Crystal Structures and Mutational Analyses of Acyl-CoA Carboxylase β Subunit of Streptomyces coelicolor

ANA ARABOLAZA, MARY ELIZABETH SHILLITO,TING-WAN LIN, LAUTARO DIACOVICH, MELROSE MELGAR, HUY PHAM, DEBORAH AMICK, HUGO GRAMAJO* AND SHIOU-CHUAN TSAI* G., AND GRAMAJO H.

BIOCHEMISTRY

AMER CHEMICAL SOC

Lugar: Washington; Año: 2010 p. 7367 - 7376

0006-2960

ABSTRACT: The first committed step of fatty acid and polyketides biosynthesis, the biotin-dependent 11 carboxylation of an acyl-CoA, is catalyzed by acyl-CoA carboxylases (ACCases) such as acetyl-CoA 12 carboxylase (ACC) and propionyl-CoA carboxylase (PCC). ACC and PCC in Streptomyces coelicolor are 13 homologue multisubunit complexes that can carboxylate different short chain acyl-CoAs. While ACC is able 14 to carboxylate acetyl-, propionyl-, or butyryl-CoA with approximately the same specificity, PCC only 15 recognizes propionyl- and butyryl-CoA as substrates. How ACC and PCC have such different specificities 16 toward these substrates is only partially understood. To further understand the molecular basis of how the 17 active site residues can modulate the substrate recognition, we mutated D422, N80, R456, and R457 of PccB, 18 the catalytic beta subunit of PCC. The crystal structures of six PccB mutants and the wild type crystal 19 structure were compared systematically to establish the sequence-structure-function relationship that 20 correlates the observed substrate specificity toward acetyl-, propionyl-, and butyryl-CoA with active site 21 geometry. The experimental data confirmed that D422 is a key determinant of substrate specificity, 22 influencing not only the active site properties but further altering protein stability and causing long-range 23 conformational changes. Mutations of N80, R456, and R457 lead to variations in the quaternary structure of 24 the beta subunit and to a concomitant loss of enzyme activity, indicating the importance of these residues in 25 maintaining the active protein conformation as well as a critical role in substrate binding