## 2 Hop Bitter Acids
### Branched-Chain Aminotransferase (BCAT)  
In plants, BCAA biosynthesis consists of eight enzymatic steps that generate leucine and valine from pyruvate, or isoleucine from threonine and pyruvate, respectively [21]. BCAT enzymes (BCATs) are essential for the production of branched-chain acyl-CoAs, as they catalyze both the final step in BCAA biosynthesis and the first step in BCAA degradation. BCATs have been identified in Arabidopsis and tomato, with six and seven isoforms, respectively [22; 23; 24].  
Two distinct BCATs were studied in _Humulus lupulus_ (HIBCAT1 and HIBCAT2) with gene expression quantification by RNA-seq and qRT-PCR [20]. The results indicated that HIBCAT1 was more highly expressed in cones than in leaves, whereas HIBCAT2 was found in glands, cones, and leaves, confirming that HIBCAT1 was glands specific [17]. HIBCAT1 and HIBCAT2 were localized to the mitochondrion and plastid, respectively. The mature forms of HIBCAT1 and HIBCAT2 were produced as his-tagged recombinant proteins in _Escherichia coli_ and purified. HIBCAT1 is a 393 amino acid protein with an estimated molecular weight of 43.2 kDa, whereas HIBCAT2 is a 44.5 kDa (408 amino acid) protein with a 44.5 kDa calculated molecular weight. Enzymatic assays indicated the hop BCATs had similar \(K_{\text{m}}\) values in both the forward (anabolic, e.g., leucine to 2-ketoisocaproate) and reverse (catabolic, e.g., 2-ketoisocaproate to leucine) directions. Unlike previous research on tomatoes, the BCATs demonstrated kinetic selectivity for anabolic or catabolic activities [23].  
The first crystal structure of BCAT in the pyridoxal 5\({}^{\prime}\)-phosphate (PLP) form was explained in _Escherichia coli_ utilizing isomorphous replacement [25], which is a homo hexamer structure with D symmetry. Each subunit's polypeptide chain is folded into two domains (small and large domains). Pyridoxal PLP, a coenzyme, is found at the domain interface, with its _re_-face facing the protein. Hereunder are the active sites in BCAT that recognize branched-chain amino acids and glutamate as substrates: (1) Phe-36, Tyr-164, Tyr-31\({}^{\star}\), and Val-109\({}^{\star}\), which creates a hydrophobic core for a branched-chain; (2) Arg-97 for an acidic side chain of glutamate; and (3) Tyr-95 and two major chains NH groups of Thr-257 and Ala-258 for a-carboxylate of substrates. Currently, there are around 50 BCAT enzyme structures in the protein databank, including ones from _Homo sapiens_ (PDB code 5 BWR, Figure 2) [26], _Mycobacterium tuberculosis_ (PDB code 3 HT5) [27], and Archaea [28]. The two-domain structure of the BCAT subunit with the active site located at the domain interface are the common features of these BCATs, and thus a dimer is a catalytically competent unit [29]. Almost all known BCATs are homodimers in solution, with the exception of those from _Escherichia coli_ and _Salmonella typhimurium_, which are homohexamers. BCATs exhibit a variety of quaternary structures in crystals, including homohexamers [30], homotetramers [30], or homodimers [27; 29; 31; 32].  
Hops are one of just a few plants that produce substantial amounts of BCAA-derived natural products in specialized secretory structures; therefore, exploring BCAA metabolism is particularly significant. Some Solanaceae species, for example, use BCAAs in trichromocalized acyl sugar synthesis [33]. However, few studies on the impact of BCATs on the synthesis of bitter acids in hops are available.