Sequencing of four overlapping DNA fragments comprising 3.527 kb isolated from a L. leichmannii genomic library revealed three complete open reading frames (ORFs) and one that was truncated. The deduced amino acid sequences of the complete ORFs showed considerable similarities with the already known sequences of the xerC, hslV, and hslU gene products of Escherichia coli: the site-specific XerC recombinase, a member of the lambda integrase family, and the HtpI resp. HtpO heat shock proteins. The deduced amino acid sequence of the fourth, incomplete ORF upstream the xerC gene showed strong homology with the gid A gene product of B. subtilis.

Nucleoside 2-deoxyribosyltransferase plays an important role in the salvage pathway of nucleotide metabolism in certain organisms, catalyzing the cleavage of beta-2'-deoxyribonucleosides and the subsequent transfer of the deoxyribosyl moiety to an acceptor purine or pyrimidine base. The kinetics describe a ping-pong-bi-bi pathway involving the formation of a covalent enzyme-deoxyribose intermediate. The enzyme is produced by a limited number of microorganisms and its functions have been exploited in its use as a biocatalyst to synthesize nucleoside analogs of therapeutic interest. We describe the crystal structure of the enzyme with and without bound ligand. The native structure was solved by the single isomorphous replacement with anomalous scattering method (SIRAS) and refined to 2.5 A resolution resulting in a crystallographic R factor of 16.6%. The enzyme comprises a single domain that belongs to the general class of doubly-wound alpha/beta proteins; it also exhibits a unique nucleoside-binding motif. X-ray analysis of enzyme-purine and enzyme-pyrimidine complexes presented here reveals that the active site lies in a cleft formed by the edge of the beta sheet and two alpha helices and contains side chains from two subunits. These results indicate residues that may be important in substrate binding and catalysis and thus may serve as a framework for elucidating the mechanism of enzyme activity. In particular, the proposed nucleophile, Glu98, lies in the nucleoside-binding pocket at an appropriate position for nucleophilic attack. A comparison of the enzyme interactions with both a purine and pyrimidine ligand provides some insight into the structural basis for enzyme specificity.

The gene encoding dihydroorotase (DHOase) of Lactobacillus leichmannii, the third enzyme of the pyrimidine biosynthetic pathway (Genbank (EMBL) accession no X78999), was cloned by phenotypic complementation of an E coli pyrC deficient mutant after transformation with Lactobacillus leichmannii genomic library DNA. The open reading frame of the L leichmannii pyrC gene spans 1281 bp and codes for a 427 amino cid polypeptide with a calculated M(r) of 46,316 Da. Primer extension showed that the initiation site for transcription is 37 bp upstream of the putative start codon ATG and Northern blot analysis confirmed its independent transcription from the adjacent pyrB gene. Comparison of the deduced amino acid sequence of L leichmannii DHOase with sequences established for other organisms yielded 46.6% identity with the corresponding Bacillus subtilis enzyme. Highly conserved protein domains suggest importance for the enzyme's function.

Ribonucleoside-triphosphate reductase (RTPR, EC 1.17.4.2) from Lactobacillus leichmannii, a monomeric adenosylcobalamin-requiring enzyme, catalyzes the conversion of nucleoside triphosphates to deoxynucleoside triphosphates. The gene for this enzyme has been cloned and sequenced. In contrast to expectations based on mechanistic considerations, there is no statistically significant sequence homology with the Escherichia coli reductase that requires a dinuclear-iron center and tyrosyl radical cofactor. The RTPR has been overexpressed and purified to homogeneity, yielding 90 mg of protein from 2.5 g of bacteria. Initial characterization of the recombinant RTPR indicates that its properties are identical to those of the RTPR isolated from L. leichmannii.

The Lactobacillus leichmannii pyrB gene, encoding pyrimidine biosynthetic enzyme aspartate transcarbamylase (ATCase), was cloned from a partial genomic library lying on a 1468 bp Sa/I/BstXI fragment. The predicted polypeptide sequence extending over 351 amino acid residues (M(r) 39 855 Da) was compared to those of various other organisms revealing clear identities towards them and important conservative stretches, implying that these proteins are closely related. Transcriptional initiation was mapped by primer extension and occurred 54 bp upstream of the pyrB open reading frame (ORF). Northern blot analysis indicates that the pyrB gene is transcribed as a single mRNA and not together with the following overlapping pyrC gene as a bicistronic mRNA. At high copy number the pyrB gene of L leichmannii seems to be lethal for its E coli host; inserted in a low copy vector it complements the uracil auxotrophy of an E coli pyrB mutant which shows distinct ATCase activity in the cell extract. With an excess of uracil in the growth medium the gene is apparently repressed and no ATCase activity can be measured.

