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Home , Biotin synthase, Molybdopterin, Molybdopterin synthase

709

Structural biology of involved in NAD and Menico Rizzi* and Hermann Schindelin†

The structural analysis of all enzymes in a metabolic pathway is as nicotinamide adenine dinucleotide (NAD) and flavin a prerequisite to answering fascinating questions, such as adenine dinucleotide (FAD), and those that contain those relating to the evolutionary relationships between organometallic groups, such as and the molybdenum enzymes within the same and related pathways. Furthermore, cofactor (Moco). the observed impressive diversity of catalytic functions displayed by these enzymes can lead to the synthesis of highly NAD is an essential cofactor for both energy complex or unstable molecules, frequently involving unusual and signal transduction, sharing this dual functionality chemical reactions. Moreover, a detailed description of the with two other important : ATP and GTP. Its of each in a pathway is of immense direct action on equilibrium in metabolism has been importance for the rational design of new drugs. The recent well known for a long time, being indeed a milestone in progress made in the structural biology of enzymes involved in every biochemistry textbook. More recently, its impact in NAD and molybdenum cofactor biosynthesis presents a processes such as DNA repair, calcium-dependent signaling significant step toward these goals. pathways and, most fascinating, life-span extension in yeast was widely demonstrated [1,2,3•]. It is therefore not Addresses surprising that NAD homeostasis must be tightly regu- *DISCAFF-INFM, University of Piemonte Orientale, Via Bovio 6, lated in all living . NAD biosynthesis can be 28100 Novara, Italy and Department of Genetics and Microbiology, accomplished either through a de novo pathway or through University of Pavia, Via Ferrata 1, 27100 Pavia, Italy; salvage pathways, with notable differences between e-mail: [email protected] †Department of Biochemistry and Center for Structural Biology, and [4,5] (Figure 1). The past years State University of New York at Stony Brook, Stony Brook, have seen an ever-increasing interest in NAD biosynthesis NY 11794-5115, USA; as an important source of new targets for the development e-mail: [email protected] of novel antibacterial agents [6]. The structural enzymology Current Opinion in Structural Biology 2002, 12:709–720 of several enzymes involved in NAD biosynthesis has provided a detailed picture of their catalytic mechanism, 0959-440X/02/$ — see front matter © 2002 Elsevier Science Ltd. All rights reserved. paving the way for the design of potent and highly selective inhibitors. Abbreviations FAD flavin adenine dinucleotide Moco [7,8] is the essential component of a diverse group of LASPO L-aspartate oxidase MGD guanine dinucleotide redox enzymes [9–12]. The cofactor consists of a mono- Moco molybdenum cofactor nuclear molybdenum coordinated by the dithiolene moiety MPT molybdopterin of a family of tricyclic pyranopterin structures, the simplest NaAD nicotinic acid adenine dinucleotide of which is commonly referred to as molybdopterin (MPT) NAD nicotinamide adenine dinucleotide NADS NAD synthetase [7]. More recently, a -containing pyranopterin NaMN nicotinic acid mononucleotide cofactor has also been discovered [13,14]. Moco biosynthesis NMN nicotinamide mononucleotide is an evolutionarily conserved pathway comprising several NMNAT NaMN/NMN adenylyltransferase novel reactions. Mutations in the human Moco biosynthetic QA quinolinic acid QAPRT quinolinic acid phosphoribosyltransferase genes lead to Moco deficiency, a severe disease that leads PDB Data Bank to premature death in early childhood [15,16]. The affected PPi pyrophosphate patients show severe neurological abnormalities, such as PRPCP 5-phosphoribosyl-1-(β-methylene)pyrophosphate attenuated growth of the brain, seizures and, frequently, PRPP 5-phosphoribosyl-1-pyrophosphate SAM S-adenosyl methionine dislocated ocular lenses. Recently, the first mutations in several human genes encoding Moco biosynthetic have been identified [17–22]. Introduction Many enzymes require cofactors for their catalytic activities This review summarizes recent progress made in the and, if these cofactors cannot be taken up from the structural biology of enzymes involved in NAD and Moco environment as , metabolic pathways are required biosynthesis. These pathways illustrate different levels of for their synthesis. An important subclass of enzymes are phylogenetic conservation of a biochemical pathway; the involved in redox transformations. Among the cofactors reactions in Moco biosynthesis are almost universally utilized by these enzymes are those that are made up of conserved across different kingdoms, whereas NAD purely inorganic molecules, such as the FeS clusters of biosynthesis shows pronounced differences in prokaryotes various compositions, those that are entirely organic, such and eukaryotes. 710 and regulation

