Identification of Genes Essential for the Biogenesis of Quinohemoprotein Amine Dehydrogenase

Abstract

The structural genes encoding quinohemoprotein amine dehydrogenase (QHNDH) in Gram-negative bacteria constitute a polycistronic operon together with several nearby genes, which are collectively termed "qhp". We previously showed that the qhpD gene, which lies between qhpA and qhpC (encoding the α and γ subunits of QHNDH, respectively), and the qhpE gene, which follows qhpB (encoding the β subunit), both encode enzymes specifically involved in the posttranslational modification of the γ subunit and are hence essential for QHNDH biogenesis in Paracoccus denitrificans [Ono, K., et al. (2006) J. Biol. Chem. 281, 13672-13684; Nakai, T., et al. (2012) J. Biol. Chem. 287, 6530-6538]. Here we further demonstrate that the qhpF gene, which follows qhpE, and the qhpG and qhpR genes, peripherally located in the complementary strand, are also indispensable for QHNDH biogenesis. The qhpF gene encodes an efflux ABC transporter, which probably translocates the γ subunit into the periplasm in a process coupled with hydrolysis of ATP. The qhpG gene encodes a putative FAD-dependent monooxygenase, which is required for the generation of the quinone cofactor in the γ subunit. Finally, the qhpR gene encodes an AraC family transcriptional regulator, which activates expression of the qhp operon in response to the addition of n-butylamine to the culture medium. Database analysis of the qhp genes reveals that they are very widely distributed, not only in many Gram-negative species but also in a few Gram-positive bacteria.

The Radical S-Adenosyl-l-methionine Enzyme QhpD Catalyzes Sequential Formation of Intra-protein Sulfur-to-Methylene Carbon Thioether Bonds

“…QhpD is essential for the biogenesis of QHNDH, most likely by participating in the Cys-to-Asp/Glu thioether bond formation in the ␥ subunit (maturated QhpC) (7). Consistent with its indispensable role in the QHNDH biogenesis, the qhpD gene is strictly conserved in all bacteria possessing the qhp operon with the common gene array, qhpADCB (and their reverse, BCDA, in the complementary strands) (4). According to recent bioinformatics analysis, QhpD is classified into the subgroup "SPASM/ twitch domain-containing," named for its biochemically characterized founding members as follows: subtilosin Asynthesizing enzyme, AlbA; pyrroloquinoline quinone biosynthetic enzyme, PqqE; anaerobic sulfatase-maturating enzyme, anSME; and mycofactocin synthesizing enzyme, MtfC (13,14).…”

“…Although the enzyme was originally reported to occur only in a limited number of Gram-negative bacteria (2,3), our recent survey with the updated genome sequence database revealed a wide distribution of the enzyme not only in more than 200 Gram-negative species but also in some Gram-positive bacteria with the associated genes constituting an operon designated "qhp" (4). QHNDH consists of three nonidentical subunits, with each subunit exhibiting distinct structural features (Fig.…”

“…Three other thioether cross-links (Cys-7-Glu-16, Cys-27-Asp-33, and Cys-41-Asp-49) are formed between the sulfur atom of a * This work was supported by Japan Society for the Promotion of Science Cys residue and the methylene carbon atom of an Asp or Glu residue, affording the structural stabilization of the subunit polypeptide chain that is otherwise a featureless coil with only two short ␣-helices. Therefore, the ␥ subunit must undergo multiple post-translational modifications, in which many gene products encoded in the qhp operon, including QhpD, QhpE, QhpF, and QhpG, are involved (4,7,8), and such events occur before or after associating with the ␣ and ␤ subunits to form the active QHNDH complex.…”

Two Different Quinohemoprotein Amine Dehydrogenases Initiate Anaerobic Degradation of Aromatic Amines in Aromatoleum aromaticum EbN1

“…5B). The presence of two different quinohemoprotein operons in parallel, as in A. aromaticum EbN1, appears to be rather rare in nature, because only a few bacteria have been found to carry two copies of the genes (15). While many of the identified bacterial quinohemoprotein gene clusters are also accompanied by genes encoding enzymes potentially involved in the further degradation pathway, such as aldehyde dehydrogenases, Mo-containing aldehyde hydroxylases, or acyl coenzyme A (acyl-CoA) synthetases ( Fig.…”

“…The so-called copper amine oxidases contain a redoxactive Cu ion in addition to their quinoid cofactors and represent two separate quinoenzyme classes containing either topaquinone (TPQ) or lysine tyrosylquinone (LTQ) as quinoid cofactors (8,10,11). Metal-independent quinoenzyme classes are represented by methylamine dehydrogenases, which contain a tryptophan tryptophylquinone (TTQ) cofactor, and by quinohemoprotein amine dehydrogenases, which contain a cysteine tryptophylquinone (CTQ) cofactor (12)(13)(14)(15).…”