Molecular characterization of an NADPH-dependent acetoin reductase/2,3-butanediol dehydrogenase from Clostridium beijerinckii NCIMB 8052

Abstract

Acetoin reductase is an important enzyme for the fermentative production of 2,3-butanediol, a chemical compound with a very broad industrial use. Here, we report on the discovery and characterization of an acetoin reductase from Clostridium beijerinckii NCIMB 8052. An in silico screen of the C. beijerinckii genome revealed eight potential acetoin reductases. One of them (CBEI_1464) showed substantial acetoin reductase activity after expression in Escherichia coli. The purified enzyme (C. beijerinckii acetoin reductase [Cb-ACR]) was found to exist predominantly as a homodimer. In addition to acetoin (or 2,3-butanediol), other secondary alcohols and corresponding ketones were converted as well, provided that another electronegative group was attached to the adjacent C-3 carbon. Optimal activity was at pH 6.5 (reduction) and 9.5 (oxidation) and around 68°C. Cb-ACR accepts both NADH and NADPH as electron donors; however, unlike closely related enzymes, NADPH is preferred (Km, 32 μM). Cb-ACR was compared to characterized close homologs, all belonging to the "threonine dehydrogenase and related Zn-dependent dehydrogenases" (COG1063). Metal analysis confirmed the presence of 2 Zn(2+) atoms. To gain insight into the substrate and cofactor specificity, a structural model was constructed. The catalytic zinc atom is likely coordinated by Cys37, His70, and Glu71, while the structural zinc site is probably composed of Cys100, Cys103, Cys106, and Cys114. Residues determining NADP specificity were predicted as well. The physiological role of Cb-ACR in C. beijerinckii is discussed.

FIG 1

Acetoin reductase activity of six potential acetoin reductases from C. beijerinckii NCIMB 8052. The genes were heterologously expressed in E. coli BL21(DE3). Acetoin reductase activity was measured in crude cell extracts.

FIG 2

Effect of temperature and pH on acetoin reductase activity. (A) Effect of temperature; 20°C to 85°C for the reduction reaction (◆) and 40°C to 80°C for the oxidation reaction (○). (B) Arrhenius plot of the temperature dependence of the reduction reaction from 20°C to 60°C. (C) Effect of pH; pH 5.5 to 7.5 for the reduction reaction (◆) and 8.0 to 10.0 for the oxidation reaction (○).

FIG 3

Multiple sequence alignment of Cb-ACR and other characterized close homologs. Gene identifiers of the genes can be found in Table 1. The sequence of the ketose reductase of Bemisia argentifolii (GI:4106363) was included because a three-dimensional structure was available (PDB 1E3J), which was used for the modeling of Cb-ACR. The sequences were aligned using the web-based T-Coffee program (22). Conserved residues involved in metal and NAD(P) binding are shaded in black. Asterisks indicate residues involved in binding of the catalytic and structural zinc. The secondary structure prediction of Cb-ACR (using the psipred program [59]) is shown above the alignment.

FIG 4

Phylogenetic tree of characterized ACRs/BDHs. Amino acid sequences used for the multiple alignment were also used to construct a phylogenetic tree, except that sequences of ACRs/BDHs that belong to a different superfamily, viz., K. pneumoniae and Corynebacterium glutamicum, were added. The tree includes sequences of archaeal, bacterial, and eukaryotic origin, revealing the broad phylogenetic spreading of this group of enzymes. The different clades of NAD- and NADP-dependent enzymes and threonine dehydrogenases are indicated.

FIG 5

Three-dimensional model of Cb-ACR. (A) Overall structure of Cb-ACR. Residues Cys₃₇, His₇₀, and Glu₇₁ of the catalytic site are indicated. The catalytic and structural zinc molecules (purple spheres) were modeled into the structure using a superposition with B. argentifolii Ba-KR ADH (PDB 1e3j) (44). The four cysteines (Cys₁₀₀, Cys₁₀₃, Cys₁₀₆, and Cys₁₁₄) binding the structural zinc and the conserved arginines (Arg₂₀₅ and Arg₂₀₉) involved in binding the adenyl phosphate moiety of NADP are indicated. (B) Surface representation of Cb-ACR model. An NADP molecule was modeled into the structure using a superposition with T. brockii TBADH (PDB 1ykf) (41), giving an indication of how NADP is bound. These images were generated using PyMOL (PyMOL Molecular Graphics System, Version 1.2r3pre; Schrödinger, LLC).