Figure 1

H2N CH COOH

L-aspartate CH2 COOH

O2 LASPO

H2O2

HN CH COOH

Iminoaspartate CH2

COOH QA synthase Dihydroxyacetone ? phosphate

2H2O + Pi

COOH QA N COOH QAPRT PRPP Mg

O PPi + CO2

OH(NH2) O

HO P O N

OH O HH NaMN H H OH OH NMNAT ATP Mg

PPi NH2 O

N N OH NaAD O O N N O P O P O N

O OH OH O H H HH H H H H OH OH OH OH

ATP Mg + NH3

NH2 PPi + AMP O N N NH2 O O N N N NADS NAD OP O P O O OH OH O H H HH H H H H OH OH OH OH

Current Opinion in Structural Biology NAD and molybdenum cofactor biosynthesis Rizzi and Schindelin 711

Figure 1 legend

De novo NAD biosynthesis in prokaryotes. The structures of the NMNAT and 2NSY for B. subtilis NADS). In eukaryotes, NAD can be proteins involved in each step are shown in ribbon representation, with synthesized de novo from tryptophan via a five-step kynurenine different subunits colored differently (PDB codes: 1KNR for E. coli pathway. The two pathways converge at the level of QA, from where a LASPO, 1QPR for M. tuberculosis QAPRT, 1F9A for M. jannaschii common route is followed in all organisms [5].

Structural biology of Moco biosynthesis cells was demonstrated only after its receptor-anchoring Genes involved in Moco biosynthesis have been identified properties had been characterized [40]. in eubacteria, and eukaryotes. Although some details of Moco biosynthesis are still unclear at present, the Additional steps pathway can be divided into three universal stages [12,23], Eubacteria typically use Moco in a dinucleotide form in as described below and summarized in Figure 2. which the pyranopterin is linked to a second via a pyrophosphate linkage [41]. In E. coli, the cofactor is Conversion of a guanosine derivative into precursor Z present as molybdopterin guanine dinucleotide (MGD) This aspect is different from other biosynthetic and the MobA protein is involved in its generation [42,43]. pathways, as C8 of the purine is inserted between the Other dinucleotide forms of the cofactor, most notably 2′ and 3′ ribose carbon during formation of precursor Z, molybdopterin cytosine dinucleotide (MCD), have also rather than being eliminated [24,25]. In , the been observed. MoaA and MoaC proteins are responsible for this conver- sion, but the detailed aspects of the reactions catalyzed by Structural studies of Moco biosynthetic enzymes these enzymes and their actual substrates are unknown. The structures of almost all of the E. coli enzymes involved in Moco biosynthesis (Figure 2), with the exception of Transformation of precursor Z into MPT MoaA, have been published [44•,45••,46,47••,48–52,53•]. This process generates the dithiolene group responsible for Of particular interest are the structures of MPT synthase coordination of the molybdenum in the cofactor and is [47••] and the MoeB–MoaD complex [45••] involved in catalyzed by MPT synthase [26]. MPT synthase is MPT biosynthesis, and the MogA [46] and MoeA [53•] composed of two subunits encoded in E. coli by the moaD proteins involved in metal incorporation. Relatively little is and moaE genes. In its active form, MoaD contains a currently known about the first step in the pathway, thiocarboxylate at its C-terminal glycine [27], which acts as whereby the MoaA and MoaC proteins catalyze the elabo- the donor for the synthesis of the dithiolene group. rate rearrangement reaction in which precursor Z is formed MoeB activates MPT synthase by transferring a sulfur atom from an uncharacterized guanosine derivative. The MoaA onto the C terminus of MoaD, generating the thiocarboxy- protein is a member of a large superfamily of proteins late. MoeB exhibits striking sequence similarity to the referred to as SAM (S-adenosyl methionine) [54], N-terminal region of the -activating enzyme (E1), which, among others, also contains anaerobic ribonu- the first component of the ubiquitin-dependent protein cleotide reductase, pyruvate formate , synthase degradation pathway, and to related enzymes catalyzing the and ThiH, a protein involved in biosynthesis. activation of ubiquitin-like protein modifiers. The sequence Members of this family contain an -labile FeS cluster, similarity between MoeB and E1, in concert with their which reacts with SAM to form 5′-deoxyadenosine and a functional relationship, has fostered speculation regarding substrate radical [54,55]. The crystal structure of MoaC an evolutionary link between the two pathways [23,28]. [52] revealed a protein with a ferredoxin fold (as classified by SCOP), which is characterized by two repeating β/α/β Metal incorporation motifs. The protein is present as a tightly packed hexamer The MogA and MoeA proteins together are responsible for with D3 symmetry. Although the active site of MoaC could metal incorporation [29,30]. Current evidence suggests be defined by biochemical studies, the unknown substrate that MogA incorporates the metal into MPT, whereas of the enzyme has hampered further characterization of MoeA has been proposed to convert molybdate into an the active site. unknown compound, which serves as the metal donor. The mammalian protein is a MogA–MoeA fusion MPT synthase comprises a heterotetramer of two MoaD and protein containing a linker region with 160 residues [31]. two MoaE subunits. The MoaE subunits dimerize and the Gephyrin is critical for the postsynaptic anchoring of MoaD subunits bind on opposite ends of the MoaE dimer. inhibitory glycine receptors [32,33] and major GABAA Each MoaD C terminus is deeply inserted into the active site, receptor subtypes [34]. Its activity appears to be regulated located primarily in the proximal MoaE subunit, but also by a variety of interactions with components of the involving residues from the distal MoaE subunit. The MoaE cytoskeleton and proteins involved in signal transduction subunit has an α/β hammerhead fold containing an additional pathways [35–39]. Direct participation of the multifunctional antiparallel three-stranded β sheet. The crystal structure gephyrin protein in Moco biosynthesis in mammalian of MPT synthase [47••] and the NMR structure of the 712 Catalysis and regulation

