Structure of the ThDP-dependent enzyme benzaldehyde lyase refined to 1.65 Å resolution

Maraite, Andy; Schmidt, Timothy R.; Ansorge‐Schumacher, Marion B.; Brzozowski, A.M.; Grogan, Gideon

doi:10.1107/s1744309107028576

Cited by 10 publications

(13 citation statements)

References 20 publications

(22 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Small organic molecules might be able to enter the active site and thus block parts of it, which would result in changes in the overall activity and selectivity. It has already been shown that a cosolvent was able to enter the active site of Pf BAL and attach to hydrophobic patches 26. On the other hand, the solubility of the substrate, intermediates and products can be enhanced or impaired by shifting kinetic parameters, equilibria and final product concentrations.…”

Section: Discussionmentioning

confidence: 99%

Influence of Organic Solvents on Enzymatic Asymmetric Carboligations

et al. 2012

View full text Add to dashboard Cite

The asymmetric mixed carboligation of aldehydes with thiamine diphosphate (ThDP)-dependent enzymes is an excellent example where activity as well as changes in chemo- and stereoselectivity can be followed sensitively. To elucidate the influence of organic additives in enzymatic carboligation reactions of mixed 2-hydroxy ketones, we present a comparative study of six ThDP-dependent enzymes in 13 water-miscible organic solvents under equivalent reaction conditions. The influence of the additives on the stereoselectivity is most pronounced and follows a general trend. If the enzyme stereoselectivity in aqueous buffer is already >99.9% ee, none of the solvents reduces this high selectivity. In contrast, both stereoselectivity and chemoselectivity are strongly influenced if the enzyme is rather unselective in aqueous buffer. For the S-selective enzyme with the largest active site, we were able to prove a general correlation of the solvent-excluded volume of the additives with the effect on selectivity changes: the smaller the organic solvent molecule, the higher the impact of this additive. Further, a correlation to log P of the additives on selectivity was detected if two additives have almost the same solvent-excluded volume. The observed results are discussed in terms of structural, biochemical and energetic effects. This work demonstrates the potential of medium engineering as a powerful additional tool for varying enzyme selectivity and thus engineering the product range of biotransformations. It further demonstrates that the use of cosolvents should be carefully planned, as the solvents may compete with the substrate(s) for binding sites in the enzyme active site.

show abstract

Section: Discussionmentioning

confidence: 99%

Influence of Organic Solvents on Enzymatic Asymmetric Carboligations

et al. 2012

View full text Add to dashboard Cite

show abstract

“…In complex with methyl benzoylphosphonate, the protein crystallizes in the trigonal space group P3 2 21 with unit cell dimensions of 152 × 152 × 98 Å. (Table 1) The asymmetric unit contains a dimer, rather than the dimer of dimers observed for the protein in the absence of MBP (13,14). A crystallographic two-fold axis recapitulates the tetrameric assembly seen in those structures.…”

Section: Structural Characterization Of Bal Co-crystallized With Mbpmentioning

confidence: 99%

“…This represents the first structure of BAL with a substrate analog bound. No large structural reorganization was detected in this complex when compared to the complex of BAL with ThDP (13,14) or, as described below, to the complex of BAL with ThDP and sulfate.…”

mentioning

confidence: 98%

Probing the Active Center of Benzaldehyde Lyase with Substitutions and the Pseudosubstrate Analogue Benzoylphosphonic Acid Methyl Ester

