Characterization of HphA: The First Enzyme in the Homologation Pathway of l-Phenylalanine and l-Tyrosine

Abstract

Homologation of amino acids is the insertion or deletion of a methylene group to their side chain, which is a relatively uncommon chemical transformation observed in peptide natural product (NP) structure. Homologated amino acids can potentially make the NP more stable in a biological system, but its biosynthesis is yet to be understood. This study biochemically characterized the first of three unexplored enzymes in the homologation pathway of l-phenylalanine and l-tyrosine. Previously proposed reactions catalyzed by HphA were confirmed by reversed-phase high-performance liquid chromatography and tandem mass spectrometry analysis. The substrate profile and kinetic parameters showed high selectivity for the natural substrates and their close analogs. The comparability of HphA to homologous enzymes in primary metabolic pathways, 2-isopropylmate synthase and homocitrate synthase which are involved in l-leucine and l-lysine biosynthesis, respectively, was validated by bioinformatical and site-directed mutagenesis studies. The knowledge obtained from this study has deepened the understanding of the homologation of amino acids, which can lead to future combinatorial biosynthesis and metabolic engineering studies.

Keywords: Anabaenopeptins; Biosynthesis; Enzymology; Homologation; Natural products.

Figure 1.

Proposed individual enzymatic reactions and intermediates in the homologation of l-Phe (R = H) and l-Tyr (R = OH). The red-colored enzymes are encoded in the biosynthetic gene cluster of anabaenopeptins produced by N. punctiforme PCC 73102.^[4] The enzyme utilized in this study HphA is bolded. The second steps of HphA (hydrolysis) and HphB (decarboxylation) are non-enzymatic spontaneous reactions. Each abbreviation depicts the following: ArAT = aromatic amino acid aminotransferase, PPA = phenylpyruvic acid, 4HPPA = 4-hydroxyphenylpyruvic acid, B2HB = 2-benzyl-2-hydroxybutanedioic acid, 4HB2HB = 2-(4-hydroxybenzyl)-2-hydroxybutanedioic acid, B3HB = 2-benzyl-3-hydroxybutanedioic acid, 4HB3HB = 2-(4-hydroxybenzyl)-3-hydroxybutanedioic acid, OPB = 2-oxo-4-phenylbutanoic acid, 4HPOB = 4-(4-hydroxyphenyl)-2-oxobutanoic acid, PLP = pyridoxal 5’-phosphate, CoA = coenzyme A, NAD(P)⁺ = nicotinamide adenine dinucleotide (phosphate).

Figure 2.

RP-HPLC analysis to observe the consumption of the substrate PPA by HphA. The solution A (the synthetic standard) contains PPA, B (reaction solution) contains HphA C-His, PPA, and AcCoA, C (reaction without HphA) contains PPA and AcCoA, and D (reaction without AcCoA) contains HphA and PPA.

Figure 3.

MS/MS analysis for the product of the reaction catalyzed by HphA C-His. A. The commercially available 4HB2HB. B. Extracted sample from the HphA reaction solution. C. Possible fragmentation patterns on 4HB2HB. Because of two hydroxy groups and two carboxyl groups in 4HB2HB structure, fragmentation of one of the two functional groups are indistinguishable. A number of hydrogens cleavable from the hydroxy and carboxyl group might make the observed mass −1 or −2 from the calculated mass. The molecular formulas with labels “a”-“e” correspond to the peaks on the chromatograms.

Figure 4.

Substrate profile of HphA. The natural substrates are colored red. Each abbreviation depicts the following: PPA = phenylpyruvic acid, 4HPPA = 4-hydroxyphenylpyruvic acid, 3HPPA = 3-hydroxyphenylpyruvic acid, 4MeOPPA = 4-methoxyphenylpyruvic acid, hPPA = homologated pyruvic acid (2-oxo-4-phenylbutanoic acid), InPA = Indole-3-pyruvic acid, ImPA = 4-imidazolepyruvic acid, MOBA = 3-methyl-2-oxobutanoic acid, PA = pyruvic acid, HPA = hydroxypyruvic acid, OAA = oxalacetic acid, αKG = α-ketoglutaric acid. The error bars are the range of the data (n = 2).

Figure 5.

A. The active site of the enzyme in the structure of homocitrate synthase-α-KG-CoA complex (PDB ID: 6KTQ).^[16] The residues in parentheses are those in HphA. The ligands and interacting residues are shown as sticks, and their polar interactions are represented by dotted lines. The catalytic Zn²⁺ and a coordinated water molecule are shown by magenta and cyan balls, respectively. B. Amino acid sequence alignment with HphA, Neisseria meningitidis LeuA (PDB ID: 3RMJ),^[18] Cytophaga hutchinsonii LeuA (PDB ID: 3EEG),^[19] and Listeria monocytogenes LeuA (PDB ID: 3EWB).^[20] The purple triangles depict conserved amino acid residues in the Zn²⁺ binding site, and the green triangles depict non-conserved amino acid residues that interact with the side chain of the substrate shown in Figure 5A.

Figure 6.

Comparison of the activity between HphA wild type (WT) and mutants. The error bar for WT is the standard deviation (n = 4), and the error bars for mutants are the range of the data (n = 2).