Nucleotide sequences of the Acinetobacter calcoaceticus benABC genes for benzoate 1,2-dioxygenase reveal evolutionary relationships among multicomponent oxygenases

Abstract

The nucleotide sequences of the Acinetobacter cakoaceticus benABC genes encoding a multicomponent oxygenase for the conversion of benzoate to a nonaromatic cis-diol were determined. The enzyme, benzoate 1,2-dioxygenase, is composed of a hydroxylase component, encoded by benAB, and an electron transfer component, encoded by benC. Comparison of the deduced amino acid sequences of BenABC with related sequences, including those for the multicomponent toluate, toluene, benzene, and naphthalene 1,2-dioxygenases, indicated that the similarly sized subunits of the hydroxylase components were derived from a common ancestor. Conserved cysteine and histidine residues may bind a [2Fe-2S] Rieske-type cluster to the oa-subunits of all the hydroxylases. Conserved histidines and tyrosines may coordinate a mononuclear Fe(ll) ion. The less conserved 13-subunits of the hydroxylases may be responsible for determining substrate specificity. Each dioxygenase had either one or two electron transfer proteins. The electron transfer component of benzoate dioxygenase, encoded by benC, and the corresponding protein of the toluate 1,2-dioxygenase, encoded by xylZ, were each found to have an N-terminal region which resembled chloroplast-type ferredoxins and a C-terminal region which resembled several oxidoreductases. These BenC and XylZ proteins had regions similar to certain monooxygenase components but did not appear to be evolutionarily related to the two-protein electron transfer systems of the benzene, toluene, and naphthalene 1,2-dioxygenases. Regions of possible NAD and flavin adenine dinucleotide binding were identified.The complete degradation of benzoate by aerobic bacteria can occur by either of two catabolic pathways. In both reaction sequences, benzoate is converted to a nonaromatic cis-diol, 2-hydro-1,2-dihydroxybenzoate, and then to catechol (51) (Fig. 1) (75).In addition to the hydroxylase component, the dioxygenases described above usually contain one or two electron transport proteins. The benzoate 1,2-dioxygenase of P. arvilla has a single iron-sulfur flavoprotein exhibiting an NADH-cytochrome c reductase activity that is responsible for the electron transfer from NADH to the aromatic ring hydroxylase. This enzyme is a 38-kDa polypeptide with one iron-sulfur cluster of the [2Fe-2S] type and one molecule of flavin adenine dinucleotide (FAD) (73,74). The involvement of similar proteins in the electron transfer reactions of benzoate 1,2-dioxygenase from A. calcoaceticus and toluate 1,2-dioxygenase from P. putida have been suggested by our previous genetic studies (19,21,42). In the benzene, toluene, and naphthalene dioxygenase systems, however, two 5385 JOURNAL

Cis‐diol dehydrogenases encoded by the TOL pWW0 plasmid xylL gene and the Acinetobacter calcoaceticus chromosomal benD gene are members of the short‐chain alcohol dehydrogenase superfamily

“…2. These genes are located downstream of the xylZ and benC genes, respectively, which have previously been characterized by us [7,10,IOa]. The first ATG codons downstream of xylZ and benC are preceded by Shine-Dalgarno-like sequences [l 11, GAGGT and GGAGA, respectively.…”

“…calcoaceticus BD413 have been described [2,7]. The DNA in a 2.2-kbp region containing benD was subcloned into M13mp19 [8].…”

Identification of Rv3230c as the NADPH Oxidoreductase of a Two-Protein DesA3 Acyl-CoA Desaturase inMycobacterium tuberculosisH37Rv

“…This unmatched region has amphipathic character, with a predicted short hydrophobic region immediately preceding the start of the flavin-binding domain (white spheres in Figure 7B). Overall, the remainder of the Rv3230c model closely matched the three domain structure of phthalate dioxygenase reductase, including conserved residues making contact with the flavin cofactor and the NAD(P)H binding domain [ (43,58,59), summarized in Figure 3C] and the spacing of the Cys ligands to the [2Fe-2S] center (e.g., Rv3230c Cys-333, Cys-338, Cys-341 and Cys-368 and 2PIA Cys-272, Cys-277, Cys-280 and Cys-308). Of note, Phe225 from phthalate dioxygenase reductase has been proposed to have configurational flexibility in order to promote nicotinamide-flavin stacking (42), and this residue is conserved in Rv3230c as Phe287.…”

Structural prototypes for an extended family of flavoprotein reductases: Comparison of phthalate dioxygenase reductase with ferredoxin reductase and ferredoxin

“…[PI refers to a pterin-binding moiety. Other proteins identified as members of the family (Neidle et al, 1991;Andrews et al, 1992) are xylene monooxygenase reductase (Suzuki et al, 1991), toluate dioxygenase reductase (XylZ) (Neidle et al, 1991), vanillate demethylase reductase (Brunel & Davison, 1988), benzoate dioxygenase reductase (Neidle et al, 1991), naphthalene dioxygenase ferredoxin reductase (Simon et al, 1993), nitric oxide synthase (Bredt et al, 1991), bacterial hemoglobin-like protein (HMP) (Andrews et al, 1992), LuxC (Swartzman et al, 1990), ferrisiderophore reductase C (Fsrc) (Spyrou et al, 1991;Andrews et al, 1992), phenol hydroxylase component 5 (Nordlund et al, 1990), and cytochrome bLX5 (Segal et al, 1992 160 170 180 190 200 210 220 230 240 250 260 27 0 280 arating equivalent main-chain atoms (Kabsch, 1976). using only atomic separations of less than 3.0 A (see Methods).…”

“…) ferredoxins. Among the reductases related to PDR, Gly follows the second Cys ligand in vanillate demethylase reductase (Brunel & Davison, 1988), benzoate dioxygenase reductase (Neidle et al, 1991), and xylene monooxygenase reductase (Suzuki et al, Table 3. 1991). An exception is methane monooxygenase component C, with Ala at the equivalent position (Stainthorpe et al, 1990) and a [2Fe-2S] potential of -220 mV (Lund & Dalton, 1985).…”