Figure 2

MoaD and MoeB

? O MoaA N OO OSH HN H H O N SH * N 2´ * 3´ O HN P HN H N N N 2 OH OPO 2− 5´ O 3 CH2 1´ 4´ NH NNO P NH2 NNO 2 n O H H 4´ 1´ Precursor Z MPT 3´ 2´ OH OH

GXP MPT synthase MoaC

MogA MoeA

Mo OS Mo H O OS N S H HN N N S NH HN O O O 2− NH2 NNO P P N OPO N NH2 3 H − − O NH2 NNO OOO O H

OHOH Moco MGD

MobA Current Opinion in Structural Biology

Moco biosynthesis in E. coli. The carbon atom at position 8 of the involved in each step are shown in ribbon representation, with different guanosine derivative of unknown composition (GXP), which is the subunits colored differently. In the case of MPT synthase, the MoaD starting structure, is incorporated into precursor Z as indicated by subunits are shown in yellow and blue, and the MoaE subunits in cyan the asterisk. In mature Moco, additional molybdenum ligands are and magenta. In the case of the MoaD–MoeB complex, the MoaD present besides the dithiolene sulfurs shown here, with the metal subunits are shown in green and red, and the MoeB subunits in yellow being either penta- or hexa-coordinated. The structures of the proteins and cyan.

MoaD-related ThiS protein [56••] demonstrated unambiguously acyladenylate intermediate [45••] have helped to elucidate the divergent evolutionary relationship between a subset of the activation reaction catalyzed by MoeB/ThiF and the E1 enzymes involved in the biosynthesis of sulfur-containing enzymes. Like MPT synthase, the MoaD–MoeB complex cofactors (e.g. Moco, thiamine and certain FeS clusters) and forms a heterotetramer in which the MoeB subunits dimerize the process of ubiquitin activation (Figure 3a). MoaD and and the two MoaD subunits do not contact each other. ThiS share the same fold as ubiquitin (Figure 3b), despite the The MoeB subunit has an α/β/α structure with an eight- absence of any detectable sequence homology. Together with stranded mostly parallel β sheet. The C terminus of each the known sequence similarities between MoeB/ThiF and MoaD subunit is deeply inserted into the MoeB active site, the E1 enzymes, this confirmed that the ubiquitin-like protein which, like its counterpart in MoaE, is composed of residues modifiers and their activating enzymes are the probable from both MoeB subunits. The reaction sequence catalyzed evolutionary offspring of the corresponding proteins involved by MoeB, ThiF and the E1 enzymes begins with a nucleo- in Moco biosynthesis. philic attack of the C-terminal carboxylate of MoaD, ThiS and ubiquitin, respectively, on the α-phosphate of an ATP The co-crystal structures of the MoaD–MoeB complex in its molecule bound at the active site of the activating enzymes, apo form, with bound ATP and after formation of the MoaD leading to the formation of a high-energy acyladenylate NAD and molybdenum cofactor biosynthesis Rizzi and Schindelin 713

