Active site comparisons and catalytic mechanisms of the hot dog superfamily.

In 1996, Leesong et al. published the structure of FabA (PDB ID:1MKA), the dehydratase–isomerase enzyme involved in the bacterial type II fatty acid synthase (FAS) system from E. coli.1 The authors described a central, long α-helix “hot dog” surrounded by a bent β-sheet “bun” and so dubbed this the “hot dog fold” (Figure 1). Since that time, roughly 60 proteins have been crystallized and found to have the hot dog fold.2 The core fold topology can be described as an antiparallel β-sheet ordered 1-3-4-5-2. The long hot dog helix is located between strands β1 and β2 (Figure 2D). Some enzymes contain additional β-strands in their “bun”. Figure 1 The Hot Dog Fold Figure 2 Three Types of Hot Dog Folds This enzyme fold serves as a scaffold to execute a variety of reactions involving fatty acid or polyketide thioesters and is found in enzymes of both fatty acid catabolism and anabolism as well as in enzymes of natural product biosynthetic pathways. Every enzyme in this family containing a single hot dog (SHD) fold dimerizes (Figure 2A and D), and two identical active sites are found at the dimeric interface, with key residues in each active site residing on both monomers. Depending on the enzyme and species, these homodimers (protomers) themselves often associate into dimers of dimers or trimers of dimers. (For an excellent review of the various oligomeric states of hot dog enzymes, see Pidugu et al.2) Some hot dog enzymes have two hot dog motifs — thought to arise from gene duplication — and these double hot dog (DHD) enzymes (or domains) have two SHD subdomains that are fused together. In DHD structures, one of the active sites, which both reside at the pseudodimeric interface, has become inactive (Figure 2B and E). The hot dog helix of the C-terminal domain is often kinked; the one in the N-terminal domain remains linear. The oligomeric states of these pseudodimeric enzymes also vary. There are even instances of triple hot dog (THD) enzymes; fungal FAS dehydratase domains, for example, contain such a fold.3,4 In a THD enzyme, the third hot dog region is a domain insertion between the first and second hot dog motifs, which associate with each other in similar manner to the two motifs of a DHD enzyme, including the bent, C-terminal helix. The added domain is highly distorted, such that the hot dog helix is much shorter. It no longer locates within the β-sheet bun and is instead found alongside the β-sheet (Figure 2C and F). THD enzymes also only have a single active site at the pseudodimeric interface of the “normal” hot dog regions. The significance of only one hot dog helix remaining linear in both DHD and THD folds will be discussed later in this review. The secondary structural arrangements of idealized SHD, DHD, and THD folds are compared in the topology diagrams depicted in Figure 2D, E, and F. Because different enzymes have extra helices and strands in addition to the core hot dog and bun shown in the topology diagrams, for the purposes of comparing hot dog folds, we have formulated the following system: Core β-strands are labeled with an Arabic numeral in the same order as they occur in the sequence. Strands found in the second subdomain of DHD folds are treated as if they reside on an SHD with the further designation of a prime symbol. The strands of the inserted third hot dog motif of the THDs are designated with a double prime. Any extra strands in addition to these may be designated with an “x” followed by an Arabic numeral. The hot dog helices of the first, second, or third motifs are labeled αHD, αHD′, αHD″, respectively; additional helices are named by “x” followed by a capital letter. This review will use this notation throughout. The hot dog superfamily includes over a thousand enzymes or domains and has been divided into at least 17 subfamilies based on primary sequence analysis — although this analysis did not capture the majority of DHD and THD enzymes, including animal and fungal FAS domains.5 Hot dog folds are found or predicted in archaea, bacteria, and eukaryotes. Functionally, however, the majority of the superfamily can be primarily divided into two groups: those enzymes performing either dehydration or hydration of thioester substrates and those performing thioester hydrolysis.