A minimal peptide substrate in biotin holoenzyme synthetase-catalyzed biotinylation

E. coli BirA transfers biotin from the biotinyl-5'-adenylate (bio-5'-AMP) intermediate to the target lysine of a 23-residue peptide (peptide 85, selected from a combinatorial library biased toward a consensus biotinylation sequence) at the same rate as to the natural substrate BCCP87, the 87-residue C-terminal fragment of E. coli biotin carboxyl carrier protein. With peptide 85 fused to the N-terminus of maltose-binding protein (MBP), the apparent first-order rate at 10 µM acceptor is 0.112 ± 0.002 s-1, against 0.107 ± 0.001 s-1 for BCCP87 (20 °C, pH 7.5, 1 µM 5-hydroxytryptophan-labeled BirA, 0.9 µM bio-5'-AMP).

Free synthetic peptide 85 is biotinylated by BirA at essentially the same apparent first-order rate as BCCP87 over 10-20 µM acceptor: 0.086 ± 0.003 vs 0.091 ± 0.003 s-1 at 10 µM, 0.126 ± 0.004 vs 0.115 ± 0.003 s-1 at 15 µM, and 0.17 ± 0.01 vs 0.148 ± 0.005 s-1 at 20 µM (20 °C, 0.9 µM BirA-bio-5'-AMP complex), so the biotin acceptor activity resides in the peptide sequence alone.

MBP fusions of peptide 37 (from a completely random library) and peptide 45 (from a library biased to resemble natural biotin acceptors) show no measurable activity in the stopped-flow assay for BirA-catalyzed biotin transfer, although both are detectably biotinylated in vitro and in vivo, so their biotinylation is much slower than that of BCCP87 or the peptide 85 fusion. A peptide sequence selected from a library biased toward the consensus biotinylation sequence (peptide 85) is the best of the three BirA substrates.

In truncations of the 23-residue peptide 85, deleting amino-terminal residues up to position -9 relative to the biotinylated lysine leaves the BirA biotin-transfer rate essentially unchanged, and removing the glycine at position -9 sharply reduces it. Deleting carboxy-terminal residues up to position +7 also leaves the rate similar, deleting the aspartate at position +6 gives a higher rate than any other peptide in the series or the natural substrate, and further C-terminal truncation gradually reduces the rate.

The 14-residue truncation of peptide 85 (peptide 85-14) is quantitatively biotinylated (fraction modified 1.00) by 1 µM BirA with 200 µM bio-5'-AMP in 1 h at 20 °C, while the truncation 85-6 is not biotinylated (fraction modified ~0) as judged by MALDI-TOF mass spectrometry. Peptides that are poor substrates in the stopped-flow kinetic assay are biotinylated to very low levels, whereas peptides biotinylated at rates comparable to BCCP are converted to the biotinylated form.

The 14-residue peptide 85-14 is a kinetic mimic of the BCCP domain for BirA: the specificity constant kcat/Km for biotin transfer is 10,000 ± 500 M-1 s-1 for peptide 85-14 and 11,900 ± 400 M-1 s-1 for BCCP87, measured with 5-hydroxytryptophan-labeled BirA by nonlinear least-squares fitting of rate against substrate concentration (20 °C, pH 7.5). The 14-mer is a minimal BirA acceptor, far smaller than the BCCP domain.

A BirA acceptor peptide containing the peptide 85 core sequence functions when fused at the C-terminus of a protein. The 14-residue minimal peptide fused to the C-terminus of MBP through a TGG linker (after the Factor Xa site of pMAL-c2) is biotinylated in E. coli, and its initial biotin-transfer rate with 5-hydroxytryptophan-labeled BirA is indistinguishable from that of the free 14-mer.

The BirA minimal acceptor peptide shares little sequence similarity with the naturally biotinylated BCCP sequence. Only four positions relative to the target lysine (-2, -3, -5 and +2) carry residues that are also frequent in natural substrates, with E at -3, A at -2 and E at +2 in the minimal peptide. In the BCCP structure these positions lie on one face of the surface formed by the beta-turn that contains the target lysine.

The 15-residue peptide GLNDIFEAQKIEWHE, based on the C-terminal sequence of peptide 85-11, is proposed as the optimal biotin acceptor fusion partner for BirA, because it shows a consistently higher biotinylation rate than the 14-mer. At a protein C-terminus it is connected through at least a GG linker, and at an N-terminus it must be preceded by a Met start codon and a second codon such as the Ala codon of the MA start (ATGGCT).

Binding of bio-5'-AMP quenches the intrinsic fluorescence of E. coli BirA by about 40%, and binding of an acceptor followed by biotin transfer restores it. This allows biotin transfer to be followed by stopped-flow fluorescence, and for tryptophan-containing acceptors such as MBP fusions the Trp residues of BirA can be replaced with 5-hydroxytryptophan so that excitation at 316 nm avoids acceptor fluorescence.