Enzymatic synthesis of Thia-L-tryptophans

Phillips, Robert S.; Cohen, Louis A.; Annby, Ulf; Wensbo, David; Gronowitz, Salo

doi:10.1016/0960-894x(95)00181-r

Cited by 21 publications

(10 citation statements)

References 4 publications

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“…6-Methyltetrahydropterin (6MPH 4 ) 1 was from B. Schircks Laboratories. L-Tryptophan, L-5-hydroxytryptophan, DL-4-methyltryptophan, DL-5-methyltryptophan, DL-6-methyltryptophan, DL-7-methyltryptophan, and DL-7azatryptophan were purchased from Sigma Chemical Co. L-4-Azatryptophan, L-5-azatryptophan, and L-6-azatryptophan were synthesized according to the methods of Sloan and Phillips (16) and Phillips et al (17). L-2-Azaisotryptophan (β-indazoylyl-L-alanine) was prepared by the method of Tanaka et al (18).…”

Section: Methodsmentioning

confidence: 99%

Influence of Steric Bulk and Electrostatics on the Hydroxylation Regiospecificity of Tryptophan Hydroxylase: Characterization of Methyltryptophans and Azatryptophans as Substrates

Moran

Phillips²,

Fitzpatrick

1999

Biochemistry

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Tryptophan hydroxylase is a pterin-dependent amino acid hydroxylase that catalyzes the incorporation of one atom of molecular oxygen into tryptophan to form 5-hydroxytryptophan. The substrate specificity and hydroxylation regiospecificity of tryptophan hydroxylase have been investigated using tryptophan analogues that have methyl substituents or nitrogens incorporated into the indole ring. The products of the reactions show that the regiospecificity of tryptophan hydroxylase is stringent. Hydroxylation does not occur at the 4 or 6 carbon in response to changes in substrate topology or atomic charge. 5-Hydroxymethyltryptophan and 5-hydroxy-4-methyltryptophan are the products from 5-methyltryptophan. These products establish that the hydroxylating intermediate is sufficiently potent to hydroxylate benzylic carbons and that the direction of the NIH shift in tryptophan hydroxylase is from carbon 5 to carbon 4. The effects on the V/K values for the amino acids indicate that the enzyme is most sensitive to changes at position 5 of the indole ring. The V(max) values for amino acid hydroxylation differ at most by a factor of 3 from that observed for tryptophan, while the efficiencies of hydroxylation with respect to tetrahydropterin consumption vary 6-fold, consistent with oxygen transfer to the amino acid being partially or fully rate limiting in productive catalysis.

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Section: Methodsmentioning

confidence: 99%

Influence of Steric Bulk and Electrostatics on the Hydroxylation Regiospecificity of Tryptophan Hydroxylase: Characterization of Methyltryptophans and Azatryptophans as Substrates

Moran

Phillips²,

Fitzpatrick

1999

Biochemistry

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“…The tryptophan synthase catalyzes the synthesis of L‐tryptophan from L‐serine and indole. The α‐subunit of this enzyme catalyzes the reversible retroaldol cleavage of indole‐3‐glycerol phosphate to produce D‐glyceraldehyde‐3‐phosphate and indole, while the β‐subunit in presence of L‐serine catalyzes the condensation of indole to give L‐tryptophan [49, 50]. Considering its potentiality, Budisa's group successfully demonstrated the metabolic transformation of azaindole into azatryptophans (4‐AzaTrp and 5‐AzaTrp) using tryptophan synthase and further without any purification directly used the cell extracts that contained the biosynthesized UAA to incorporate the Trp analogs into human annexin V protein (Fig.…”

Section: Uaa Synthesis For Biosynthetic Incorporationmentioning

confidence: 99%

Incorporating unnatural amino acids to engineer biocatalysts for industrial bioprocess applications

Ravikumar

Nadarajan

Yoo

et al. 2015

Biotechnology Journal

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The bioprocess engineering with biocatalysts broadly spans its development and actual application of enzymes in an industrial context. Recently, both the use of bioprocess engineering and the development and employment of enzyme engineering techniques have been increasing rapidly. Importantly, engineering techniques that incorporate unnatural amino acids (UAAs) in vivo has begun to produce enzymes with greater stability and altered catalytic properties. Despite the growth of this technique, its potential value in bioprocess applications remains to be fully exploited. In this review, we explore the methodologies involved in UAA incorporation as well as ways to synthesize these UAAs. In addition, we summarize recent efforts to increase the yield of UAA engineered proteins in Escherichia coli and also the application of this tool in enzyme engineering. Furthermore, this protein engineering tool based on the incorporation of UAA can be used to develop immobilized enzymes that are ideal for bioprocess applications. Considering the potential of this tool and by exploiting these engineered enzymes, we expect the field of bioprocess engineering to open up new opportunities for biocatalysis in the near future.

