Advances in 4-Hydroxyphenylacetate-3-hydroxylase Monooxygenase

Abstract

Catechols have important applications in the pharmaceutical, food, cosmetic, and functional material industries. 4-hydroxyphenylacetate-3-hydroxylase (4HPA3H), a two-component enzyme system comprising HpaB (monooxygenase) and HpaC (FAD oxidoreductase), demonstrates significant potential for catechol production because it can be easily expressed, is highly active, and exhibits ortho-hydroxylation activity toward a broad spectrum of phenol substrates. HpaB determines the ortho-hydroxylation efficiency and substrate spectrum of the enzyme; therefore, studying its structure-activity relationship, improving its properties, and developing a robust HpaB-conducting system are of significance and value; indeed, considerable efforts have been made in these areas in recent decades. Here, we review the classification, molecular structure, catalytic mechanism, primary efforts in protein engineering, and industrial applications of HpaB in catechol synthesis. Current trends in the further investigation of HpaB are also discussed.

Keywords: 4-hydroxyphenylacetate-3-hydroxylase monooxygenase; biocatalysis; catechols; protein engineering; structural property.

Figure 4

Tetramer and monomer structures of (a) TtHpaB (PDB: 2YYJ), (b) EcHpaB (PDB: 6QYI), and (c) AbHpaB (PDB: 2JBT). The N-terminal domain is pale cyan, intermediate domain is pink, C-terminal domain is light blue, additional C-terminal α-helix is orange, and FAD and FMN is magenta.

Figure 5

Structure-based sequence alignment of HpaBs from different bacteria. The secondary structural elements are indicated at the bottom of the alignment (Alignment was performed by using ESPript 3 [50]).

Figure 6

Proposed mechanism of the TtHpaB catalytic reaction.

Figure 1

Typical examples of natural products and drugs with catechol substructure (The red represents the position of ortho-hydroxylation.).

Figure 2

Scheme of the 4HPA3H-intiated reaction.

Figure 3

Phylogenetic tree of different HpaB sources. The red triangle represents the HpaB whose crystal structure has been resolved. (Phylogenetic tree was constructed by using MEGAX based on the neighbor-joining algorithm [48]).

Figure 7

HpaB-involved synthetic molecular pathways for caffeic acid. TAL, tyrosine ammonia-lyase.

Figure 8

HpaB-involved synthetic molecular pathways for caffeic alcohol. TAL, tyrosine ammonia lyase; 4CL1, p-coumarate-CoA ligase; CCR, cinnamoyl-CoA reductase; ADH6, alcohol dehydrogenase.

Figure 9

HpaB-involved synthetic molecular pathways for hydroxytyrosol. Route 1 (blue), Route 2 (yellow), and Route 3 (green). TyrB, aromatic-amino-acid aminotransferase; KDC, ketoacid decarboxylase; ADH, alcohol dehydrogenase; EER, endogenous E. coli reductase; YahK, aldehyde reductase; TDC; tyrosine decarboxylase; TYO, tyramine oxidase.

Figure 10

HpaB-involved synthetic molecular pathways for hydroxytyrosol acetate. AroG, 3-deoxy-D-arabinoheptulosonate-7-phosphate synthase; TyrA, chorismate mutase/prephenate dehydrogenase; KDC, 2-keto acid decarboxylase; YeaE, aldehyde reductases; ATF1, alcohol acetyltransferase.

Figure 11

HpaB-involved synthetic molecular pathways for other catechol-containing compounds. The synthesis route of eriodictyol (blue zone): TAL, tyrosine ammonia lyase; 4CL, 4-coumaroyl-CoA ligase; CHS, chalcone synthase; and CHI, chalcone isomerase. The synthesis route of L-dopa (rose zone). The synthesis route of 3,4-dihydroxymandelic acid (yellow zone): HmaS, hydroxymandelate synthase. The synthesis route of salvianic acid A (green zone): D-LDH, D-lactate dehydrogenase.