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

Journal Article · Thu Jan 01 00:00:00 EST 2009 · Journal of Biological Chemistry

DOI:https://doi.org/10.1074/jbc.M805783200· OSTI ID:980100

Chou, C; Yu, L; Tong, L

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 {angstrom} resolution of wild-type Escherichia coli BC in complex with its substrates biotin, bicarbonate, and Mg-ADP. The structure suggests that Glu{sup 296} is the general base that extracts the proton from bicarbonate, and Arg{sup 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-{angstrom} 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.

• Research Organization: Brookhaven National Lab. (BNL), Upton, NY (United States). National Synchrotron Light Source
• Sponsoring Organization: Doe - Office Of Science
• DOE Contract Number: DE-AC02-98CH10886
• OSTI ID: 980100
• Report Number(s): BNL-93018-2010-JA; JBCHA3; TRN: US201015%%1485
• Journal Information: Journal of Biological Chemistry, Vol. 284; ISSN 0021-9258
• Country of Publication: United States
• Language: English

Related Subjects

36 MATERIALS SCIENCE
59 BASIC BIOLOGICAL SCIENCES
99 GENERAL AND MISCELLANEOUS//MATHEMATICS, COMPUTING, AND INFORMATION SCIENCE
ACID CARBONATES
ADENINES
ATOMS
ATP
AVAILABILITY
BASES
BIOTIN
CARBOXYLASE
CARBOXYLATION
CATALYSIS
CRYSTAL STRUCTURE
DECOMPOSITION
ENZYMES
ESCHERICHIA COLI
FUNCTIONS
INFORMATION
KINETICS
MUTAGENESIS
NUCLEOTIDES
OXYGEN
PHOSPHATES
PROTONS
RESIDUES
RESOLUTION
SUBSTRATES
national synchrotron light source