Mycobacterium tuberculosis (Mtb) depends on biotin synthesis for survival during infection. In the absence of biotin, disruption of the biotin biosynthesis pathway results in cell death rather than growth arrest, an unusual phenotype for an Mtb auxotroph. Humans lack the enzymes for biotin production, making the proteins of this essential Mtb pathway promising drug targets. To this end, we have determined the crystal structures of the second and third enzymes of the Mtb biotin biosynthetic pathway, 7,8-diaminopelargonic acid synthase (DAPAS) and dethiobiotin synthetase (DTBS), at respective resolutions of 2.2 Å and 1.85 Å. Superimposition of the DAPAS structures bound either to the SAM analog sinefungin or to 7-keto-8-aminopelargonic acid (KAPA) allowed us to map the putative binding site for the substrates and to propose a mechanism by which the enzyme accommodates their disparate structures. Comparison of the DTBS structures bound to the substrate 7,8-diaminopelargonic acid (DAPA) or to ADP and the product dethiobiotin (DTB) permitted derivation of an enzyme mechanism. There are significant differences between the Mtb enzymes and those of other organisms; the Bacillus subtilis DAPAS, presented here at a high resolution of 2.2 Å, has active site variations and the Escherichia coli and Helicobacter pylori DTBS have alterations in their overall folds. We have begun to exploit the unique characteristics of the Mtb structures to design specific inhibitors against the biotin biosynthesis pathway in Mtb.

Phosphoglycerate dehydrogenases exist in at least three different structural motifs. The first D-3-phosphoglycerate dehydrogenase structure to be determined was from Escherichia coli and is a tetramer composed of identical subunits that contain three discernable structural domains. The crystal structure of D-3-phosphoglycerate dehydrogenase from Mycobacterium tuberculosis has been determined at 2.3 Å. This enzyme represents a second structural motif of the D-3-phosphoglycerate dehydrogenase family, one that contains an extended Cterminal region. This structure is also a tetramer of identical subunits, and the extended motif of 135 amino acids exists as a fourth structural domain. This intervening domain exerts quite a surprising characteristic to the structure by introducing significant asymmetry in the tetramer. The asymmetric unit is composed of two identical subunits that exist in two different conformations characterized by rotation of ϳ180°around a hinge connecting two of the four domains. This asymmetric arrangement results in the formation of two different and distinct domain interfaces between identical domains in the asymmetric unit. As a result, the surface of the intervening domain that is exposed to solvent in one subunit is turned inward in the other subunit toward the center of the structure where it makes contact with other structural elements. Significant asymmetry is also seen at the subunit level where different conformations exist at the NAD-binding site and the putative serinebinding site in the two unique subunits. 1 catalyzes the first committed step in this conversion of D-3-phosphoglycerate to phosphohydroxypyruvate utilizing NAD ϩ as a cofactor. Subsequently, phosphohydroxypyruvate is converted to phosphoserine by phosphoserine transaminase and then to L-serine by phosphoserine phosphatase (1, 2). PGDH also belongs to a family of proteins classified as 2-hydroxy acid dehydrogenases that are generally specific for substrates with a D-configuration (3). It shares sequence homology with other members of this family such as formate dehydrogenase and glycerate dehydrogenase whose structures are also known (4, 5).PGDH appears to be ubiquitously found in all organisms. It exists in at least three different basic structural motifs that do not appear to be strictly specific for organism type (6). The PGDH of some bacteria and some lower eukaryotes, such as yeast and Neurospora are similar to the Escherichia coli enzyme. In addition to substrate and nucleotide-binding domains, they possess a homologous C-terminal domain that, in E. coli, is involved in regulation of activity through binding the effector, L-serine. Other bacteria such as Mycobacterium, Bacillus subtilis, Corynebacterium, plants such as Arabidopsis, and higher order eukaryotes, including mammals, possess a large polypeptide insertion (ϳ150 -200 amino acids) in their C-terminal segment immediately following the substrate-binding domain and before the C-terminal domain. A third motif, which lacks the C-terminal regulatory domain altog...

