Stereoelectronic control of bond formation in Escherichia coli tryptophan synthase: substrate specificity and enzymatic synthesis of the novel amino acid dihydroisotryptophan

Biochemistry ◽  
1988 ◽  
Vol 27 (18) ◽  
pp. 6698-6704 ◽  
Author(s):  
Melinda Roy ◽  
Samir Keblawi ◽  
Michael F. Dunn
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Enzymatic Synthesis ◽  
Bond Formation ◽  
Tryptophan Synthase ◽  
The Novel ◽  
Stereoelectronic Control

scholarly journals Enzymic synthesis of N-acetyl-l-phenylalanine in Escherichia coli K12

1971 ◽  
Vol 124 (5) ◽  
pp. 905-913 ◽  
Author(s):  
R. V. Krishna ◽  
P. R. Krishnaswamy ◽  
D. Rajagopal Rao
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Substrate Specificity ◽  
Acetyl Coa ◽  
Ph Optimum ◽  
E Coli ◽  
Enzymic Synthesis ◽  
Escherichia Coli K12

1. Cell-free extracts of Escherichia coli K12 catalyse the synthesis of N-acetyl-l-phenylalanine from acetyl-CoA and l-phenylalanine. 2. The acetyl-CoA–l-phenylalanine α-N-acetyltransferase was purified 160-fold from cell-free extracts. 3. The enzyme has a pH optimum of 8 and catalyses the acetylation of l-phenylalanine. Other l-amino acids such as histidine and alanine are acetylated at slower rates. 4. A transacylase was also purified from E. coli extracts and its substrate specificity studied. 5. The properties of both these enzymes were compared with those of other known amino acid acetyltransferases and transacylases.

scholarly journals Inhibitor-resistant TEM (IRT) beta-lactamases with different substitutions at position 244.

1997 ◽  
Vol 41 (11) ◽  
pp. 2547-2549 ◽  
Author(s):  
L Bret ◽  
E B Chaibi ◽  
C Chanal-Claris ◽  
D Sirot ◽  
R Labia ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Amino Acid Replacement ◽  
Beta Lactamase ◽  
The Novel ◽  
Beta Lactamases ◽  
Coli Isolate ◽  
Amoxicillin Clavulanate

A novel inhibitor-resistant TEM (IRT) beta-lactamase was detected in an Escherichia coli isolate resistant to amoxicillin-clavulanate and susceptible to cephalothin. The substrate and inhibitor profiles of this beta-lactamase were similar to those of IRT-1 and IRT-2. The novel IRT's bla gene was sequenced, and the deduced amino acid sequence showed the amino acid replacement Arg for His-244 of the TEM-1 sequence. Substitutions for Arg-244 have been reported in three TEM-1 mutants: IRT-1 (which corresponds to TEM-31) (Cys), IRT-2/TEM-30 (Ser), and TEM-41 (Thr). We designated this novel beta-lactamase, which corresponds to TEM-51, IRT-15.

scholarly journals Lipase and Its Modulator fromPseudomonas sp. Strain KFCC 10818: Proline-to-Glutamine Substitution at Position 112 Induces Formation of Enzymatically Active Lipase in the Absence of the Modulator

2001 ◽  
Vol 183 (20) ◽  
pp. 5937-5941 ◽  
Author(s):  
Eun Kyung Kim ◽  
Won Hee Jang ◽  
Jung Ho Ko ◽  
Jong Seok Kang ◽  
Moon Jong Noh ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Future Research ◽  
The Novel ◽  
Wild Type ◽  
Functional Interaction ◽  
Lipase Gene ◽  
Efficient Expression ◽  
Active Lipase

ABSTRACT A lipase gene, lipK, and a lipase modulator gene,limK, of Pseudomonas sp. strain KFCC 10818 have been cloned, sequenced, and expressed in Escherichia coli. The limK gene is located immediately downstream of the lipK gene. Enzymatically active lipase was produced only in the presence of the limK gene. The effect of the lipase modulator LimK on the expression of active lipase was similar to those of the Pseudomonas subfamily I.1 and I.2 lipase-specific foldases (Lifs). The deduced amino acid sequence of LimK shares low homology (17 to 19%) with the knownPseudomonas Lifs, suggesting thatPseudomonas sp. strain KFCC 10818 is only distantly related to the subfamily I.1 and I.2 Pseudomonasspecies. Surprisingly, a lipase variant that does not require LimK for its correct folding was isolated in the study to investigate the functional interaction between LipK and LimK. When expressed in the absence of LimK, the P112Q variant of LipK formed an active enzyme and displayed 63% of the activity of wild-type LipK expressed in the presence of LimK. These results suggest that the Pro112residue of LipK is involved in a key step of lipase folding. We expect that the novel finding of this study may contribute to future research on efficient expression or refolding of industrially important lipases and on the mechanism of lipase folding.

scholarly journals Directed evolution  improves the enzymatic synthesis of L-5-hydroxytryptophan by an  engineered tryptophan synthase

2021 ◽  
Author(s):  
Xu Lisheng ◽  
Tingting Li ◽  
Ziyue Huo ◽  
Qiong Chen ◽  
Qiuxia Xia ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Directed Evolution ◽  
Enzymatic Synthesis ◽  
High Throughput Screening ◽  
Screening Method ◽  
Mutant Enzyme ◽  
Tryptophan Synthase ◽  
Effective Strategy ◽  
Reaction Conditions ◽  
Important Amino Acid