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“…The noncanonical amino acids [2,3]Tpa and [3,2]Tpa were enzymatically synthesized according to protocols described elsewhere (Phillips et al 1995). All other chemicals were purchased from Sigma or Aldrich unless stated otherwise.…”

Section: Methodsmentioning

confidence: 99%

“…Moreover, occurrence of Trp is rare as it represents only about 1% of all residues of globular proteins (McCaul and Ludescher 1999), and thus it may well provide a quasi‐site‐specific intrinsic probe for studying protein structure, dynamics, and function. Apart from its role as a basic building block in ribosome‐mediated protein synthesis, Trp plays an essential role in the metabolism of living cells, as it is involved in the biosynthesis of hormones such as serotonin or melatonin in animals, and of indole alkaloids in plants (Phillips et al 1995). Thus, noncanonical amino acids that mimic Trp could be of great interest as potential antagonists, drugs, or antibiotics, and their incorporation into proteins could be a promising approach for production of therapeutic agents (Budisa et al 1998b; Minks et al 2000b).…”

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confidence: 99%

Proteins with β‐(thienopyrrolyl)alanines as alternative chromophores and pharmaceutically active amino acids

et al. 2001

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L-␤-(Thieno [3,2-b]pyrrolyl)alanine and L-␤-(thieno [2,3-b]pyrrolyl)alanine are mutually isosteric and pharmaceutically active amino acids that mimic tryptophan with the benzene ring in the indole moiety replaced by thiophene. Sulfur as a heteroatom causes physicochemical changes in these tryptophan surrogates that bring about completely new properties not found in the indole moiety. These synthetic amino acids were incorporated into recombinant proteins in response to the Trp UGG codons by fermentation in a Trpauxotrophic Escherichia coli host strain using the selective pressure incorporation method. Related protein mutants expectedly retain the secondary structure of the native proteins but show significantly changed optical and thermodynamic properties. In this way, new spectral windows, fluorescence, polarity, thermodynamics, or pharmacological properties are inserted into proteins. Such an engineering approach by translational integration of synthetic amino acids with a priori defined properties, as shown in this study, proved to be a novel and useful tool for protein rational design.Keywords: UV absorbance; fluorescence; bioincorporation; protein engineering; thia-surrogates of tryptophanThe immense functional diversity of naturally occurring proteins is based on the simple concept of varying the combination of the 20 canonical amino acids in polypeptide chains. In this context, the possibility of artificially expanding this diversity by in vivo protein synthesis is a challenging and attractive goal because newly incorporated noncanonical amino acids could deliver novel properties into existing protein structures. An a priori design of proteins with desired properties such as altered spectral windows or pharmacological activities should be feasible if some of the coded (i.e., canonical) amino acid chromophores are exchangeable with noncanonical ones in template-directed protein synthesis.For the in vivo translational incorporation of a variety of noncanonical amino acids, an efficient method was developed that is based on the use of auxotrophic Escherichia coli host strains submitted to strong selective pressure (selective pressure incorporation; SPI) (Budisa et al. 1995(Budisa et al. , 1998aRoss et al. 1997). It enables the expansion of the scope of protein biosynthesis, primarily because the main principle of this basically in vivo methodology is based on codon reassignments at the translational level under stringent fermentation conditions. Tryptophan represents an attractive target for such replacements because it is the main source of the absorption and fluorescence of proteins. Moreover, occurrence of Trp is rare as it represents Article and publication are at

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Enzymatic synthesis of Thia-L-tryptophans

Cited by 21 publications

References 4 publications

Influence of Steric Bulk and Electrostatics on the Hydroxylation Regiospecificity of Tryptophan Hydroxylase: Characterization of Methyltryptophans and Azatryptophans as Substrates

Influence of Steric Bulk and Electrostatics on the Hydroxylation Regiospecificity of Tryptophan Hydroxylase: Characterization of Methyltryptophans and Azatryptophans as Substrates

Incorporating unnatural amino acids to engineer biocatalysts for industrial bioprocess applications

Proteins with β‐(thienopyrrolyl)alanines as alternative chromophores and pharmaceutically active amino acids

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