Figure 3 legend Figure 3

MPT biosynthesis and relationship to the ubiquitin activation step. (a) Schematic diagram indicating the relationship between the MoeB, (a) ThiF and E1 enzymes. The reaction sequence catalyzed by the E1, Ubiquitin E1 MoeB and ThiF enzymes initially leads to the formation of a high-energy – acyladenylate intermediate. Subsequently, a thiocarboxylate is formed at C O E1 C AMP E1 C S Cys the C terminus of MoaD and ThiS, with the sulfur ultimately derived from O O O cysteine with the aid of an IscS/NifS-like protein. In the case of ATP PPi ubiquitin, a thioester is formed, leading to the formation of a covalent complex between the protein modifier and the E1 enzyme via the MoaD sidechain of a conserved cysteine residue in the E1 active site. (b) A structural comparison of MoaD (yellow) and ubiquitin (red). C O– MoeB C AMP C S– (c) MoeB-catalyzed reaction. Close-up view of the acyladenylate O O O intermediate formed during MoaD activation, in which AMP is covalently CSD/IscS/ ATP PP CsdB + Cys linked at Gly81 to the MoaD C terminus, of which residues 78–81 i are shown together with AMP in all-bonds representation. The subunits ThiS of the MoeB dimer are shown in dark and light gray, with residues conserved in the MoeB/ThiF/E1 superfamily highlighted in green C O– ThiF C AMP ThiI C S– and yellow. O O IscS + Cys O ATP PPi intermediate (Figure 3c). Subsequently, a thiocarboxylate is formed at the C termini of MoaD and ThiS, with the sulfur (b) N ultimately derived from cysteine with the aid of an N IscS/NifS-like protein. These proteins are PLP-dependent enzymes and, in E. coli, three members of this family, CSD, IscS and CsdB, can provide the sulfur atoms for MPT synthesis, with CSD showing the highest activity [57]. In the case of ubiquitin and related protein modifiers, a thioester is formed instead of the thiocarboxylate, leading to the formation of a covalent complex between the protein modifier and the cognate E1 enzyme via the sidechain of a conserved cysteine residue in the E1 active site. The MoeB crystal structure suggests that the E1 enzymes contain a C tandem repeat of MoeB-like domains, which together form C one functional active site (Figure 3c). (c) Together with biochemical data, the crystal structures of the single-domain MogA protein [46] and the related G-domains of gephyrin and the plant ortholog Cnx1 [49] identified the MPT- in these enzymes and two conserved aspartic acid residues, which are crucial for their activities. All three proteins form highly stable trimers, in G81 which the MPT-binding site appears to be restricted to just the monomer. MogA folds into an α/β/α structure contain- ing a mostly parallel five-stranded β sheet. The larger MoeA protein consists of four domains grouped together to form a highly extended molecule [48,53•]. The fold of domain III, the largest of the domains, is closely related to that of MogA (Figure 4). The structural similarity is restricted not only to the overall fold, but also to the putative active sites, where two negatively charged residues are conserved. Domains I Current Opinion in Structural Biology and II form the stalk of the extended molecule, and are characterized by unusual folds containing mostly β sheets, whereas domain IV contains two sharply bent β ribbons. The oligomeric state of both proteins (i.e. MogA is a very stable trimer, whereas MoeA forms a less stable dimer) is of trimerization of its MogA-related G-domain and dimerization particular relevance to gephyrin, the MogA–MoeA fusion of its MoeA-related E-domain [53•]. This proposed structure protein found in mammals. The oligomeric states of the would be crucial for the receptor-anchoring function of this bacterial proteins suggest that gephyrin can form a hexagonal protein by providing evenly spaced binding sites for the scaffold underneath the postsynaptic membrane through glycine receptor and tubulin to which it binds. 714 Catalysis and regulation

Figure 4

Proteins involved in metal incorporation. Structural comparison of domain III of MoeA (yellow) and MogA (green). The two active site aspartic acid residues of MogA (Asp49 and Asp82) and their structural counterparts in MoeA are shown in all-bonds representation. A signature sequence motif common to all MogA and MoeA proteins (TTGGT in E. coli MogA and SSGGVS in E. coli MoeA) is highlighted in red.

