1792 Biochemical Society Transactions (2014) Volume 42, part 6 Fructose-1,6-bisphosphate aldolase (FBA) – a conserved glycolytic enzyme with virulence functions in bacteria: ‘ill met by moonlight’ Fariza Shams*, Neil J. Oldfield*, Karl G. Wooldridge* and David P.J. Turner* 1 *Molecular Bacteriology and Immunology Group, School of Life Sciences, Centre for Biomolecular Sciences, University of Nottingham, Nottingham NG7 2RD, U.K. Abstract Moonlighting proteins constitute an intriguing class of multifunctional proteins. Metabolic enzymes and chaperones, which are often highly conserved proteins in bacteria, archaea and eukaryotic organisms, are among the most commonly recognized examples of moonlighting proteins. Fructose-1,6-bisphosphate aldolase (FBA) is an enzyme involved in the Embden–Meyerhof–Parnas (EMP) glycolytic pathway and in gluconeogenesis. Increasingly, it is also recognized that FBA has additional functions beyond its housekeeping role in central metabolism. In the present review, we summarize the current knowledge of the moonlighting functions of FBA in bacteria. Introduction Fructose-1,6-bisphosphate aldolase (FBA) (EC 4.1.2.13) catalyses the cleavage of fructose 1,6-bisphosphate (FBP) into glyceraldehyde 3-phosphate and dihydroxyacetone phosphate (DHAP) or the reverse aldol condensation reaction. This is an indispensable enzyme for glycolysis, gluconeogenesis and the Calvin cycle [1]. FBAs are classified according to the reaction mechanism employed. Class I FBAs act by forming a Schiff-base intermediate between the amino group of a lysine residue in the active site and the C-2 carbonyl group of the substrate (DHAP or FBP). Class I enzymes are mainly found in animals, plants and green algae and only occasionally in bacteria [1–3]. The enzyme forms tetramers in eukaryotes but more variable forms in bacteria [4–6]. Conversely, class II FBAs are mainly found in bacteria and require a divalent metal ion at the active site [7–9]. Mycobacterium tuberculosis and Escherichia coli are among the few organisms containing both classes of FBAs [10,11], whereas two distinct class II enzymes are encoded on the chromosome and on a plasmid respectively, of the thermo- tolerant Gram-positive methylotroph Bacillus methanolicus [12]. On the basis of the amino acid sequence, class II aldolases can be further divided into type A and type B [4]. Type A enzymes are dimeric in contrast with the type B enzymes, which may be dimeric, tetrameric or octameric [2,13]. It has been reported that type A enzymes are mostly involved in glycolysis and gluconeogenesis, while diverse metabolic roles and substrate specificities have been reported for type B Key words: adhesion, bacteria, fructose bisphosphate aldolase (FBA), glycolytic enzyme, moonlighting protein, plasminogen. Abbreviations: CAM, cell-adhesion molecule; DHAP, dihydroxyacetone phosphate; EACA, ε-aminocaproic acid; FBA, fructose-1,6-bisphosphate aldolase; FBA-nm, FBA of Neisseria meningitidis; FBA-tb, FBA of Mycobacterium tuberculosis; FBP, fructose-1,6-bisphosphate; FCR, flamingo cadherin receptor; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; rFBA, recombinant fructose-1,6-bisphosphate aldolase. 1 To whom correspondence should be addressed (email david.turner@nottingham.ac.uk). aldolases [2]. Although both class I and class II aldolases have the same (α/β ) 8 barrel-fold (also known as a triose phosphate isomerase or TIM barrel), similar molecular masses (∼40 kDa), and catalyse the same enzymatic reactions, they do not have significant amino acid sequence homology [14,15] or conserved catalytic residues [16]. Moreover, their active sites are located in different parts of the TIM barrel, suggestive of independent evolution from a common ancestor with the possibility of some large insertions or deletions separating the two groups [17,18]. A common evolutionary history is further suggested by the aldolases found in the archaea, which belong to class I based on their reaction mechanism, but lack significant amino acid sequence homology with other class I or class II aldolases [6]. Hyperthermophilic archaea also possess a bifunctional FBA/phosphatase enzyme which is a structurally unique class V enzyme with FBA activity [19]. Overview of moonlighting proteins Moonlighting proteins are a group of proteins which perform two or more autonomous, often dissimilar functions, using a single polypeptide chain, rather than distinct protein domains [20,21]. There are examples of moonlighting proteins in yeast [22], plants [23], vertebrates [24] and bacteria [25]. Many of these proteins are conserved in bacteria and eukaryotic organisms and are often recognized as enzymes of central metabolism or as molecular chaperones, but can be involved in an array of intracellular biochemical functions [25]. It is now recognized that bacterial moonlighting pro- teins may impart virulence properties to bacterial and fungal pathogens including M. tuberculosis, Streptococcus pyogenes, Streptococcus pneumoniae, Staphylococcus aureus, Helicobacter pylori and Candida albicans. Moonlighting proteins initially thought to be solely located in the cytoplasm are increasingly being found on the surface of C  The Authors Journal compilation C  2014 Biochemical Society Biochem. Soc. Trans. (2014) 42, 1792–1795; doi:10.1042/BST20140203 Biochemical Society Transactions www.biochemsoctrans.org