Title: Structural Analysis of Substrate, Reaction Intermediate, and Product Binding in Haemophilus influenzae Biotin Carboxylase

Abstract

Acetyl-CoA carboxylase catalyzes the first and regulated step in fatty acid synthesis. In most Gram-negative and Gram-positive bacteria, the enzyme is composed of three proteins: biotin carboxylase, a biotin carboxyl carrier protein (BCCP), and carboxyltransferase. The reaction mechanism involves two half-reactions with biotin carboxylase catalyzing the ATP-dependent carboxylation of biotin-BCCP in the first reaction. In the second reaction, carboxyltransferase catalyzes the transfer of the carboxyl group from biotin-BCCP to acetyl-CoA to form malonyl-CoA. In this report, high-resolution crystal structures of biotin carboxylase from Haemophilus influenzae were determined with bicarbonate, the ATP analogue AMPPCP; the carboxyphosphate intermediate analogues, phosphonoacetamide and phosphonoformate; the products ADP and phosphate; and the carboxybiotin analogue N1'-methoxycarbonyl biotin methyl ester. The structures have a common theme in that bicarbonate, phosphate, and the methyl ester of the carboxyl group of N1'-methoxycarbonyl biotin methyl ester all bound in the same pocket in the active site of biotin carboxylase and as such utilize the same set of amino acids for binding. Here, this finding suggests a catalytic mechanism for biotin carboxylase in which the binding pocket that binds tetrahedral phosphate also accommodates and stabilizes a tetrahedral dianionic transition state resulting from direct transfer of CO₂ from the carboxyphosphate intermediate tomore » biotin.« less

Authors:

Broussard, Tyler C. ^[1]; Pakhomova, Svetlana ^[1]; Neau, David B. ^[2]; Bonnot, Ross ^[1]; Waldrop, Grover L. ^[1]

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+ Show Author Affiliations

1. Louisiana State Univ., Baton Rouge, LA (United States)
2. Argonne National Lab. (ANL), Argonne, IL (United States)

Publication Date:

Thu May 28 00:00:00 EDT 2015

Research Org.:

Argonne National Laboratory (ANL), Argonne, IL (United States)

Sponsoring Org.:

USDOE; National Inst. of Health

OSTI Identifier:

1186930

Grant/Contract Number:  

AC02-06CH11357; P41 GM103403; S10 RR029205

Resource Type:

Accepted Manuscript

Journal Name:

Biochemistry

Additional Journal Information:

Journal Volume: 54; Journal Issue: 24; Journal ID: ISSN 0006-2960

Publisher:

American Chemical Society (ACS)

Country of Publication:

United States

Language:

ENGLISH

Subject:

59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; nutrition; phosphates; organic compounds; peptides and proteins; alkyls