Site-directed Mutagenesis Provides Insight into Racemization and Transamination of Alanine Catalyzed by Treponema denticola Cystalysin

Abstract

In addition to ␣, ␤-elimination of L-cysteine, Treponema denticola cystalysin catalyzes the racemization of both enantiomers of alanine accompanied by an overall transamination. Lys-238 and Tyr-123 or a water molecule located on the si and re face of the cofactor, respectively, have been proposed to act as the acid/base catalysts in the proton abstraction/donation at C␣/C4 of the external aldimine. In this investigation, two site-directed mutants, K238A and Y123F, have been characterized. The Lys 3 Ala mutation results in the complete loss of either lyase activity or racemase activity in both directions or transaminase activity toward L-alanine. However, the K238A mutant is able to catalyze the overall transamination of D-alanine, and only D-alanine is the product of the reverse transamination. For Y123F the k cat /K m is reduced 3.5-fold for ␣,␤-elimination, whereas it is reduced 300 -400-fold for racemization. Y123F has ϳ18% of wild type transaminase activity with L-alanine and an extremely low transaminase activity with D-alanine. Moreover, the catalytic properties of the Y124F and Y123F/Y124F mutants rule out the possibility that the residual racemase and transaminase activities displayed by Y123F are due to Tyr-124. All these data, together with computational results, indicate a two-base racemization mechanism for cystalysin in which Lys-238 has been unequivocally identified as the catalyst acting on the si face of the cofactor. Moreover, this study highlights the importance of the interaction of Tyr-123 with water molecules for efficient proton abstraction/donation function on the re face.Treponema denticola cystalysin catalyzes the pyridoxal 5Ј-phosphate (PLP) 1 -dependent ␣,␤-elimination of L-cysteine to generate pyruvate, ammonia, and H 2 S (1). It has been shown that several sulfur-and non-sulfur-containing amino acids as well as disulfidic amino acids also serve as substrates for this reaction (2). The protein is composed of two identical subunits, each of which comprises 399 amino acid residues. The threedimensional structure of the enzyme has been solved to 1.9 Å (3). The PLP cofactor in the active site forms a Schiff base with the ⑀-amino group of Lys-238 of cystalysin (3). Through sitedirected mutagenesis, spectroscopic, and kinetic studies, we have shown recently that Lys-238 is an essential residue for ␣,␤-elimination catalyzed by the enzyme cystalysin. In addition to strengthening the coenzyme binding and facilitating transimination, this residue seems to participate as a general base abstracting the C␣ proton from the substrate and possibly as a general acid, protonating the ␤-leaving group (4).In a recent paper we have shown that, in addition to the lyase activity, the enzyme exhibits an alanine racemase activity accompanied by an overall transaminase activity toward both enantiomers of alanine (5). Considering that racemization is a side reaction of cystalysin, it occurs with a significant k cat (ϳ1 s Ϫ1 ), even if it is ϳ1000-fold lower than that of alanine racemase (6). The structure of cy...

Upregulation of MetC Is Essential for d -Alanine-Independent Growth of an alr/dadX -Deficient Escherichia coli Strain

“…This proves that both MetC and MalY have cystathionine beta-lyase activity (52) and thus potentially similar catalytic mechanisms. Furthermore, it was recently reported that cystalysin from Treponema denticola possesses alanine racemase activity (4,7). E. coli MalY shows 51% sequence similarity to Treponema denticola cystalysin, and we found that a plasmid expressing E. coli MalY was indeed able to complement the alr/dadX deletion (data not shown).…”

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

“…The system was stable after 90 ns of MD simulation. Moreover, to validate the behavior of the equilibrated system, interactions for PLP binding during PLP‐dependent lyase activity were verified to be well maintained along the MD simulations. In this regard, it is known that certain interactions are needed for proper functioning of the enzyme that are assumed to be maintained during MD simulations.…”

“…Moreover, to validate the behavior of the equilibrated system, interactions for PLP binding during PLP‐dependent lyase activity were verified to be well maintained along the MD simulations. In this regard, it is known that certain interactions are needed for proper functioning of the enzyme that are assumed to be maintained during MD simulations. These include: (a) a H‐bond between the phenolic hydroxyl group of Tyr55 and the phosphate group of PLP; (b) stacking between the aromatic side chain of Tyr114 with the pyridine ring of PLP; (c) a salt bridge between Arg351 and the carboxyl group of the ligand (Fig.…”

Pyridoxal 5-Phosphate Enzymes as Targets for Therapeutic Agents

“…Additionally, it has been demonstrated that cystalysin has a high catalytic versatility and is able to catalyze the racemization and transamination of both enantiomers of alanine [319,320], the β-desulfination of L-cysteine sulfinic acid and the β-decarboxylation of L-aspartate and oxalacetate [321]. Recently, important hints were obtained on a possible prodrug that is activated by the parasite-specific enzyme and has anti-trichomonal activity in vivo.…”