The crystal structure of the E. coli MobA protein involved (Figure 1). The structures of the wild-type E. coli apo form in MGD formation has been determined in its apo state and the R386L mutant in complex with FAD and with [44•,51] and with bound Mn2+•GTP [44•]. On the basis of succinate were reported [58•,59]. LASPO consists of three the MobA–GTP structure, a model for the MGD product domains: a classic FAD-binding domain exhibiting the complex has been postulated. The catalytic mechanism of ‘Rossmann fold’ topology, a C-terminal three-helical-bundle this enzyme appears to involve the attack of the terminal domain and an α+β capping domain [59] (Figure 1). phosphate of MPT on the α-phosphate of GTP, leading to The most striking aspect emerging from the structural the release of pyrophosphate (PPi). The MobA-catalyzed comparison between apo and holo forms of LASPO is a reaction is similar in stoichiometry and mechanism to that remarkable conformational change observed upon FAD of the NaMN/NMN adenylyltransferases (NMNATs), binding [58•]. Two long polypeptide stretches, which are which catalyze the dinucleotide formation step during fully disordered in the apo form, become fully structured NAD biosynthesis (see below); however, the proteins are in the holo form, sealing the active site cavity and partici- in different protein superfamilies. MobA belongs to the pating in substrate binding. Moreover, the entire cap nucleotide-diphospho-sugar superfamily, which domain rotates 27° with respect to the conformation is characterized by a mixed seven-stranded β sheet, and the observed in the apo form. The major effect resulting from NMNATs belong to the nucleotidylyl transferase super- the observed structural changes is an effective shielding of family, which is characterized by a parallel five-stranded the product, which is totally solvent inaccessible when β sheet. Because of the exclusive presence of MobA in bound to the active site of LASPO [58•]. eubacteria, including many pathogenic organisms, this enzyme potentially represents an interesting target for the In the second step of prokaryotic de novo NAD biosynthesis, rational design of antibiotics. iminoaspartate is condensed with dihydroxyacetone phos- phate to form quinolinic acid (QA) by the action of QA Structural studies on NAD biosynthesis synthase [4,5] (Figure 1). Unfortunately, no structural data This pathway can be divided into two parts: the first part have been reported for QA synthase, yet. It has been yields nicotinic acid mononucleotide (NaMN), formally argued that LASPO and QA synthase could function as a representing the first half of the final target NAD. In the multienzyme complex, because of the extreme chemical second part, the mononucleotide is transformed into NAD instability of iminoaspartate [60]. Such a molecule would by two subsequent reactions [5]. not survive for a long time in solution and it is proposed to be directly channeled from LASPO to the QA synthase From aspartic acid to nicotinic acid mononucleotide active site, where QA is formed. The first step in prokaryotic de novo NAD biosynthesis is catalyzed by L-aspartate oxidase (LASPO), which carries The only route for QA metabolism is represented by the out the oxidation of L-aspartate to iminoaspartate using action of the ubiquitous quinolinic acid phosphoribosyltrans- FAD as a noncovalently bound [4,5] ferase (QAPRT). QAPRT catalyses the Mg-dependent NAD and molybdenum cofactor biosynthesis Rizzi and Schindelin 715