et al. 2008

View full text Add to dashboard Cite

Benzaldehyde lyase (BAL) catalyzes the reversible cleavage of (R)-benzoin to benzaldehyde utilizing thiamin diphosphate and Mg 2+ as cofactors. The enzyme is important for the chemoenzymatic synthesis of a wide range of compounds via its carboligation reaction mechanism. In addition to its principal functions, BAL can slowly decarboxylate aromatic amino acids such as benzoylformic acid. It is also intriguing mechanistically due to the paucity of acid-base residues at the active center that can participate in proton transfer steps thought to be necessary for these types of reactions. Here methyl benzoylphosphonate, an excellent electrostatic analogue of benzoylformic acid, is used to probe the mechanism of benzaldehyde lyase. The structure of benzaldehyde lyase in its covalent complex with methyl benzoylphosphonate was determined to 2.49 Å (Protein Data Bank entry 3D7K) and represents the first structure of this enzyme with a compound bound in the active site. No large structural reorganization was detected compared to the complex of the enzyme with thiamin diphosphate. The configuration of the predecarboxylation thiamin-bound intermediate was clarified by the structure. Both spectroscopic and X-ray structural studies are consistent with inhibition resulting from the binding of MBP to the thiamin diphosphate in the active centers. We also delineated the role of His29 (the sole potential acid-base catalyst in the active site other than the highly conserved Glu50) and Trp163 in cofactor activation and catalysis by benzaldehyde lyase.

show abstract

“…Lyases are a class of enzymes that catalyze the cleavage of CC, CO, CN, and other bonds by other means than by hydrolysis or oxidation, which have aroused great interest in the food and cosmetic industries owing to their application as biocatalysts . Benzaldehyde lyase (BAL, EC 4.1.2.38) is a thiamin diphosphate (THDP)‐dependent enzyme, which catalyzes the reversible conversion of aromatic 2‐hydroxy ketones like (R)‐benzoin to benzaldehyde, as shown in Scheme . The ability of BAL to cleave acyloin linkages with strict R‐selectivity, which can be used in the kinetic resolution of racemic benzoins, has not yet been observed in other enzymes .…”

Section: Introductionmentioning

confidence: 99%

“…Up to now, several high‐resolution X‐ray data have provided deep insight into the structures of BAL . For example, Maraite et al reported the high resolution (1.65 Å) crystal structure of BAL from Pseudomonas fluorescens (PDB code: 2UZ1). In general, the enzyme is a homotetramer of 4 × 563 amino acid residues with a molecular mass of 58,919 Da/monomer.…”

Section: Introductionmentioning

confidence: 99%

Theoretical investigation on the dissociation of (R)-benzoin catalyzed by benzaldehyde lyase

Zhang

Sheng

Hou

et al. 2013

Int. J. Quantum Chem.

View full text Add to dashboard Cite

Benzaldehyde lyase (BAL) is a versatile thiamin diphosphate (THDP)‐dependent enzyme with widespread synthetic applications in industry. Besides lyase activity, BAL also performs the functions as carboligase and decarboxylase. Unlike many other THDP‐dependent enzymes, the active center of BAL is devoid of any acid‐base amino acid residues except Glu50 and His29, and therefore, the catalytic mechanism of BAL is unusual. In this article, the dissociation mechanism of (R)‐benzoin to benzaldehyde catalyzed by BAL has been studied by using density functional theory method. The calculation results indicate that the whole reaction consists of four elementary steps, and at least two steps contribute to rate‐limiting. A big difference with other THDP‐dependent enzymes is that, in the first stage of the reaction, the ligation of substrate and THDP ylide is not companied by proton transfer, and in the subsequent transition states and intermediates, the carbonyl oxygen always exists in the form of anion. Gln113, His29, and 4′‐amino group of THDP are found to have the function to stabilize the transition states and intermediates. His29 acts as the proton acceptor in step 2 and proton donor in step 3 using one water molecule as mediator. © 2013 Wiley Periodicals, Inc.

show abstract

Structure of the ThDP-dependent enzyme benzaldehyde lyase refined to 1.65 Å resolution

Cited by 10 publications

References 20 publications

Influence of Organic Solvents on Enzymatic Asymmetric Carboligations

Influence of Organic Solvents on Enzymatic Asymmetric Carboligations

Probing the Active Center of Benzaldehyde Lyase with Substitutions and the Pseudosubstrate Analogue Benzoylphosphonic Acid Methyl Ester

Theoretical investigation on the dissociation of (R)-benzoin catalyzed by benzaldehyde lyase

Contact Info

Product

Resources

About