Rol protein. Hence, we interpret these data to indicate that the Rol protein is anchored into the cytoplasmic membrane via its amino- and carboxy-terminal ends but that the majority of the protein is located in the periplasmic space. To confirm that rol is responsible for the effects on O-antigen chain length observed with the cloned rfb genes in E. coli K-12, it was mutated in S. flexneri by insertion of a kanamycin resistance cartridge. The resulting strains produced LPS with O antigens of nonmodal chain length, thereby confirming the function of the rol gene product. We propose a model for the function of Rol protein in which it acts as a type of molecular chaperone to facilitate the interaction of the O-antigen ligase (RfaL) with the O-antigen polymerase (Rfc) and polymerized, acyl carrier lipid-linked, O-antigen chains. Analysis of the DNA sequence of the region identified a number of ORFs corresponding to the well-known gnd and hisIE genes. The rol gene was located immediately downstream of two ORFs with sequence similarity to the gene encoding UDPglucose dehydrogenase (HasB) of Streptococcus pyogenes. The ORFs arise because of a deletion or frameshift mutation within the gene we have termed udg (for UDPglucose dehydrogenase).     

D-alanine-D-alanine ligase (ADP) from Salmonella typhimurium. Overproduction, purification, crystallization and preliminary X-ray analysis

The ddlA gene from Salmonella typhimurium coding for D-alanine-D-alanine ligase (ADP-forming) has been subcloned behind the tac promotor in the plasmid pKK223-3, with expression in Escherichia coli JM105. The overexpression system yields 58 mg of active enzyme from 12 g of wet cell paste after 40-fold purification to homogeneity. 5,5'-Dithiobis-(2-nitrobenzoic acid) titrations indicate that all four cysteine residues exist as free thiols. Two crystal forms of the 39,300 Mr enzyme have been produced. A tetragonal form grows at 21 degrees C from 10 to 15% (w/v) polyethylene glycol 8000 in space group P4(1)2(1)2, with two molecules in the asymmetric unit; it has cell constants a = b = 83.8 A, c = 220.0 A, and diffracts to 2.9 A. A monoclinic form grows from 30% (w/v) ammonium sulfate in space group P2(1), with two molecules in the asymmetric unit; it has cell constants a = 60.4 A, b = 102.1 A, c = 64.3 A, beta = 115.7 degrees, and diffracts to 2.2 A resolution.     

Glutamyl-tRNA reductase from Escherichia coli and Synechocystis 6803. Gene structure and expression.

In the cyanobacterium Synechocystis sp. PCC 6803 and in the enterobacterium Escherichia coli delta-amino-levulinic acid (ALA) is formed from glutamyl-tRNA by the sequential action of two enzymes, glutamyl-tRNA reductase (GluTR) and glutamate-1-semialdehyde aminotransferase. E. coli has two GluTR proteins with sizes of 45 kDa (GluTR45) and 85 kDa (GluTR85) (Jahn, D., Michelsen, U., and S?ll, D. (1991) J. Biol. Chem. 266, 2542-2548). The hemA gene, isolated from E. coli and several other eubacteria, has been proposed to encode a structural component of GluTR. Because of the inability to overexpress this gene in E. coli, we demonstrate directly GluTR function for the E. coli hemA gene product by its expression and functional analysis in yeast, which does not form ALA from Glu-tRNA. Gel filtration experiments demonstrated definitively that the yeast-expressed HemA protein corresponded to GluTR45. Furthermore, analysis of GluTR activity in an E. coli strain with a disrupted hemA gene displayed GluTR85, but not GluTR45 activity. The hemA gene from Synechocystis 6803 was cloned by functional complementation in E. coli. DNA sequence analysis revealed an open reading frame capable of encoding a 427-amino acid polypeptide (molecular mass of 47,525 Da). The Synechocystis 6803 amino acid sequence shows significant similarity upon alignment with HemA sequences from E. coli, Bacillus subtilis, Salmonella typhimurium, and Chlorobium vibrioforme but does not contain the amino acid sequence derived from the N terminus of the previously purified GluTR protein (Rieble, S., and Beale, S. I. (1991) J. Biol. Chem. 266, 9740-9745). These experiments are the first direct demonstration of GluTR activity of the HemA protein and provide further evidence for two pathways of ALA formation in prokaryotes.     

The primary structure of Salmonella typhimurium HPr, a phosphocarrier protein of the phosphoenolpyruvate:glycose phosphotransferase system. A correction

The protein HPr is a low-molecular-weight phosphocarrier protein of the bacterial phosphoenolpyruvate:glycose phosphotransferase system. We have recently reported the complete primary amino acid sequence of HPr isolated from Salmonella typhimurium (Weigel, N., Powers, D.A., and Roseman, S. (1982) J. Biol. Chem. 257, 14499-14509). This sequence is incorrect at certain residues; the correct primary structure of the protein is presented in this report. The corrected structure generally agrees with the primary sequence predicted for HPr from Escherichia coli (based on the nucleotide sequence of the corresponding ptsH gene). The one apparent ambiguity is at the carboxyl terminus.     

Molecular characterization of D-amino acid oxidase from common carp Cyprinus carpio and its induction with exogenous free D-alanine

A full-length cDNA encoding D-amino acid oxidase (DAO, EC 1.4.3.3) was cloned and sequenced from the hepatopancreas of carp fed a diet supplemented with D-alanine. This clone contained an open reading frame encoding 347 amino acid residues. The deduced amino acid sequence exhibited about 60 and 19-29% identity to mammalian and microbial DAOs, respectively. The expression of full-length carp DAO cDNA in Escherichia coli resulted in a significant level of protein with DAO activity. In carp fed the diet with D-alanine for 14 days, DAO mRNA was strongly expressed in intestine followed by hepatopancreas and kidney, but not in muscle. During D-alanine administration, DAO gene was expressed quickly in hepatopancreas with the increase of DAO activity. The inducible nature of carp DAO indicates that it plays an important physiological role in metabolizing exogenous D-alanine that is abundant in their prey invertebrates, crustaceans, and mollusks.