transfer of the phosphoribosyl moiety from 5-phosphoribosyl-1- The intriguing capability of NMNAT from different pyrophosphate (PRPP) to QA, yielding NaMN, PPi and sources to strictly recognize either NaMN or NMN has CO2. The reaction is the third step in de novo NAD biosyn- been unraveled thanks to the structural studies carried out thesis in all organisms [5] (Figure 1). The structures of on the E. coli and Bacillus subtilis NMNAT–NaAD com- QAPRTs from Salmonella typhimurium and Mycobacterium plexes [66,67•] and on archaeal Methanobacterium tuberculosis were reported in different states, covering almost thermoautotrophicum NMNAT in complex with NAD and all the stages along the reaction coordinate [61,62]. The NMN [68•]. Large conformational changes are observed in QAPRT fold consists of a two-domain structure, with a E. coli NMNAT upon NaAD binding [67•]. Several loop mixed α/β N-terminal domain and a unique α/β-barrel- regions surrounding the enzyme active site move substan- like domain containing seven β strands [61] (Figure 1). tially on ligand binding, forming a highly specific binding The structure determination of the nonproductive site for NaAD. The eubacterial enzyme specifically recog- Michaelis ternary complex with phthalic acid (a QA analog) nizes the deamidated pyridine through a few important and with the substrate analog 5-phosphoribosyl-1-(β-methyl- hydrogen bonds between the nicotinate carboxylate and ene)pyrophosphate (PRPCP) allowed the description of the mainchain amides [67•]. On the other hand, NMN recogni- catalytic stage in M. tuberculosis QAPRT, revealing the tion in archaeal NMNAT is achieved through several presence of two octahedrally coordinated Mg2+ ions located interactions with protein sidechains, with a key role played • on either side of the PPi moiety of PRPCP [62]. Structural by an aspartic acid residue [68 ]. Moreover, although no apo comparison of M. tuberculosis QAPRT in complex with the form NMNAT from archaeal species has been reported so substrate QA, with the product NaMN and in its free form far, no dramatic structural changes are expected upon ligand revealed remarkable structural differences, with a striking binding in archaeal NMNAT. It is noteworthy that the conformational flexibility featuring Lys172, the key residue significant differences in the pyridine-nucleotide-binding for QA recognition. QAPRT appears to exist in two major site observed between eubacterial and archaeal NMNATs conformers: a relaxed conformer characterizing the apo form are mainly concentrated around the pyridine carboxamide/ and the complex with NaMN; and an active conformer carboxylate moiety, with the remaining parts of the observed in the complex with the substrate QA and in the mononucleotide being recognized through a series of con- nonproductive ternary complex [62]. served interactions. Human NMNAT shows a striking dual specificity, being able to promptly recognize both forms of From NaMN to NAD the pyridine nucleotide without any dramatic conforma- The NaMN produced by the first three reactions is now tional change [65•,69••]. The NaAD and NAD binding transformed into nicotinic acid adenine dinucleotide modes are very similar, and the pyridine carboxamide/ (NaAD) by the ubiquitous NMNAT [5] (Figure 1). The carboxylate group establishes few hydrogen bonds with enzyme catalyzes the nucleophilic attack by the 5′ phos- mainchain protein atoms and with structurally conserved phate of NaMN on the α-phosphoryl of ATP, yielding solvent molecules [69••,70]. Subtle changes of the electro- 2+ NaAD and PPi, in a strictly Mg -dependent reaction [5]. static distribution in the nucleotide-binding site are likely Because the reaction is fully reversible, NaAD can be recon- to be responsible for the relaxed substrate specificity of verted to ATP and NaMN. Because NMNAT is located at human NMNAT, allowing the enzyme to accommodate the merging point between de novo and salvage routes, the substrates with different charge properties [69••]. enzyme developed an intriguing dual specificity, being able to recognize either NaMN or nicotinamide mononu- A set of conserved interactions are employed by all cleotide (NMN), depending on the source [5]; this reflects NMNATs to recognize ATP and the pyridine mononu- the relative importance of de novo against recycling routes in cleotide, with the exception of its carboxamide/carboxylate different organisms. The eubacterial enzyme has a marked moiety, which is recognized in different NMNATs by a set preference for NaMN, whereas archaeal NMNAT strongly of unique interactions. The requirement to control different prefers NMN. Interestingly, human NMNAT displays a NaMN/NMN metabolic fluxes and to balance de novo unique dual specificity, being able to recognize with the against recycling routes in different organisms has been same efficiency both NaMN and NMN. satisfied by evolution by sculpting the required specificity on a highly conserved catalytic core, which guarantees Notably, in Haemoplilus influenzae, NMNAT has been efficient catalysis. reported to feature the N-terminal domain of NadR, a bifunctional protein also endowed with ribosylnicoti- The last step in NAD biosynthesis consists of an amide namide kinase activity [63]. The NMNAT architecture transfer onto NaAD yielding the final product, in a two- consists of a single α/β domain, closely resembling the step reaction catalyzed by the ubiquitous enzyme NAD ‘Rossmann fold’ topology, with a central parallel β sheet synthetase (NADS) [5] (Figure 1). Detailed structural flanked by several α helices on both sides [64••] (Figure 1). studies have been performed on the strictly ammonia- The ATP-binding site is well conserved in all NMNATs dependent B. subtilis NAD+ synthetase [71–73,74•]. The reported so far, with two structurally conserved motifs, enzyme is a functional homodimer with α/β subunit topology, GXXXPX(T/H)XXH and SXTXXR, providing the essential resembling the well-known ‘Rossmann fold’ [73]. Three residues for ATP recognition and stabilization [65•]. Mg2+ ions were reported to feature the ATP-binding site, 716 Catalysis and regulation

