Role of active‐site residues Tyr55 and Tyr114 in catalysis and substrate specificity of Corynebacterium diphtheriae C‐S lyase

Abstract

In recent years, there has been increased interest in bacterial methionine biosynthesis enzymes as antimicrobial targets because of their pivotal role in cell metabolism. C-S lyase from Corynebacterium diphtheriae is a pyridoxal 5'-phosphate-dependent enzyme in the transsulfuration pathway that catalyzes the α,β-elimination of sulfur-containing amino acids, such as L-cystathionine, to generate ammonia, pyruvate, and homocysteine, the immediate precursor of L-methionine. In order to gain deeper insight into the functional and dynamic properties of the enzyme, mutants of two highly conserved active-site residues, Y55F and Y114F, were characterized by UV-visible absorbance, fluorescence, and CD spectroscopy in the absence and presence of substrates and substrate analogs, as well as by steady-state kinetic studies. Substitution of Tyr55 with Phe apparently causes a 130-fold decrease in K(d)(PLP) at pH 8.5 providing evidence that Tyr55 plays a role in cofactor binding. Moreover, spectral data show that the mutant accumulates the external aldimine intermediate suggesting that the absence of interaction between the hydroxyl moiety and PLP-binding residue Lys222 causes a decrease in the rate of substrate deprotonation. Mutation of Tyr114 with Phe slightly influences hydrolysis of L-cystathionine, and causes a change in substrate specificity towards L-serine and O-acetyl-L-serine compared to the wild type enzyme. These findings, together with computational data, provide useful insights in the substrate specificity of C-S lyase, which seems to be regulated by active-site architecture and by the specific conformation in which substrates are bound, and will aid in development of inhibitors.

Cystathionine β-synthase is involved in cysteine biosynthesis and H2S generation in Toxoplasma gondii

“…To test the usefulness of the coupled-coupled enzyme assay to detect CBS activity, we first investigated the kinetic parameters of our recombinant CBL in catalyzing the β-elimination of l-Cth to pyruvate, l-Hcys and NH 3 (k cat = 93 ± 2 s −1 , K m = 0.8 ± 0.1 mM, k cat /K m = 116 mM −1 s −1 ) and of l-Ser to pyruvate and NH 3 (k cat = 1.2 ± 0.2 s −1 , K m = 7.5 ± 1.4 mM, k cat /K m = 0.16 mM −1 s −1 ) under the conditions used for the CBS coupled-coupled assay. The obtained values agree with those previously published by our laboratory 31,32 . Importantly, the catalytic efficiency of CBL toward l-Ser was < 0.2% compared to l-Cth, and thus this low activity does not interfere with the accuracy of the assay.…”

“…The steady-state kinetic parameters of TgCBS in the canonical reactions were determined by applying a highly sensitive continuous assay based on recombinant cystathionine beta-lyase (CBL) from Corynebacterium diphtheriae produced in our laboratory 31,32 and on commercial lactate dehydrogenase (LDH) as coupling enzymes, following a method described in Ref. 33 (see "Materials and methods").…”

Structural insight into the unique conformation of cystathionine β-synthase from Toxoplasma gondii

“…The oligomeric state of the Tg CBS construct was determined by gel filtration analysis employing a GE Healthcare Superdex 200 10/300 GL column in 20 mM sodium phosphate buffer pH 8.5, 150 mM NaCl and 0.1 mM DTT as described elsewhere [7] , [33] . A calibration curve was constructed using the GE Healthcare high molecular weight gel filtration calibration kit, following protocols in [33] The CBS activity in the canonical reactions was determined by a previously described continuous assay for Cth production [7] , which employs recombinant cystathionine β-lyase (CBL) from C. diphtheriae, produced in our lab [34] , [35] and lactate dehydrogenase (LDH) from rabbit muscle (Sigma-Aldrich) as coupling enzymes. The activity assay was performed using the dimeric fraction of the enzyme isolated by gel filtration.…”

Functional Characterization and Structure-Guided Mutational Analysis of the Transsulfuration Enzyme Cystathionine γ-Lyase from Toxoplasma gondii

“…Chromatography was performed using 50 mM sodium phosphate buffer pH 8.5, 150 mM NaCl, 0.1 mM DTT. The calibration curve was obtained as described [ 38 , 39 , 40 ].…”