The Rv0948c gene from Mycobacterium tuberculosis H37Rv encodes a 90 amino acid protein as the natural gene product with chorismate mutase (CM) activity. The protein, 90‐MtCM, exhibits Michaelis–Menten kinetics with a kcat of 5.5 ± 0.2 s−1 and a Km of 1500 ± 100 μm at 37 °C and pH 7.5. The 2.0 Å X‐ray structure shows that 90‐MtCM is an all α‐helical homodimer (Protein Data Bank ID: 2QBV) with the topology of Escherichia coli CM (EcCM), and that both protomers contribute to each catalytic site. Superimposition onto the structure of EcCM and the sequence alignment shows that the C‐terminus helix 3 is shortened. The absence of two residues in the active site of 90‐MtCM corresponding to Ser84 and Gln88 of EcCM appears to be one reason for the low kcat. Hence, 90‐MtCM belongs to a subfamily of α‐helical AroQ CMs termed AroQδ. The CM gene (y2828) from Yersinia pestis encodes a 186 amino acid protein with an N‐terminal signal peptide that directs the protein to the periplasm. The mature protein, *YpCM, exhibits Michaelis–Menten kinetics with a kcat of 70 ± 5 s−1 and Km of 500 ± 50 μm at 37 °C and pH 7.5. The 2.1 Å X‐ray structure shows that *YpCM is an all α‐helical protein, and functions as a homodimer, and that each protomer has an independent catalytic unit (Protein Data Bank ID: 2GBB). *YpCM belongs to the AroQγ class of CMs, and is similar to the secreted CM (Rv1885c, *MtCM) from M. tuberculosis.

The protein has an all alpha-helical structure, and the active site is formed within a single chain without any contribution from the second chain in the dimer. Analysis of the structure shows a novel fold topology for the protein with a topologically rearranged helix containing Arg 134 . We provide evidence by site-directed mutagenesis that the residues Arg 49 , Lys 60 , Arg 72 , Thr 105 , Glu 109 , and Arg 134 constitute the catalytic site; the numbering of the residues includes the signal sequence. Our investigation on the effect of phenylalanine, tyrosine, and tryptophan on *MtCM shows that *MtCM is not regulated by the aromatic amino acids. Consistent with this observation, the X-ray structure of *MtCM does not have an allosteric regulatory site.

The cyclic AMP receptor protein (CRP, also called catabolite gene activator protein or CAP) plays a key role in metabolic regulation in bacteria and has become a widely studied model allosteric transcription factor. On binding its effector cAMP in the N-terminal domain, CRP undergoes a structural transition to a conformation capable of specific DNA binding in the C-terminal domain and transcription initiation. The crystal structures of Escherichia coli CRP (EcCRP) in the cAMP-bound state, both with and without DNA, are known, although its structure in the off state (cAMP-free, apoCRP) remains unknown. We describe the crystal structure at 2.0 Å resolution of the cAMPfree CRP homodimer from Mycobacterium tuberculosis H 37 R v (MtbCRP), whose sequence is 30% identical with EcCRP, as the first reported structure of an off-state CRP. The overall structure is similar to that seen for the cAMP-bound EcCRP, but the apo MtbCRP homodimer displays a unique level of asymmetry, with a root mean square deviation of 3.5 Å between all C␣ positions in the two subunits. Unlike structures of on-state EcCRP and other homologs in which the C-domains are asymmetrically positioned but possess the same internal conformation, the two C-domains of apo MtbCRP differ both in hinge structure and in internal arrangement, with numerous residues that have completely different local environments and hydrogen bond interactions, especially in the hinge and DNA-binding regions. Comparison of the structures of apo MtbCRP and DNA-bound EcCRP shows how DNA binding would be inhibited in the absence of cAMP and supports a mechanism involving functional asymmetry in apoCRP. CRP2 belongs to the large CRP/FNR family of bacterial transcription factors that link a molecular sensor function to gene expression modulation (1-3). The best studied of these is CRP from Eschericia coli (EcCRP), although many homologous proteins have also been analyzed (4 -6). The mechanism by which effector binding in the N-terminal domain controls DNA binding in the C-terminal domain over 30 Å away, with subsequent recruitment of RNA polymerase, has been a subject of extensive study (7-10). A general goal of these studies has been to infer allosteric mechanism by comparing the inactive and active states, ideally for the same protein. As yet, there is no single protein for which structures of both inactive and DNA-bound states are known.Mycobacterium tuberculosis H 37 R v (Mtb) is one of the world's most lethal microbes, currently infecting about onethird of human beings and killing about 5000 per day. The Mtb genome encodes 15 isoforms of adenylyl cyclase (11-14); thus cAMP is likely produced under a variety of metabolic conditions including interactions with host cells. Considering the known virulence role of adenylyl cyclase in other pathogens such as Bacillus anthracis (15, 16), cAMP signaling is likely involved in Mtb pathogenesis. The CRP ortholog in Mtb (Mtb-CRP, gene O69644_MYCTU (UniProt), Rv3676) has been biochemically characterized (17) and linked with 73 different prom...

High-level expression of recombinant human growth hormone (hGH) in Escherichia coli (E. coli) leads to the formation of insoluble aggregates as inclusion bodies devoid of biological activity. Until recently, significant efforts have been made to improve the recovery of active hGH from inclusion bodies. Here, we developed an efficient procedure for the production of completely soluble hGH by minimizing the formation of inclusion bodies and optimizing protein purification conditions. Under the newly established conditions we were able to obtain most of the total hGH in the soluble fraction. We show that the soluble protein can be efficiently purified in high yield by a series of chromatographic procedures. We analyzed the resulting hGH using various analytical techniques such as reversed-phase high-performance liquid chromatography (RP-HPLC), size-exclusion chromatography (SEC), matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry, and circular dichroism (CD). These multiple analyses support the conclusion that we obtained highly pure hGH with the expected molecular mass and intact secondary structure. The biological activity of purified hGH was also confirmed by evaluating its growth-promoting effect using a cell proliferation assay. Taken together, we describe a straightforward strategy for the production of completely soluble and biologically active hGH in E. coli.