Resistance to bacterial speck disease in tomato (Solanum lycopersicum) is activated upon recognition by the host Pto kinase of either one of two sequence-unrelated effector proteins, AvrPto or AvrPtoB, from Pseudomonas syringae pv tomato (Pst). Pto induces Pst immunity by acting in concert with the Prf protein. The recently reported structure of the AvrPto-Pto complex revealed that interaction of AvrPto with Pto appears to relieve an inhibitory effect of Pto, allowing Pto to activate Prf. Here, we present the crystal structure of the Pto binding domain of AvrPtoB (residues 121 to 205) at a resolution of 1.9Å and of the AvrPtoB121-205–Pto complex at a resolution of 3.3 Å. AvrPtoB121-205 exhibits a tertiary fold that is completely different from that of AvrPto, and its conformation remains largely unchanged upon binding to Pto. In common with AvrPto-Pto, the AvrPtoB-Pto complex relies on two interfaces. One of these interfaces is similar in both complexes, although the primary amino acid sequences from the two effector proteins are very different. Amino acid substitutions in Pto at the other interface disrupt the interaction of AvrPtoB-Pto but not that of AvrPto-Pto. Interestingly, substitutions in Pto affecting this unique interface also cause Pto to induce Prf-dependent host cell death independently of either effector protein.

Mycobacterium smegmatis MshC catalyzes the ATP-dependent condensation of GlcN-Ins and Lcysteine to form L-Cys-GlcN-Ins, the penultimate step in mycothiol biosynthesis. Attempts to crystallize the native, full-length MshC have been unsuccessful. However, incubation of the enzyme with the cysteinyl adenylate analogue, 5′-O-[N-(L-cysteinyl)-sulfamonyl]adenosine (CSA), followed by a 24-hour limited trypsin proteolysis yielded an enzyme preparation that readily crystallized. The three-dimensional structure of MshC with CSA bound in the active site was solved and refined to 1.6 Å. The refined structure exhibited electron density corresponding to the entire 47 kDalton MshC molecule, with the exception of the KMSKS loop (residues 285-297), a loop previously implicated in the formation of the adenylate in related tRNA synthases. The overall tertiary fold of MshC is similar to that of cysteinyl-tRNA synthetase, with a Rossmann fold catalytic domain. The interaction of the thiolate of CSA with a zinc ion at the base of the active site suggests that the metal ion participates in amino acid binding and discrimination. A number of active site residues were observed to interact with the ligand, suggesting a role in substrate binding and catalysis. Analysis utilizing modeling of the proteolyzed loop and GlcN-Ins docking, as well as the examination of sequence conservation in the active site suggests similarities and differences between cysteinyl-tRNA synthetases and MshC in recognition of the substrates for their respective reactions. KeywordsMycobacterium; mycothiol; cysteine ligase; limited proteolysis; three-dimensional structure Actinomycetes produce mycothiol (MSH, acetyl-cys-GlcN-Ins) as the predominant low molecular weight thiol to minimize oxidative stress and protect against electrophilic toxins (1-4). Among actinomycetes, mycobacteria generate the highest intracellular levels of MSH (5). Mycobacterium smegmatis mutants which are deficient in MSH production become more sensitive towards oxidizing agents, electrophiles, and antibiotics (1-3), indicating the critical role of MSH in the survival and pathogenicity of mycobacteria (1). In contrast, eukaryotes and many eubacteria produce glutathione (GSH). This suggests that the enzymes involved in the mycothiol biosynthetic pathway may be potential targets for selective antimicrobial chemotherapy.MSH is synthesized via a series of four unique enzyme-catalyzed reactions (6-9), as illustrated in Scheme 1. Mycothiol is reversibly oxidized by cellular oxidants and can react with electrophiles to form the S-conjugates. A detailed biochemical characterization of the cysteine ligase (MshC) from Mycobacterium smegmatis, which is the penultimate enzyme in mycothiol (15), and acyl-and aryl-CoA synthetases/ligases (16-18) catalyze reactions that generally can be divided into two halves. In the first halfreaction, one substrate reacts with ATP to form an adenylate and inorganic pyrophosphate; in the second half-reaction the second substrate reacts with the adenylated substrate...