Catalytic and structural properties of ATP‐dependent caprolactamase from Pseudomonas jessenii

Abstract

Caprolactamase is the first enzyme in the caprolactam degradation pathway of Pseudomonas jessenii. It is composed of two subunits (CapA and CapB) and sequence‐related to other ATP‐dependent enzymes involved in lactam hydrolysis, like 5‐oxoprolinases and hydantoinases. Low sequence similarity also exists with ATP‐dependent acetone‐ and acetophenone carboxylases. The caprolactamase was produced in Escherichia coli, isolated by His‐tag affinity chromatography, and subjected to functional and structural studies. Activity toward caprolactam required ATP and was dependent on the presence of bicarbonate in the assay buffer. The hydrolysis product was identified as 6‐aminocaproic acid. Quantum mechanical modeling indicated that the hydrolysis of caprolactam was highly disfavored (ΔG0'= 23 kJ/mol), which explained the ATP dependence. A crystal structure showed that the enzyme exists as an (αβ)2 tetramer and revealed an ATP‐binding site in CapA and a Zn‐coordinating site in CapB. Mutations in the ATP‐binding site of CapA (D11A and D295A) significantly reduced product formation. Mutants with substitutions in the metal binding site of CapB (D41A, H99A, D101A, and H124A) were inactive and less thermostable than the wild‐type enzyme. These residues proved to be essential for activity and on basis of the experimental findings we propose possible mechanisms for ATP‐dependent lactam hydrolysis.

Interactome analysis identifies MSMEI_3879 as a substrate ofMycolicibacterium smegmatisClpC1

“…The MSMEI_3879 locus encodes a 446 amino acid product with homology to ATP-hydrolyzing hydantoinase/oxoprolinase enzymes, which catalyze ring-opening reactions on lactam substrates (76–78). Orthologs with >90% sequence identity to MSMEI_3879 are present in members of the genus Mycolicibacterium , but absent in other groups of the family Mycobacteriaceae , including M. tuberculosis (79).…”

Mild hydrolysis of chemically stable valerolactams by a biocatalytic ATP-dependent system fueled by metaphosphate

“…The heterologous over-expression of both proteins carrying a N-terminal His-tag in E. coli BL21 (DE3) in lysogeny Fig. 1 State-of-the art in the biohydrolysis of lactams and lactam-like compounds: (A) cyclic amide-bond containing substrates cleaved by hydrolytic enzymes; 7,15,16 (B) ATP-dependent hydrolysis of ε-caprolactam by PjCapAB; 18,19 (C) hydrolysis of oxoproline by ATP-dependent oxoprolinase. 20 broth under isopropyl β-D-1-thiogalactopyranoside (IPTG) induction at 30 °C resulted in poor soluble expression, as was the case in trials at lower temperatures (20 °C, data not shown), despite good overall over-expression (see ESI, Fig.…”

“…Since the presence of bicarbonate was found necessary for the formation of the product, and highest velocity was obtained in the presence of a large excess of bicarbonate (50 mM), bicarbonate was postulated to be involved in the reaction mechanism through the formation of a carboxyphosphate intermediate from ATP. 19 This hypothesis was derived from analogy with related enzymes acetone and acetophenone carboxylases. ATPdependent phosphorylation of bicarbonate is indeed a vital process in a number of biological processes, through which carboxyphosphate can transfer the carboxy group to a variety of acceptors, including acetone and acetophenone, 21,22 or ammonia.…”

In silico prediction of the enzymes involved in the degradation of the herbicide molinate by Gulosibacter molinativorax ON4T

“…BS4, were described as capable of caprolactam degradation 56,[68][69][70] . Among these organisms, only the enzyme acting upon the cleavage of caprolactam in Pseudomonas jessenii GO3 was described 56,71 . This caprolactamase, which consists of two subunits with high sequence identity to 5-oxoprolinases, hydrolyzes caprolactam into 6-aminohexanoic acid in an ATP-dependent manner 56,71 .…”

“…Among these organisms, only the enzyme acting upon the cleavage of caprolactam in Pseudomonas jessenii GO3 was described 56,71 . This caprolactamase, which consists of two subunits with high sequence identity to 5-oxoprolinases, hydrolyzes caprolactam into 6-aminohexanoic acid in an ATP-dependent manner 56,71 . Such information suggested the possible involvement of the GMOLON4_3203 and GMOLON4_3204 gene products in the breakdown of caprolactam by strain ON4 T .…”