## 2 Hop Bitter Acids
### Carboxyl CoA Ligase (CCL)  
BCAAs are degraded in the mitochondria of plant cells, which has been extensively established [44]. However, because these branched short-chain acyl-CoAs must migrate to the cytosol from the mitochondria, they must be reliant on the downstream type III polyketide synthase's cytosolic localization. A CoA ligase is considered to be essential in hop trichomes to create and maintain a pool of branched short-chain acyl-CoA for bitter acid synthesis [45]. A putative CoA ligase unigene was originally found which assembled with 32 expressed sequence tags (ESTs) [46], and it potentially could be involved in the production of branched short-chain acyl-CoAs. The in vivo activity of the HICCL2/HICCL4 (carboxyl CoA ligase in _Humulus Lupulus_) was verified by a successful reconstruction of  
Figure 2: Crystal structure of BCAT enzyme catalyzing the BCAA degradation (PDB: 5 BWR). The different colors indicate the two-domain structures of the BCAT subunit. The arrow interface represents the position of the domain interface.  
the initial steps of the bitter acid pathway by co-introducing HLCCL2/HLCCL4 and HIVPS (valerophenone synthase in _Humulus Lupulus_) in a yeast system. HLCCL2 had a high specific activity for isovaleric acid; however, HLCCL4 had a high specific activity for isobutyric acid (\(K_{\rm cat}/K_{\rm m}>10^{3}\) s\({}^{-1}\) M\({}^{-1}\)). Simultaneously, an engineered yeast that generated \(\beta\)-bitter acids was established by expressing the complete pathway genes for bitter acid biosynthesis, including HLCCL2 and HLCCL4 [47]. Besides the enzymes CCL2 and CCL4 involved in the bitter acid biosynthesis pathway, CCL1 (\(\rho\)-coumaroyl-CoA ligase) catalyzed the conversion of 4-coumaric acid to 4-coumaroyl-CoA.  
The acyl activating enzymes (AAEs) superfamily of carboxyl-CoA ligases and associated proteins has diversified in higher plants to supply enzymes for numerous primary and secondary metabolic pathways, as well as hormone conjugation to amino acids [48]. The catalytic mechanism of carboxylate compounds is activated by CoA ligases in two stages [49]: the carboxylic acid group is adenylated to generate the acyl-adenosine 5\({}^{\prime}\)-monophosphate (acyl-AMP) intermediary, then AMP is displaced with CoA to generate the matching thioester and AMP. Although there is no precise information on the structure of this enzyme in hops, crystal structures from other plants are available. In _Populus lometose_, the enzyme (PDB: 3 NI2, Figure 4) consists of two globular domains connected by a flexible linker region [50] (Table 1). The larger N-domain contains a substrate-binding pocket, while the C-domain contains catalytic residues. Residues essential for catalytic activities (Lys-438, Gln-443, and Lys-523) and substrate binding (Tyr-236, Gly-306, Gly-331, Pro-337, and Val-338) were identified. The size of the binding site has been shown to be the most  
Figure 3: Crystal structure of branched-chain keto-acid dehydrogenase catalyzing oxidative decarboxylation (PDB: 2 BP7). The different colors indicate the different chains. While the black circle represents the position of the PSBD.  
critical determinant in defining CCL's substrate specificities [50]. Crystal structures of CCL in both conformations have been obtained in _Nicotiana tabacum_, with the adenylation conformation structure revealing an organized P loop and bound nucleotide. In addition, designing a Val-341 deletion gives the enzyme the capacity to use inapinate, and structural data provide a mechanistic explanation for this flip in selectivity [51].