Crystal structures of a meta‐cleavage product hydrolase from Pseudomonas fluorescens IP01 (CumD) complexed with cleavage products

Abstract

Abstract2-Hydroxy-6-oxo-7-methylocta-2,4-dienoate hydrolase (CumD) from Pseudomonas fluorescens IP01 hydrolyzes a meta-cleavage product generated in the cumene (isopropylbenzene) degradation pathway. The crystal structures of the inactive S103A mutant of the CumD enzyme complexed with isobutyrate and acetate ions were determined at 1.6 and 2.0 Å resolution, respectively. The isobutyrate and acetate ions were located at the same position in the active site, and occupied the site for a part of the hydrolysis product with CumD, which has the key determinant group for the substrate specificity of related hydrolases. One of the oxygen atoms of the carboxyl group of the isobutyrate ion was hydrogen bonded with a water molecule and His252. Another oxygen atom of the carboxyl group was situated in an oxyanion hole formed by the two main-chain N atoms. The isopropyl group of the isobutyric acid was recognized by the side-chains of the hydrophobic residues. The substrate-binding pocket of CumD was long, and the inhibition constants of various organic acids corresponded well to it. In comparison with the structure of BphD from Rhodococcus sp. RHA1, the structural basis for the substrate specificity of related hydrolases, is revealed.

A Product Analog Bound Form of 3-Oxoadipate-enol-Lactonase (PcaD) Reveals a Multifunctional Role for the Divergent Cap Domain

“…The role of the cap domain in promoting oligomerization is consistent with previous observations among members of the α/β-hydrolase (ABH) fold family, yet the size of the cap domain varies considerably and those comprising fewer than four helices tend to result in monomeric enzymes. 23 In contrast, some ABH family members oligomerize through β-strands that form a topological antiparallel β-sheet [24][25][26] highlighting the diversity of structural solutions employed by this important family of enzymes in directing higher-order assembly.…”

Structure and Action of a CC Bond Cleaving α/β-Hydrolase Involved in Nicotine Degration

“…12 The overall sequence similarity to the established C-C bond cleaving hydrolases of the meta-cleavage pathway of aromatic compounds, such as DmpD, 13 TodF, 14 BphD, 11,[15][16][17] XylF, 18 CumD, 19,20 CarC 21 and MhpC 22 was insignificant. The known structures of BphD, 11,17 CumD, 19,20 CarC 21 and MhpC 22 demonstrate, however, that they belong to the α/β-hydrolase family and are therefore distantly related to the DHPON hydrolase. In contrast to the oligomerization by β-sheet extension in the other C-C bond cleaving α/β-hydrolases, DHPON hydrolase dimerizes via the N-terminal domains (Figure 2(a)).…”

Biochemical Evidence That phaZ Gene Encodes a Specific Intracellular Medium Chain Length Polyhydroxyalkanoate Depolymerase in Pseudomonas putida KT2442

“…The binding surface of the core domain is highly hydrophobic, and the only polar residues surrounding the substrate are the catalytic triad (Fig. 8D) and Asn 35 , which may take part in substrate recognition as described for the analogous Ser 34 in CumD (46). Finally, it should be pointed out that the side chain of the only cysteine residue in PhaZ (Cys 119 ) is buried and facing off solvent, so that the possibility of disulfide bridge formation is reduced, a result that is in agreement with the lack of effect of reducing reagents on the enzymatic activity of PhaZ (Table 2).…”