ArticleThe Journal of biological chemistry2016
Mechanistic and Evolutionary Insights from the Reciprocal Promiscuity of Two Pyridoxal Phosphate-dependent Enzymes.
Article in The Journal of biological chemistry, 2016. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 15 papers.
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15 citing papers in PubMed, 35 citations in OpenAlex.
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- Profiling of Diverse Pyridoxal-5'-Phosphate Dependent Enzymes Reveals Promiscuous Aldolase Activity with (2-Azaaryl)methanamines.Journal of the American Chemical Society · 2025Article
- One substrate many enzymes virtual screening uncovers missing genes of carnitine biosynthesis in human and mouse.Nature communications · 2024Article
- Dan Tawfik's Lessons for Protein Engineers about Enzymes Adapting to New Substrates.Biochemistry · 2023Review
- How to Recruit a Promiscuous Enzyme to Serve a New Function.Biochemistry · 2023Article
- Engineering DszC Mutants from Transition State Macrodipole Considerations and Evolutionary Sequence Analysis.Journal of chemical information and modeling · 2023Article
- d-Serine and d-Alanine Regulate Adaptive Foraging Behavior inThe Journal of neuroscience : the official journal of the Society for Neuroscience · 2020Article
- Biochemical Characteristics of Microbial Enzymes and Their Significance from Industrial Perspectives.Molecular biotechnology · 2019Review
- Bioinformatic analysis of the fold type I PLP-dependent enzymes reveals determinants of reaction specificity in l-threonine aldolase fromFEBS open bio · 2018Article
- Review: Engineering of thermostable enzymes for industrial applications.APL bioengineering · 2018Review
- Human serine racemase structure/activity relationship studies provide mechanistic insight and point to position 84 as a hot spot for β-elimination function.The Journal of biological chemistry · 2017Article
- Multifunctional enzymes from reduced genomes - model proteins for simple primordial metabolism?Molecular microbiology · 2017Article
- Glucosylceramide transferase in Giardia preferentially catalyzes the synthesis of galactosylceramide during encystation.Molecular and biochemical parasitology · 2017Article
- Structures and kinetics of Thermotoga maritima MetY reveal new insights into the predominant sulfurylation enzyme of bacterial methionine biosynthesis.The Journal of biological chemistryArticle
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4 authors at 3 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Enzymes that utilize the cofactor pyridoxal 5'-phosphate play essential roles in amino acid metabolism in all organisms. The cofactor is used by proteins that adopt at least five different folds, which raises questions about the evolutionary processes that might explain the observed distribution of functions among folds. In this study, we show that a representative of fold type III, the Escherichia coli alanine racemase (ALR), is a promiscuous cystathionine β-lyase (CBL). Furthermore, E. coli CBL (fold type I) is a promiscuous alanine racemase. A single round of error-prone PCR and selection yielded variant ALR(Y274F), which catalyzes cystathionine β-elimination with a near-native Michaelis constant (Km = 3.3 mm) but a poor turnover number (kcat ≈10 h(-1)). In contrast, directed evolution also yielded CBL(P113S), which catalyzes l-alanine racemization with a poor Km (58 mm) but a high kcat (22 s(-1)). The structures of both variants were solved in the presence and absence of the l-alanine analogue, (R)-1-aminoethylphosphonic acid. As expected, the ALR active site was enlarged by the Y274F substitution, allowing better access for cystathionine. More surprisingly, the favorable kinetic parameters of CBL(P113S) appear to result from optimizing the pKa of Tyr-111, which acts as the catalytic acid during l-alanine racemization. Our data emphasize the short mutational routes between the functions of pyridoxal 5'-phosphate-dependent enzymes, regardless of whether or not they share the same fold. Thus, they confound the prevailing model of enzyme evolution, which predicts that overlapping patterns of promiscuity result from sharing a common multifunctional ancestor.
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