Abstract L-5-Hydroxytryptophan is an important amino acid that is widely used in food and medicine. In this study, L-5-hydroxytryptophan was synthesized by a modified tryptophan synthase. A direct evolution strategy was applied to engineer tryptophan synthase from Escherichia coli to improve the efficiency of L-5-hydroxytryptophan synthesis. Tryptophan synthase was modified by error-prone PCR. A high activity mutant enzyme (V231A/K382G) was obtained by a high-throughput screening method. The activity of mutant enzyme (V231A/K382G) is 3.79 times higher than that of its parent, and kcat/Km of the mutant enzyme (V231A/K382G) was 4.36 mM− 1∙s− 1. The mutant enzyme (V231A/K382G) reaction conditions for the production of L-5-hydroxytryptophan were 100 mmol/L L-serine at pH 8.5 and 35°C for 15 h, reaching a yield of L-5-hydroxytryptophan of 86.7%. Directed evolution is an effective strategy to increase the activity of tryptophan synthase.

scholarly journals Haloalkane Dehalogenase LinB Is Responsible for β- and δ-Hexachlorocyclohexane Transformation in Sphingobium indicum B90A

2006 ◽  
Vol 72 (9) ◽  
pp. 5720-5727 ◽  
Author(s):  
Poonam Sharma ◽  
Vishakha Raina ◽  
Rekha Kumari ◽  
Shweta Malhotra ◽  
Charu Dogra ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Amino Acid Sequence ◽  
Substrate Specificity ◽  
Incubation Mixture ◽  
Resting Cells ◽  
Haloalkane Dehalogenase ◽  
Hch Isomers ◽  
Hexachlorocyclohexane Isomers

ABSTRACT Incubation of resting cells of Sphingobium indicum B90A, Sphingobium japonicum UT26, and Sphingobium francense Sp+ showed that they were able to transform β- and δ-hexachlorocyclohexane (β- and δ-HCH, respectively), the most recalcitrant hexachlorocyclohexane isomers, to pentachlorocyclohexanols, but only resting cells of strain B90A could further transform the pentachlorocyclohexanol intermediates to the corresponding tetrachlorocyclohexanediols. Moreover, experiments with resting cells of Escherichia coli expressing the LinB proteins of strains B90A, UT26, and Sp+ indicated that LinB was responsible for these transformations. Purified LinB proteins from all three strains also effected the formation of the respective pentachlorocyclohexanols. Although the three LinB enzymes differ only marginally with respect to amino acid sequence, they showed interesting differences with respect to substrate specificity. When LinB from strain B90A was incubated with β- and δ-HCH, the pentachlorocyclohexanol products were further transformed and eventually disappeared from the incubation mixtures. In contrast, the LinB proteins from strains UT26 and Sp+ could not catalyze transformation of the pentachlorocyclohexanols, and these products accumulated in the incubation mixture. A mutant of strain Sp+ lacking linA and linB did not degrade any of the HCH isomers, including β-HCH, and complementation of this mutant by linB from strain B90A restored the ability to degrade β- and δ-HCH.

Enzymatic Synthesis of l - threo -β-Hydroxy-α-Amino Acids via Asymmetric Hydroxylation Using 2-Oxoglutarate-Dependent Hydroxylase from Sulfobacillus thermotolerans Y0017

2021 ◽  
Author(s):  
Ryotaro Hara ◽  
Yuta Nakajima ◽  
Hiroaki Yanagawa ◽  
Ryo Gawasawa ◽  
Izumi Hirasawa ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Amino Acid ◽  
Enzymatic Synthesis ◽  
Scale Production ◽  
Dependent Manner ◽  
Amino Acid Synthesis ◽  
Preparative Scale ◽  
Whole Cell ◽  
Pharmaceutical Development

β-Hydroxy-α-amino acids are useful compounds for pharmaceutical development. Enzymatic synthesis of β-hydroxy-α-amino acids has attracted considerable interest as a selective, sustainable, and environmentally benign process. In this study, we identified a novel amino acid hydroxylase, AEP14369, from Sulfobacillus thermotolerans Y0017, which is included in a previously constructed CAS-like superfamily protein library, to widen the variety of amino acid hydroxylases. The detailed structures determined by nuclear magnetic resonance and X-ray crystallography analysis of the enzymatically produced compounds revealed that AEP14369 catalyzed threo -β-selective hydroxylation of l -His and l -Gln in a 2-oxoglutarate-dependent manner. Furthermore, the production of l - threo -β-hydroxy-His and l - threo -β-hydroxy-Gln was achieved using Escherichia coli expressing the gene encoding AEP14369 as a whole-cell biocatalyst. Under optimized reaction conditions, 137 mM (23.4 g L −1 ) l - threo -β-hydroxy-His and 150 mM l - threo -β-hydroxy-Gln (24.3 g L −1 ) were obtained, indicating that the enzyme is applicable for preparative-scale production. AEP14369, an l -His/ l -Gln threo -β-hydroxylase, increases the availability of 2-oxoglutarate-dependent hydroxylase and opens the way for the practical production of β-hydroxy-α-amino acids in the future. The amino acids produced in this study would also contribute to the structural diversification of pharmaceuticals that affect important bioactivities. Importance Owing to an increasing concern for sustainability, enzymatic approaches for producing industrially useful compounds have attracted considerable attention as a powerful complement to chemical synthesis for environment-friendly synthesis. In this study, we developed a bioproduction method for β-hydroxy-α-amino acid synthesis using a newly discovered enzyme. AEP14369 from the moderate thermophilic bacterium Sulfobacillus thermotolerans Y0017 catalyzed the hydroxylation of l -His and l -Gln in a regioselective and stereoselective fashion. Furthermore, we biotechnologically synthesized both l - threo -β-hydroxy-His and l - threo -β-hydroxy-Gln with a titer of over 20 g L −1 through whole-cell bioconversion using recombinant Escherichia coli cells. As β-hydroxy-α-amino acids are important compounds for pharmaceutical development, this achievement would facilitate future sustainable and economical industrial applications.

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