D-3-Phosphoglycerate dehydrogenase (PGDH) from Mycobacterium tuberculosis has been isolated to homogeneity and displays an unusual relationship to the Escherichia coli and mammalian enzymes. In almost all aspects investigated, the M. tuberculosis enzyme shares the characteristics of the mammalian PGDHs. These include an extended C-terminal motif, substrate inhibition kinetics, dependence of activity levels and stability on ionic strength, and the inability to utilize ␣-ketoglutarate as a substrate. The unique property that the M. tuberculosis enzyme shares with E. coli PGDH that it is very sensitive to inhibition by L-serine, with an I 0.5 ‫؍‬ 30 M. The mammalian enzymes are not inhibited by L-serine. In addition, the cooperativity of serine inhibition appears to be modulated by chloride ion, becoming positively cooperative in its presence. This is modulated by the gain of cooperativity in serine binding for the first two effector sites. The basis for the chloride modulation of cooperativity is not known, but the sensitivity to serine inhibition can be explained in terms of certain amino acid residues in critical areas of the structures. The differential sensitivity to serine inhibition by M. tuberculosis and human PGDH may open up interesting possibilities in the treatment of multidrug-resistant tuberculosis.D-3-Phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) 1 has been investigated in a variety of organisms (1-4) and is a member of a family of proteins classified as 2-hydroxyacid dehydrogenases that are generally specific for substrates with a D-configuration (5). PGDH catalyzes the first committed step in the phosphorylated pathway of L-serine biosynthesis by converting D-3-phosphoglycerate to hydroxypyruvic acid phosphate utilizing NAD ϩ as a cofactor (1, 6). Subsequently, hydroxypyruvic acid phosphate is converted to phosphoserine by phosphoserine transaminase and then to L-serine by phosphoserine phosphatase (6). The reaction catalyzed by PGDH occurs spontaneously in vitro in the direction of conversion of hydroxypyruvic acid phosphate to phosphoglycerate.In some organisms, such as Escherichia coli (7), PGDH is strongly inhibited by L-serine (I 0.5 ϭ ϳ2-4 M), the end product of the pathway. E. coli PGDH is the most studied and is classified as a V-type enzyme (8) because L-serine regulates catalysis by altering the velocity of the reaction rather than the affinity of the substrate. In Bacillus subtilis, inhibition of PGDH by L-serine is less sensitive (I 0.5 ϳ 0.6 mM) but appears to lose its sensitivity to L-serine under oxidizing conditions (9). PGDH from Corynebacterium glutamicum has also been reported to be inhibited by L-serine only at very high concentrations (I 0.5 ϳ 10 mM) but has not been studied in homogeneous form (10). In addition, L-serine inhibition of both the B. subtilis and C. glutamicum PGDH require extensive preincubation of the enzyme with the inhibitor before appreciable inhibition can be measured. In the pea (Pisum sativum), the sensitivity to L-serine has been reported to be cold ...

Diversity-generating retroelements (DGRs) are a unique family of retroelements that confer selective advantages to their hosts by facilitating localized DNA sequence evolution through a specialized error-prone reverse transcription process. We characterized a DGR in Legionella pneumophila , an opportunistic human pathogen that causes Legionnaires disease. The L. pneumophila DGR is found within a horizontally acquired genomic island, and it can theoretically generate 10 26 unique nucleotide sequences in its target gene, legionella determinent target A ( ldtA ), creating a repertoire of 10 19 distinct proteins. Expression of the L. pneumophila DGR resulted in transfer of DNA sequence information from a template repeat to a variable repeat (VR) accompanied by adenine-specific mutagenesis of progeny VRs at the 3′end of ldtA . ldtA encodes a twin-arginine translocated lipoprotein that is anchored in the outer leaflet of the outer membrane, with its C-terminal variable region surface exposed. Related DGRs were identified in L. pneumophila clinical isolates that encode unique target proteins with homologous VRs, demonstrating the adaptability of DGR components. This work characterizes a DGR that diversifies a bacterial protein and confirms the hypothesis that DGR-mediated mutagenic homing occurs through a conserved mechanism. Comparative bioinformatics predicts that surface display of massively variable proteins is a defining feature of a subset of bacterial DGRs.

Summary Diversity-generating retroelements (DGRs) are the only known source of massive protein sequence variation in prokaryotes. These elements transfer coding information from a template region (TR) through an RNA intermediate to a protein-encoding variable region (VR). This retrohoming process is accompanied by unique adenine-specific mutagenesis, and in the prototypical BPP-1 DGR, requires a reverse transcriptase (bRT) and an accessory variability determinant (bAvd) protein. To understand the role of bAvd, we determined its 2.69 Å resolution structure, which revealed a highly positively charged pentameric barrel. In accordance with its charge, bAvd bound both DNA and RNA, albeit without a discernable sequence preference. We found that the coding sequence of bAvd functioned as part of TR, but identified means to mutate bAvd without affecting TR. This mutational analysis revealed a strict correspondence between retrohoming and interaction of bAvd with bRT, suggesting that the bRT-bAvd complex is important for DGR retrohoming.