The crystal structure of adenylate kinase from maize ligated with an inhibitor has been determined by molecular replacement and refined to 3.5-Å resolution. The enzyme keeps the ATP/ADP/AMP equilibrium in the cell. In the C 4 plant maize, it has the special task to recycle the AMP produced in large amounts in primary CO 2 assimilation. The established structure explains the side reaction with CMP. Moreover, it shows infinite rods that can be readily discerned in the crystal packing. In comparison with homologues, two structural differences that are crucial for this supramolecular assembly are evident. We propose that the rods represent a natural inactive storage form that assembles at night when maize stops CO 2 assimilation and thus most of the AMP production in its C 4 cycle. The enzyme is particularly abundant in mesophyll chloroplasts, where such an assembly would release appreciable amounts of water that can be used in other processes during the night.Keywords : C 4 plant metabolism; crystal contact; dual substrate specificity; supramolecular structure ; X-ray structure analysis.Adenylate kinases are ubiquitous monomeric enzymes that tity) to the mammalian isoenzyme-2 from the mitochondrial intermembrane space. catalyze the reaction AKM has been isolated from leaves [3, 4, 10] as a homogeMg 2ϩ ATP ϩ AMP < Mg 2ϩ ADP ϩ ADP.neous protein containing 222 residues with a molecular mass of 24 867 Da [9]. Besides AMP, the enzyme accepts also CMP at They belong to the well-characterized family of nucleosidemonophosphate kinases [1,2]. Maize (Zea mays) is a C 4 plant a relative activity of 10% [4]. Here, we report the crystal structure of AKM in complex with the inhibitor [11] adenosineand contains a particularly large amount of adenylate kinase (AKM), i.e. about 50-times more than C 3 plants [3Ϫ5]. Like the (5′)pentaphospho(5′)adenosine (Ap 5 A), explain the CMP activity, and deduce a putative natural supramolecular assembly. other C 4 plants, maize needs a high catalytic efficiency because its CO 2 assimilation produces stoichiometric amounts of AMP in the mesophyll chloroplasts, which have to be recycled quickly EXPERIMENTAL PROCEDURES [5Ϫ7]. The respective reactions are:For crystallization, we used a screening system [12] with ATP ϩ P i ϩ pyruvate < phosphoenolpyruvate ϩ AMP ϩ PP i hanging drops. Crystals grew only with one of the enzyme prepwhere phosphoenolpyruvate fixes CO 2 according to the arations and only at one condition, where the drop was a 1 :1 following reaction: mixture of 7 mg/ml enzyme with 2 mM Ap 5 A in 10 mM Hepes, pH 7.5 and 1 M Li 2 SO 4 with about 10% polyethylene glycolphosphoenolpyruvate ϩ CO 2 < oxaloacetate ϩ P i .8000. The latter solution was also used as the reservoir, which Among the about 50 known sequences of this kinase family was not completely air-tight, however, when the crystals apthere are only two from plants: rice [8] and maize [9]. The AKM peared. X-ray data were collected at room temperature using a sequence finds its closest homologues (42% identity) in Escheri-multi-wire ...

This paper describes the sequence of adenylate kinase (Mg-ATP + AMP * Mg-ADP + ADP) from maize chloroplasts. This light-inducible enzyme is important for efficient CO, fixation in the C, cycle, by removing and recycling AMP produced in the reversible pyruvate phosphate dikinase reaction.The complete sequence was determined by analyzing peptides from cleavages with trypsin, Asp-N protease and CNBr and subcleavage of a major CNBr peptide with chymotrypsin. N-terminal Edman degradation and carboxypeptidase digestion established the terminal residues. Electrospray mass spectrometry confirmed the final sequence of 222 residues (Mr = 24867) including one cysteine and one tryptophan.The sequence shows this enzyme to be a long-variant-type adenylate kinase, the nearest relatives being adenylate kinases from Enterobacteriaceae. Alignment of the sequence with the adenylate kinase from Escherichiu coli reveals 44% identical residues. Since the E. coli structure has been published recently at 0.19-nm resolution with the inhibitor adenosine(5')pentaphospho(5')adenosine Mol. Biol. 224, 159-1771, catalytically essential residues could be compared and were found to be mostly conserved. Surprisingly, in the nucleotidebinding Gly-rich loop Gly-Xaa-Pro-Gly-Xaa-Gly-Lys the middle Gly is replaced by Ala. This is, however, compensated by an Ile+Val exchange in the nearest spatial neighborhood. A Thr-Ala exchange explains the unusual tolerance of the enzyme for pyrimidine nucleotides in the acceptor site.Adenylate kinases are small monomeric enzymes (21 -27 kDa) which catalyze the reversible transfer of a phosphoryl group from ATP to AMP according to the reaction:Mg-ATP + AMP * Mg-ADP + ADP [l]. Thereby, they perform the recycling of AMP produced in biosynthetic processes and enhance the energy charge signal in the cell. Although the sequences of some 30 adenylate kinases are known, as well as five well-resolved X-ray structures with different substrates in different conformations [2], so far we have little structural information on the enzymes from plants. Only recently two very similar cDNA sequences from rice were cloned, both showing the highest similarity to mammalian mitochondria1 AK2 [3].C, plants, like maize, have a particularly efficient CO, fixation by the C, cycle at the expense of ATP to AMP conversion in the pyruvate phosphate dikinase reaction. Because of the reversibility of the latter reaction, these plants need an efficient AMP recycling ; not surprisingly, an approximately 50-fold higher adenylate kinase (AK) content than in C, plants was found [4, 51. Genetic studies have shown that maize has only one AK gene, localized on chromosome 6Correspondence to E. Schiltz, Institut fur Organische Chemie und Biochemie, Albertstrasse 21, D-79104 Freiburg, GermanyAbbreviations. AK, adenylate kinase ; Ap5A, adenosine(5')pentaphospho(5')adenosine. Note. The sequence data will appear in the PIR Sequence Database under accession number S43039.[6] and that more than 90% of the light-inducible enzyme is found in mesophyll chloroplast...