Crystal Structure of Biotin Carboxylase in Complex with Substrates and Implications for Its Catalytic Mechanism

Chi-Yuan Chou,Linda P.C Yu,Liang Tong

doi:10.1074/jbc.m805783200

Chi-Yuan Chou, Linda P.C Yu + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m805783200

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Abstract

Biotin-dependent carboxylases are widely distributed in nature and have important functions in many cellular processes. These enzymes share a conserved biotin carboxylase (BC) component, which catalyzes the ATP-dependent carboxylation of biotin using bicarbonate as the donor. Despite the availability of a large amount of biochemical and structural information on BC, the molecular basis for its catalysis is currently still poorly understood. We report here the crystal structure at 2.0 A resolution of wild-type Escherichia coli BC in complex with its substrates biotin, bicarbonate, and Mg-ADP. The structure suggests that Glu(296) is the general base that extracts the proton from bicarbonate, and Arg(338) is the residue that stabilizes the enolate biotin intermediate in the carboxylation reaction. The B domain of BC is positioned closer to the active site, leading to a 2-A shift in the bound position of the adenine nucleotide and bringing it near the bicarbonate for catalysis. One of the oxygen atoms of bicarbonate is located in the correct position to initiate the nucleophilic attack on ATP to form the carboxyphosphate intermediate. This oxygen is also located close to the N1' atom of biotin, providing strong evidence that the phosphate group, derived from decomposition of carboxyphosphate, is the general base that extracts the proton on this N1' atom. The structural observations are supported by mutagenesis and kinetic studies. Overall, this first structure of BC in complex with substrates offers unprecedented insights into the molecular mechanism for the catalysis by this family of enzymes.

Highlights

27.2 Ϯ 4.3 0.59 Ϯ 0.08 0.10 Ϯ 0.01 our structure, and this conformational flexibility of the B domain could be stabilized upon the binding of BCCP
The further closure of the B domain in our structure has a dramatic impact on the bound position of the adenine nucleotide (Fig. 2C)
35.1 Ϯ 4.0 0.58 Ϯ 0.02 16.6 Ϯ 2.0 22.7 Ϯ 3.6 0.028 Ϯ 0.002 1.2 Ϯ 0.2 41.8 Ϯ 6.7 0.0025 Ϯ 0.0001 0.06 Ϯ 0.01 with that in the structure of the ATP complex [18], and the bound position of ADP is shifted by about 2 Å relative to that of ATP (Fig. 2C)

Summary

MATERIALS AND METHODS

Crystals of wild-type E. coli BC in complex with biotin, bicarbonate, and Mg-ADP were obtained at room temperature by. The reaction mixture contained 100 mM HEPES (pH 8.5), 0.5 mM ATP, 8 mM MgCl2, 40 mM biotin, 0.2 mM NADH, 0.5 mM phosphoenolpyruvate, 7 units of lactate dehydrogenase, 4.2 units of pyruvate kinase, and varying concentrations of bicarbonate. For assays that varied biotin concentration, the reaction mixture contained 100 mM HEPES (pH 7.4), 0.5 mM ATP, 8 mM MgCl2, 40 mM bicarbonate, 0.2 mM NADH, 0.5 mM phosphoenolpyruvate, 7 units of lactate dehydrogenase, and 4.2 units of pyruvate kinase. The crystallization condition was essentially the same as that for the wild-type BC, and the crystal was soaked overnight in a solution containing 2.5 mM Mg-ADP, 200 mM biotin, 10 mM bicarbonate, 23% (w/v) polyethylene glycol 3350, 0.05 M Li2SO4, and 2.1% (w/v) sorbitol.

RESULTS AND DISCUSSION

Km mM

With biotin as the varying substrate