Genetic differences in the Chlamydia trachomatis tryptophan synthase α-subunit can explain variations in serovar pathogenesis

Enzymes in heteromeric, allosterically regulated complexes catalyze a rich array of chemical reactions. Separating the subunits of such complexes, however, often severely attenuates their catalytic activities, because they can no longer be activated by their protein partners. We used directed evolution to explore allosteric regulation as a source of latent catalytic potential using the β-subunit of tryptophan synthase from Pyrococcus furiosus (PfTrpB). As part of its native αββα complex, TrpB efficiently produces tryptophan and tryptophan analogs; activity drops considerably when it is used as a stand-alone catalyst without the α-subunit. Kinetic, spectroscopic, and X-ray crystallographic data show that this lost activity can be recovered by mutations that reproduce the effects of complexation with the α-subunit. The engineered PfTrpB is a powerful platform for production of Trp analogs and for further directed evolution to expand substrate and reaction scope.

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Characterization of a slow folding reaction for the α subunit of tryptophan synthase

Proteins Structure Function and Bioinformatics ◽

10.1002/prot.340020107 ◽

1987 ◽

Vol 2 (1) ◽

pp. 54-63 ◽

Cited By ~ 34

Author(s):

Mark R. Hurle ◽

Greg A. Michelotti ◽

Mark M. Crisanti ◽

C. Robert Matthews

Keyword(s):

Tryptophan Synthase ◽

Α Subunit

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Cloning and characterization of indole synthase (INS) and a putative tryptophan synthase α-subunit (TSA) genes from Polygonum tinctorium

Plant Cell Reports ◽

10.1007/s00299-016-2046-3 ◽

2016 ◽

Vol 35 (12) ◽

pp. 2449-2459 ◽

Cited By ~ 6

Author(s):

Zhehao Jin ◽

Jin-Hee Kim ◽

Sang Un Park ◽

Soo-Un Kim

Keyword(s):

Tryptophan Synthase ◽

Α Subunit ◽

Polygonum Tinctorium

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Comparative Genomic Analysis of Chlamydia trachomatis Oculotropic and Genitotropic Strains

Infection and Immunity ◽

10.1128/iai.73.10.6407-6418.2005 ◽

2005 ◽

Vol 73 (10) ◽

pp. 6407-6418 ◽

Cited By ~ 155

Author(s):

John H. Carlson ◽

Stephen F. Porcella ◽

Grant McClarty ◽

Harlan D. Caldwell

Keyword(s):

Chlamydia Trachomatis ◽

Protein A ◽

Transmitted Disease ◽

Genomic Analysis ◽

Hla Class I ◽

Comparative Genomic ◽

Toxin Gene ◽

Snp Analysis ◽

Tryptophan Synthase ◽

T Cell Epitopes

ABSTRACT Chlamydia trachomatis infection is an important cause of preventable blindness and sexually transmitted disease (STD) in humans. C. trachomatis exists as multiple serovariants that exhibit distinct organotropism for the eye or urogenital tract. We previously reported tissue-tropic correlations with the presence or absence of a functional tryptophan synthase and a putative GTPase-inactivating domain of the chlamydial toxin gene. This suggested that these genes may be the primary factors responsible for chlamydial disease organotropism. To test this hypothesis, the genome of an oculotropic trachoma isolate (A/HAR-13) was sequenced and compared to the genome of a genitotropic (D/UW-3) isolate. Remarkably, the genomes share 99.6% identity, supporting the conclusion that a functional tryptophan synthase enzyme and toxin might be the principal virulence factors underlying disease organotropism. Tarp (translocated actin-recruiting phosphoprotein) was identified to have variable numbers of repeat units within the N and C portions of the protein. A correlation exists between lymphogranuloma venereum serovars and the number of N-terminal repeats. Single-nucleotide polymorphism (SNP) analysis between the two genomes highlighted the minimal genetic variation. A disproportionate number of SNPs were observed within some members of the polymorphic membrane protein (pmp) autotransporter gene family that corresponded to predicted T-cell epitopes that bind HLA class I and II alleles. These results implicate Pmps as novel immune targets, which could advance future chlamydial vaccine strategies. Lastly, a novel target for PCR diagnostics was discovered that can discriminate between ocular and genital strains. This discovery will enhance epidemiological investigations in nations where both trachoma and chlamydial STD are endemic.

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