which is entirely located within a single subunit effective protection from solvent of the extremely labile [72,73,74•]. All three Mg2+ ions are octahedrally coordinated, iminoaspartate product, which is proposed to be channeled with a relevant contribution to the coordination sphere directly to the QA synthase active site, in a putative multi- from the ATP phosphate . A comparison of the enzyme complex. A different chemical demand is likely to free form and the ATP complex revealed large conforma- be met by the conformational changes reported for QAPRT tional changes, with two long loops disordered in the free and NMNAT. Here, the structural changes appear to be enzyme becoming fully structured upon ATP binding critical for determining substrate specificity. Moreover, in [71,73]. NaAD was reported to bind to an extended and QAPRT, such a structural rearrangement is proposed to fully solvent accessible cleft located at the subunit inter- control the order of substrate admission, to ensure product face. In NADS, the ATP-binding site is located at the release and to elicit productive catalysis. The same consid- classic topological switch point [where a dinucleotide is erations can be made for NADS, in which the large usually observed in Rossmann-fold-based NAD(P)- conformational changes observed upon ATP binding are binding protein] and a novel dinucleotide-binding site is thought to control product release and to represent the present [72,73]. An ammonium ion is proposed to be the rate-limiting step of the overall reaction. primary binding species, which subsequently undergoes a deprotonation process, delivering ammonia in situ through In the second part of the pathway, significant structural a still unclear mechanism [73,74•]. The catalytic strategy similarities can be detected between NADS and NMNAT. employed by NADS relies on a fundamental contribution These two enzymes adopt the dinucleotide-binding fold of the electron-withdrawing trimetallic constellation pre- (Rossmann fold), in keeping with the need to recognize sent in the active site for NaAD adenylation and on the mononucleotides (ATP and/or NaMN/NMN) and dinu- capability to efficiently discriminate between ammonium cleotides (NAD or NaAD). Moreover, the chemistry and water binding, allowing the reaction to proceed in carried out by the two enzymes is intriguingly similar in aqueous solution. The major conformational change that they both catalyze the strictly Mg2+-dependent observed upon substrate binding is likely to represent transfer of an AMP moiety onto a mononucleotide or the rate-limiting step of the overall reaction, ultimately dinucleotide substrate, yielding PPi as the leaving group controlling product release. (Figure 1). However, if NMNAT and NADS are super- posed based on the core structure of the ‘Rossmann fold’, Conclusions a striking difference in the ATP-binding mode becomes The structural studies carried out on NAD biosynthetic immediately evident (Figure 5a). The adenosine moiety of enzymes do not reveal conservation of a common fold ATP is located at two extreme opposites with respect to along the pathway, although the dinucleotide-binding the classic topological switch point in the two enzymes domain is present in three structures: LASPO, NMNAT (Figure 5a). On the other hand, the ATP phosphates and NADS. The absence of a recurring common fold along nicely overlap in NADS and NMNAT (Figures 5a). In the pathway is not surprising, as the chemical nature of the particular, a careful analysis of the PPi-binding site in the intermediates, as well as the chemistry carried out by the two enzymes reveals the conservation of a network of different enzyme, is extremely different along the pathway interactions stabilizing the diphosphate unit (Figure 5b,c). (Figure 1). However, some positive correlations can be In both enzymes, the PPi unit establishes several hydrogen detected for QAPRT, NMNAT and NADS, which show bonds with mainchain protein atoms belonging to the loop analogy in their chemical reactivity. All three enzymes connecting the first and the fourth parallel β strands 2+ yield PPi as a leaving group and need Mg for activity. (Figure 5b,c). Strikingly, both the SGGXDS signature fin- Strikingly, a binuclear Mg2+ cluster with octahedrally gerprint characterizing all NADSs [73] and the T/HXXH 2+ coordinated Mg ions located on either side of a PPi and SXTXXR signature fingerprints characterizing all moiety has been observed in both QAPRT and NADS. NMNATs play a remarkable role in PPi stabilization Such conservation of a binuclear cluster stresses the (Figure 5b,c). Considering that the chemical hotspot for major role played by the Mg2+ ions in the catalytic process, the catalytic event is centered in both enzymes on the facilitating intermediate formation or stabilizing the α-phosphate of ATP, it is tempting to speculate that the transition state by their electron-withdrawing power. two enzymes not only share their three-dimensional architecture but also a common strategy for catalysis. The A recurring theme throughout the NAD biosynthetic two enzymes seem to provide a well-suited active site con- pathway is the high degree of conformational flexibility figuration, fixing the conformation of the reactant groups, displayed by all four enzymes whose three-dimensional without any direct involvement of protein residues in cova- structures have been reported so far. Remarkable conforma- lent or acid/base catalysis. NMNAT and NADS could have tional changes mainly affecting loop regions lining the active evolved from a common ancestor, preserving the PPi-bind- site were observed in LASPO, QAPRT, NMNAT and ing site to preserve the chemical core of the original NADS. The most dramatic example is provided by LASPO, activity (i.e. the transfer of AMP onto a second substrate). in which two long loops and an entire domain move upon Subsequently, the specificity for a different substrate and cofactor binding. The most likely chemical reason for such a the chemistry necessary in NADS for the second step of dramatic conformational change is the requirement for the overall reaction were engineered. NAD and molybdenum cofactor biosynthesis Rizzi and Schindelin 717

Figure 5 legend Figure 5

Structural comparison between M. jannaschii NMNAT in complex with ATP (PDB code 1F9A; [64••]) and B. subtilis NADS in complex with (a) AMP-CPP, a nonhydrolyzable ATP analog (PDB code 1IHB; [71]). The ATP and the protein residues are represented in ball-and-stick form. (a) Superposition of NMNAT and NADS based on the Cα atoms of the five-stranded parallel β sheet of the ‘Rossmann fold’. The five parallel β strands in ribbon representation are depicted in yellow for NMNAT and in green for NADS. The ATP binds at the topological switch point in both structures, but with a completely different orientation (colored in yellow and green for NMNAT and NADS, respectively). (b) The ATP-binding site in M. jannaschii NMNAT. The key residues for ATP phosphate stabilization are shown. The two histidine residues, which are part of the strictly conserved HXXH sequence fingerprint, are colored in cyan. Arg121 and His16 are structurally equivalent to NADS Lys186 and Ser51, respectively. (c) The ATP-binding site in B. subtilis NADS. The key residues contacting the ATP phosphates are drawn. The two serine residues, which are part of the strictly conserved SGGXDS signature fingerprint, are colored in cyan.

