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Improved Synthesis of 4‑Cyanotryptophan and Other Tryptophan Analogues in Aqueous Solvent Using Variants of TrpB from Thermotoga maritima
journal contribution
posted on 2018-04-13, 00:00 authored by Christina
E. Boville, David K. Romney, Patrick J. Almhjell, Michaela Sieben, Frances H. ArnoldThe use of enzymes has become increasingly
widespread in synthesis
as chemists strive to reduce their reliance on organic solvents in
favor of more environmentally benign aqueous media. With this in mind,
we previously endeavored to engineer the tryptophan synthase β-subunit
(TrpB) for production of noncanonical amino acids that had previously
been synthesized through multistep routes involving water-sensitive
reagents. This enzymatic platform proved effective for the synthesis
of analogues of the amino acid tryptophan (Trp), which are frequently
used in pharmaceutical synthesis as well as chemical biology. However,
certain valuable compounds, such as the blue fluorescent amino acid
4-cyanotryptophan (4-CN-Trp), could only be made in low yield, even
at elevated temperature (75 °C). Here, we describe the engineering
of TrpB from Thermotoga maritima that improved synthesis
of 4-CN-Trp from 24% to 78% yield. Remarkably, although the final
enzyme maintains high thermostability (T50 = 93 °C), its temperature profile is shifted such that high
reactivity is observed at ∼37 °C (76% yield), creating
the possibility for in vivo 4-CN-Trp production. The improvements
are not specific to 4-CN-Trp; a boost in activity at lower temperature
is also demonstrated for other Trp analogues.