Structure of the Acetophenone Carboxylase Core Complex
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www.nature.com/scientificreports OPEN Structure of the acetophenone carboxylase core complex: prototype of a new class of Received: 16 September 2016 Accepted: 24 November 2016 ATP-dependent carboxylases/ Published: 05 January 2017 hydrolases Sina Weidenweber1, Karola Schühle2, Ulrike Demmer1, Eberhard Warkentin1, Ulrich Ermler1 & Johann Heider2 Degradation of the aromatic ketone acetophenone is initiated by its carboxylation to benzoylacetate catalyzed by acetophenone carboxylase (Apc) in a reaction dependent on the hydrolysis of two ATP to ADP and Pi. Apc is a large protein complex which dissociates during purification into a heterooctameric Apc(αα′βγ)2 core complex of 482 kDa and Apcε of 34 kDa. In this report, we present the X-ray structure of the Apc(αα′βγ)2 core complex from Aromatoleum aromaticum at ca. 3 Å resolution which reveals a unique modular architecture and serves as model of a new enzyme family. Apcβ contains a novel domain fold composed of two β-sheets in a barrel-like arrangement running into a bundle of eight short polyproline (type II)-like helical segments. Apcα and Apcα′ possess ATP binding modules of the ASKHA superfamily integrated into their multidomain structures and presumably operate as ATP-dependent kinases for acetophenone and bicarbonate, respectively. Mechanistic aspects of the novel carboxylation reaction requiring massive structural rearrangements are discussed and criteria for specifically annotating the family members Apc, acetone carboxylase and hydantoinase are defined. Aromatic hydrocarbons are one of the most abundant classes of organic compounds in nature. They are primarily produced by plants as soluble secondary metabolic products or as components of the structural polymer lignin1. Moreover, considerable amounts of toxic aromatic hydrocarbons, in particular, of the BTEX (benzene, toluol, ethylbenzene, xylene) group accumulate during industrial petroleum processing as ground water contaminants2,3. Their biodegradation is of formidable biological/environmental importance and is essentially performed by spe- cial microorganisms in aerobic and anaerobic habitats. For that purpose, nature developed various pathways and enzymatic machineries with novel catalytic capabilities to enable chemically challenging dearomatization, C-H or C-C cleavage reactions. Ethylbenzene is aerobically or anaerobically catabolized via different pathways in various bacteria4,5. In most degradation pathways, the ethyl group of ethylbenzene is initially hydroxylated to 1-phenylethanol, either by a monooxygenase in aerobic bacteria6 or by the molybdenum enzyme ethylbenzene dehydrogenase in anaerobic bacteria7. The alcohol is subsequently oxidized further to acetophenone by an alcohol dehydrogenase8, and the ketone is carboxylated to benzoylacetate by acetophenone carboxylase (Apc) (Fig. 1a). Finally, benzoylacetate is activated to the CoA-thioester, which is then thiolytically cleaved to the products benzoyl-CoA and acetyl-CoA5,9. Apc is the key enzyme of acetophenone metabolism and has, so far, only been characterized from Aromatoleum aromaticum strain EbN110, where it is specifically induced in ethylbenzene and acetophenone-grown cells. Apc consists of five subunits and dissociates into a highly stable, yet inactive Apcα α ′ β γ core complex and the Apcε subunit upon purification. The Apc core complex is organized as an Apc(αα ′ β γ )2 heterooctamer with a molecu- lar mass of 482 kDa. It contains 2 Zn2+, although the activity of Apc is strongly inhibited by further added Zn2+ 1Max-Planck-Institut für Biophysik, Max-von-Laue-Str. 3, 60438, Frankfurt am Main, Germany. 2Laboratorium für Mikrobiologie, Fachbereich Biologie and SYNMIKRO, Philipps-Universität, 35032, Marburg, Germany. Correspondence and requests for materials should be addressed to U.E. (email: [email protected]) or J.H. (email: [email protected]) SCIENTIFIC REPORTS | 7:39674 | DOI: 10.1038/srep39674 1 www.nature.com/scientificreports/ Figure 1. Reactions of Apc, Acx and methyl-hydantoinase. Apc requires the hydrolysis of 2 ATP to 2 ADP + 2 Pi (a), whereas most Acx hydrolyses one ATP to AMP + 2 Pi per carboxylase reaction (b). Hydantoinase hydrolyses ATP to ADP + Pi for imidate phosphorylation (c). In all three reactions the keto/enol equilibrium is shifted towards the enolate/imidate side by phosphorylation. ions. Kinetic studies further indicated the requirement of the Apc core complex, Apcε, and ATP, but not of biotin, for catalytic activity. 