Degenerate oligodeoxyribonucleotides complementary to sequences encoding conserved amino acid (aa) motifs in D-alanine:D-alanine ligases (Ddl) were used to amplify approx. 600-bp fragments from glycopeptide-resistant strains of Leuconostoc mesenteroides (Lm), Lactobacillus plantarum, La. salivarius and La. confusus, and from a susceptible strain of La. leichmannii. Comparison of the deduced aa sequences of the PCR products revealed that the Ddl-related enzymes of resistant Lm and Lactobacillus spp. are more akin to each other (47-63% aa identity) than to that of susceptible La. leichmannii (33-37% aa identity), indicating that the Ddl-related enzymes in these intrinsically resistant species of Gram+ bacteria exhibit structural differences with those in susceptible species. The Ddl-related enzymes, VanA and VanB, implicated in acquired resistance to glycopeptides in enterococci, were not closely related to their counterparts in Lm and Lactobacillus spp., as they displayed only 26-32% aa identity.

2'-Fluoro-2'-deoxyarabinonucleosides are time-dependent inhibitors of nucleoside 2-deoxyribosyltransferase. 2,6-Diamino-9-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-9H-purine (dFDAP) inhibited the enzyme by formation of a primary complex (Kd = 140 microM) that isomerized to a secondary complex with a first-order rate constant of 0.2 min-1. Inhibited enzyme contained stoichiometric amounts of covalently bound 2'-fluoro-2'-deoxyarabinosyl moiety, recovered less than 5% of its activity after storage for a week at 5 degrees C, but regained over 70% of the lost activity by treatment with 600 microM Ade. 6-Amino-9-(2'-deoxy-2'-fluoro-beta-D-arabinofuranosyl)-9H-purine (dFAdo) was a product of the reactivation reaction. Proteolysis of inhibited enzyme identified a modified fragment that spanned residues 82-107 which could not be sequenced past Gly-96. dFDAP-inhibited enzyme and enzyme reacted with normal substrates (i.e. dThd and dAdo) were hydrolyzed between Met-97 and Glu-98 by 0.1 M NaOH. These findings and model studies on the base lability of peptides containing glutamyl esters suggested that the gamma-carboxylate of Glu-98 was esterfied during catalysis. The role of Glu-98 was confirmed by changing this residue to alanine. The specific activity of wild-type enzyme was 3 orders of magnitude greater than that of the mutant enzyme. Collectively, chemical modification and mutagenesis studies have identified Glu-98 as the active site nucleophile of nucleoside 2-deoxyribosyltransferase.

The redox-active thiols of Escherichia coli ribonucleoside diphosphate reductase and of Lactobacillus leichmannii ribonucleoside triphosphate reductase have been located by a procedure involving (1) prereduction of enzyme with dithiothreitol, (2) specific oxidation of the redox-active thiols by treatment with substrate in the absence of exogenous reductant, (3) alkylation of other thiols with iodoacetamide, and (4) reduction of the disulfides with dithiothreitol and alkylation with [1-14C]iodoacetamide. The dithiothreitol-reduced E. coli B1 subunit is able to convert 3 equiv of CDP to dCDP and is labeled with 5.4 equiv of 14C. Sequencing of tryptic peptides shows that 2.8 equiv of 14C is on cysteines-752 and -757 at the C-terminus of B1, while 1.0-1.5 equiv of 14C is on cysteines-222 and -227. It thus appears that two sets of redox-active dithiols are involved in substrate reduction. The L. leichmannii reductase is able to convert 1.1 equiv of CTP to dCTP and is labeled with 2.1 equiv of 14C. Sequencing of tryptic peptides shows that 1.4 equiv of 14C is located on the two cysteines of C-E-G-G-A-C-P-I-K. This peptide shows remarkable and unexpected similarity to the thiol-containing region of the C-terminal peptide of E. coli B1, C-E-S-G-A-C-K-I.