Although several positive correlations were observed along (b) the route for NAD biosynthesis, with remarkable similarities Arg121 in the second part, it is hard to detect a common theme in the pathway. Similar molecules can bind to different binding sites and only when similarity in the chemical His16 reactivity is detected can common features emerge.

His13 Even more so than the enzymes involved in NAD biosyn- thesis, the proteins participating in Moco biosynthesis reveal a surprising degree of structural diversity. The only Gly126 clear structural relationship is between MogA and Arg8 domain III of MoeA, which most likely arose from a gene Thr127 duplication event. A third member sharing this fold is the MoaB protein, which, based on sequence similarities Arg130 alone, is predicted to have the same fold as MogA. There is, however, doubt whether this protein is actually func- tional, as insertion mutagenesis of the E. coli moaB gene (c) does not result in a clear phenotype. In addition, there is a very weak structural similarity between MoaC and MoaE, Ser51 the large subunit of MPT synthase. Another noteworthy aspect of the proteins involved in Moco biosynthesis is the Ser46 apparent requirement for the proteins to form oligomers (Figure 2). With the exception of MobA, all the other structurally characterized proteins are present in solution in higher oligomeric forms, including dimers in the case of Asn49 MoaE, MoeB and MoeA, a trimer in the case of MogA and Thr208 a hexamer in the case of MoaC. Although the active sites of these proteins have not been characterized in comparable detail to the enzymes involved in NAD biosynthesis, it appears that the oligomerization often reflects the require- ment to bring catalytic residues from different subunits into close spatial proximity at the respective active sites. Lys186

Current Opinion in Structural Biology The proteins involved in Moco biosynthesis demonstrate nicely how novel pathways can evolve based on related reactions, as in the case of the activation of MoaD and activating enzyme found exclusively in eukaryotes. A ubiquitin. The simple two-component system of MoaD MoaD/MoeB-like system is also involved in the incorpora- and MoeB, which is present in all kingdoms of life, appears tion of the thiazole sulfur during thiamine biosynthesis. In to represent the evolutionary ancestor of ubiquitin and its addition, the fact that MoaA and ThiH both belong to the 718 Catalysis and regulation

radical SAM superfamily indicates another aspect common cofactor in four enzymes from hyperthermophilic Archaea. J Biol Chem 1993, 268:4848-4852. to Moco and thiamine biosynthesis. A second evolutionary 14. Johnson MK, Rees DC, Adams MW: Tungstoenzymes. Chem Rev mechanism becomes evident from a phylogenetic compar- 1996, 96:2817-2840. ison of the proteins involved in Moco biosynthesis. This 15. Johnson JL, Wadman SK: Molybdenum cofactor deficiency. In The aspect is illustrated by the fusion of the MogA and MoeA Metabolic Basis Of Inherited Disease, edn 6. Edited by Scriver CR, proteins in eukaryotes, which resulted in the plant Cnx1 Beaudet AL, Sly WS, Valle DL. McGraw-Hill; 1989:1463-1475. and mammalian gephyrin proteins. Among those, gephyrin 16. Reiss J: Genetics of molybdenum cofactor deficiency. Hum Genet most dramatically demonstrates how novel functions can 2000, 106:157-163. be integrated into evolutionarily ancient proteins, in this 17. Johnson JL, Coyne KE, Rajagopalan KV, Van Hove JL, Mackay M, Pitt J, Boneh A: mutations in a mild case case leading to a multifunctional protein carrying out of molybdenum cofactor deficiency. Am J Med Genet 2001, essential functions during Moco biosynthesis and in the 104:169-173. anchoring of inhibitory neuronal receptors. 18. Reiss J, Christensen E, Kurlemann G, Zabot MT, Dorche C: Genomic structure and mutational spectrum of the bicistronic MOCS1 gene defective in molybdenum cofactor deficiency type A. Hum Genet Acknowledgements 1998, 103:639-644. H Schindelin thanks his co-workers Michael W Lake, Michael J Rudolph and Song Xiang for their important contributions, as well as MM Wuebbens 19. Reiss J, Cohen N, Dorche C, Mandel H, Mendel RR, Stallmeyer B, Mutations in a polycistronic nuclear gene and KV Rajagopalan for their remarkable collaborative efforts. M Rizzi Zabot MT, Dierks T: associated with molybdenum cofactor deficiency. 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