Crystal structures and mechanisms of two enzymes involved in the degradation of tyrosine and biphenyl

Jiyuan Ke, Purdue University

Abstract

Homogentisate 1,2-dioxygenase (HGO), the third enzyme in tyrosine degradation, is an extradiol-type dioxygenase that utilizes nonheme Fe(II) and dioxygen to catalyze the ring cleavage of homogentisate (HGA). Its chemical mechanism may be studied by structural and biochemical approaches. Crystal structures of anaerobically prepared human HGO and the HGO:HGA complex have been determined to 1.6 and 1.8 Å resolution. The structure of the HGO:HGA complex shows that the substrate binds at the active site in a mode that differs from that predicted by a prior modeling study. Based on the structure, a revised mechanism is proposed. HGO is a potential target of inhibition to treat hepatorenal tyrosinemia type 1 (HT1). Biochemical data indicated that 3-Cl HGA is an effective inhibitor of human HGO and the structure of HGO:3-Cl HGA complex has been determined to 1.7 Å resolution to understand the inhibition. 3-Cl HGA binds differently than the substrate, which suggests that the slow catalysis may be due to the predominant non-productive binding mode in the crystal structure. The activity of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) hydrolase (BphD), a C-C bond hydrolase, is a key determinant in the aerobic transformation of polychlorinated biphenyls (PCBs). The failure of the bph pathway to efficiently process PCBs and PCB metabolites limits strategies for bioremediation. We study mechanisms of BphD catalysis and catalytic failure using structural and biochemical approaches. Crystal structures of BphD LB400 (BphD from Burkholderia strain LB400), its S112C mutant, the S112C:HOPDA complex, and S112A mutant have been determined at 1.6 Å resolution. BphDLB400 is a tetramer. Each monomer has a typical α/β hydrolase fold and is divisible into core and lid domains. The active site is located between the two domains and consists of polar and non-polar parts including a catalytic triad, S112-H265-D237. The mechanism of C-C bond hydrolases is a subject of debate. Recent biochemical evidence favors a mechanism that generates a gem-diol intermediate following base-catalyzed attack by water. In the S112C:HOPDA structure, C112 covalently binds HOPDA at the C6 position, forming a complex that resembles a tetrahedral intermediate consistent with a protein-nucleophile mechanism.

Degree

Ph.D.

Advisors

Bolin, Purdue University.

Subject Area

Molecular biology

COinS