Structural And Enzymatic Insights Into The Yeast Ubiquinone Biosynthesis Enzymes Abz2and Coq5

Coenzyme Q (ubiquinone or CoQ) is a lipid-soluble compound who serves as one of the most important electron carriers of respiratory chain in both prokaryotes and eukaryotes. It plays a crucial role in transferring electrons from NADH-CoQ reductase complex or succinate-CoQ reductase complex to cytochrome reductase complex. In addition, the reduced ubiquinol form of CoQ is a recyclable lipophilic antioxidant protecting membrane phospholipid layers, mitochondrial DNA and membrane proteins against oxidative damage. Therefore, CoQ10is an essential micronutrient in human nutrition due to its antioxidant function.Generally, demand of CoQ in cells relies on its de novo synthesis, which has been proved quite conserved in many kinds of species from E. colito human being.The knowledge of CoQ biosynthesis pathway in eukaryotes is mostly based on the findings in yeast. Nine genes designated COQ1-COQ9have been identified involving in CoQ6biosynthesis. It usually starts with the formation of the aromatic precursor para-hydroxybenzoic acid (PHB) and the polyprenyl chain. Afterward, the modifications of the aromatic ring including O-methylation, decarboxylation, and C-methylation and so on take place in order to generate the mature CoQ6.Alternatively,para-aminobenzoic acid (PABA) could enter the CoQ6biosynthesis pathway, as a competitor of PHB, which has long been recognized as an aromatic precursor of the benzoquinone ring of CoQ6. Besides, PABA is required for the biosynthesis of folate which is also a crucial cofactor during biological processes. Unlike CoQ which is synthesized in virtually all living organisms, de novo biosynthesis of folate only exists in most prokaryotes, microbial eukaryotes, and plants. The biosynthesis pathway of PABA has been identified in a few species of bacteria, microbial eukaryotes, and plants. In most cases, PABA is synthesized from chorismate via two steps:first,4-amino-4-deoxyisochorismate (ADC) is synthesized from chorismate and glutamine; second, ADC is converted to PABA. For instance, Saccharomyces cerevisiae ABZ1/YNR033W encodes a bifunctional ADC synthase of two domains, whereas ABZ2/YMR289w encodes an ADC lyase (EC4.1.3.38). However, the biosynthesis pathway of PHB in eukaryotes is still not clear. To our current knowledge, there are at least11genes including ABZ1,ABZ2,and COQ1-COQ9participating in the biosynthesis of CoQ6. (1) Here we present the crystal structure of yeast Abz2complexed with PLP at1.9A resolution using the method of SAD (Single-wavelength Anomalous Diffraction), representing the first eukaryotic ADC lyase structure. To date, four crystal structures of prokaryotic ADC lyases have been deposited in the PDB (http://www.pdb.org), including E. coli PabC (EcPabC, PDB code:1ET0), Pseudomonas aeruginosa PabC (PDB code:2Y4R), Thermus thermophiles PabC (PDB code:2ZGI), and Legionella pneumophila PabC (LpPabC, PDB code:3LUL). All of them display a similar dimeric overall fold, but with differences in the active-site pocket. In addition, a protein of unknown function from Mesorhizobium loti (PDB code:3QQM) has been annotated as a putative amino-acid aminotransferase. However, comparisons of both the overall structure and active site indicated that it is most likely an ADC lyase.The overall structure of Abz2(Pro23-Tyr374) is a monomer composed of an N-terminal domain (Domain I, Pro23-Ser229)and a C-terminal domain (Domain II, Thr230-Tyr374). A part texture of DomainI displays as a uniquefold referred to as the auxiliary subdomain, which is absentfrom all previous PabC structures. Further structural analysis of the auxiliary subdomain demonstrated that this fold is unnecessary for the formation of activesites, and no significant outputs were obtained when a DALI search wasperformed. However, it is probably required to maintain theintegrity and stability of Abz2.Abz2shares no significant sequence homology with PabC enzymes of known structure. Unlike E.coli ADC lyase whose dimerization is critical to the formation of the active site, the active site of Abz2is at the interdomain cleft, where a molecule of cofactor PLP is deeply buried andform a Schiff base with catalytic residue Lys251. Structure-based computational simulations using program HADDOCK with ADC-PLP intermediate defined a basic clamp composingArg182and Arg255to orientate the substrate ADC in a proper pose.In combination with site-directed mutageneses and enzymatic activity assays,we proposed a putativemechanism of the catalytic cycle of Abz2following a previously hypothesized scheme. Further structural analyses enabled us to group the current ADC lyases of known structure into two classes, with Abz2leading a unique group of monomeric ADC lyases. (II) It is suggested that a large complex comprising the Coq polypeptides (Coq1-Coq9) is peripherally associated with the inner mitochondrial membrane to catalyze these successive reactions. One of the Coq enzymes, Coq5, is shown to be a core component since it is required for stabilizing other componentsof this complex. Coq5is a S-adenosyl-methionine (SAM)-dependent methyltransferase (SAM-MTase) catalyzing the only C-methylation step that converts2-methoxy-6-polyprenyl-1,4-benzoquinol(DDMQH2)to2-methoxy-5-methyl-6-polypr enyl-1,4-benzoquinol(DMQH2).Here we report the crystal structures of yeast Coq5in the apo-form (Coq5-△N60-apo) and SAM-bound form (Coq5-△N60-SAM) at2.2and2.4A resolution, respectively. The overall structure of Coq5-△N60-apo exhibits a typical topology of Class I SAM-MTase incorporating a mixed α/β topology as the core region which represents a Rossmann-like fold. There are two minor variations to the core region of Coq5-△N60-apo. The first one is the addition of one helices (al) at the N terminus, which is usually a "lid" covering the active site correspondently in othersmall-molecule SAM-MTase. The second one is an insertion of two helices (a6and a7) connecting β5and α8. In Coq5, both two variable segments participate in the dimeric interface.The overall structure of Coq5-△N60-SAM is also a dimer as Coq5-△N60-apo. There are no significant structural differences between these two structures. We analysed the residues contributing to the formation of the dimeric interface and SAM-binding region. Multiple-sequence alignment showed that these residues are highly conserved through Coq5and homologs from diverse species.