2 ATP are hydrolyzed to 2 ADP and 2 Pi per one acetophenone carboxylated. In addition, uncoupled ATPase activity with either bicarbonate or acetophenone was detected in the absence of the second substrate10. Apc of A. aromaticum is encoded by the genes apcA-E (coding for Apcα ′ (70 kDa), Apcγ (15 kDa), Apcα (80 kDa), Apcβ (75 kDa) and Apcε (32 kDa), respectively) which are located in an apparent operon together with the gene for the following enzyme of the metabolic pathway, benzoylacetate-CoA ligase. Apcα and Apcα ′ are structurally related and have a sequence identity of 30%. Sequence comparison studies revealed phylogenetic relationships between Apc, acetone carboxylases (Acx) and ATP-dependent hydantoinases/oxoprolinases (Hyd) with sequence identities between 20 and 30%11,12. However, the subunit compositions of the three enzyme types differ. Acetone carboxylases consist of three subunits and hydantoinases/oxoprolinases of only two. To work with a uniform terminology we define the two core subunits present in all enzymes of the apparent family asα and β , the short subunit present in Apc and Acx as γ and the additional subunits of Apc as α ′ and ε , resulting in an α β protomer for Hyd (from genes hydA and hydB), an α β γ protomer for Acx (from genes acxA, acxB and acxC) and an α α ′ β γ ε protomer for Apc (from genes apcC, apcA, apcD, apcB, apcE). Acetone carboxylases (Acx) are organized as Acx(α β γ )2 heterohexamers. Upon carboxylation of acetone to − acetoacetate by HCO3 , one ATP is either stepwise hydrolyzed to ADP and AMP plus 2 Pi by the enzymes from 13–15 Xanthobacter autotrophicus or Rhodobacter capsulatus , or 2 ATP are hydrolyzed to 2 AMP and 4 Pi without the transient formation of ADP by the enzyme from A. aromaticum12 (Fig. 1b). Hydantoinases/oxoprolinases occur as Hyd(α β )2 heterotetramers that act on diverse substrates as cyclic amidohydrolases with concomitant 16,17 hydrolysis of ATP to ADP and Pi (Fig. 1c). These three types of enzymes, grouped to the hydantoinase/ketone carboxylase family, share the capability to activate a carbonyl group to a phosphoenol (or phosphoimidate) inter- mediate with ATP. In Apc and Acx the generated energy-rich phosphoenol intermediates are subsequently used to accomplish a new type of carboxylation and C-C bond forming reaction. For a more profound understanding of the complex carboxylation reactions, detailed structural data are indis- pensable. In this report, we describe the first X-ray structure of a member of the hydantoinase/ketone carboxylase family, the 482 kDa Apc core complex, at ca. 3 Å resolution. The structural details allow to outline a first proposal of the catalytic reaction cycle of Apc. Carboxylation of inert methylketones is a basic chemical process which makes the Apc reaction also attractive for biotechnological applications. SCIENTIFIC REPORTS | 7:39674 | DOI: 10.1038/srep39674 2 www.nature.com/scientificreports/ Figure 2. Structure of the Apc(αα′βγ)2 core structure. The interface subunit Apcβ is drawn in blue, the potential ATP binding subunits Apcα and Apcα ′ in yellow and green and the small Apcγ in red. Apcα ′ has no contact with Apcβ or Apcγ . The two potential ATP binding sites (black circles) are more than 50 Å apart from each other. Results and Discussion Structure of the Apc core complex. The Apc core complex reveals an impressive novel protein structure with a size of ca. 250 Å × 100 Å × 100 Å (Fig. 2). It is present in the crystals as an Apc(α α ′ β γ )2 heterooctamer which confirms the previously reported oligomeric state derived from a gel filtration–based molecular mass determination in solution10. The heterooctamer is built up by a crystallographic two-fold axis from an asymmetric unit containing one Apcαα ′ β γ protomer. A relatively small protomer interface is formed exclusively between two Apcβ (1130 Å2; 4% of the surface area18). The Apcα α ′ β γ protomer is mainly held together by large contact areas between Apcα and Apcβ (3540 Å2). Apcα ′ is located at the periphery of the Apc core complex and only attached to Apcα . The small Apcγ partly envelops Apcβ and is itself embraced by the extended C-terminal arm of Apcβ (Fig. 2). One Apcαα ′ β γ protomer is expected to contain one bound Zn2+ ion as detected by quantitative elemen- tal analysis10. Although Zn2+ was not detected in the X-ray structure, two of the found mercury binding sites are also plausible for binding of Zn2+ ions. One mercury was found to be coordinated to Asp65, His123, Asp126 and His148 in Apcβ and a second to Cys22, Cys25, Cys75 and Cys78 in Apcγ (sFig. 1). We favour the latter as more probable physiological Zn2+-binding site in Apc because of the high conservation of the cysteines in Apc and Acx, its location in a classical Zn finger motif19 and the absence of Zn2+ in Hyd, which are lacking orthologs of Apcγ . As both potential Zn2+ binding sites are located far away from the proposed active site, the role of the Zn2+ ion is probably more structural and not directly related to the catalytic reaction. The subunits of Apc are modularly built up of several domains arranged in a new topological manner. Based on the DALI20 and SCOP servers21 Apcα reveals a high structural relationship to Apcα ′ reflected in a rmsd of 4.1 Å between the two subunits (96% of the Cα used) suggesting an evolution from a simpler common ancestor by gene duplication (Fig. 3). Their most prominent structural elements (Apcα : 1–90, 250–275; 275–510; Apcα ′ : 1–80, 215–465) belong to the ASKHA (acetate and sugar kinase/heat shock cognate/actin) superfamily22.