OleB from Bacterial Hydrocarbon Biosynthesis Is a
β‑Lactone Decarboxylase That Shares Key Features with
Haloalkane Dehalogenases
James
K. Christenson
Serina L. Robinson
Tiffany A. Engel
Jack E. Richman
An N. Kim
Larry P. Wackett
10.1021/acs.biochem.7b00667.s001
https://acs.figshare.com/articles/journal_contribution/OleB_from_Bacterial_Hydrocarbon_Biosynthesis_Is_a_Lactone_Decarboxylase_That_Shares_Key_Features_with_Haloalkane_Dehalogenases/5421670
OleB
is an α/β-hydrolase found in bacteria that biosynthesize
long-chain olefinic hydrocarbons, but its function has remained obscure.
We report that OleB from the Gram-negative bacterium <i>Xanthomonas
campestris</i> performs an unprecedented β-lactone decarboxylation
reaction, to complete <i>cis</i>-olefin biosynthesis. OleB
reactions monitored by <sup>1</sup>H nuclear magnetic resonance spectroscopy
revealed a selectivity for decarboxylating <i>cis</i>-β-lactones
and no discernible activity with <i>trans</i>-β-lactones,
consistent with the known configuration of pathway intermediates.
Protein sequence analyses showed OleB proteins were most related to
haloalkane dehalogenases (HLDs) and retained the canonical Asp-His-Asp
catalytic triad of HLDs. Unexpectedly, it was determined that an understudied
subfamily, denoted as HLD-III, is comprised mostly of OleB proteins
encoded within <i>oleABCD</i> gene clusters, suggesting
a misannotation. OleB from <i>X. campestris</i> showed very
low dehalogenase activity only against haloalkane substrates with
long alkyl chains. A haloalkane substrate mimic alkylated wild-type <i>X. campestris</i> OleB but not OleB<sub>D114A</sub>, implicating
this residue as the active site nucleophile as in HLDs. A sequence-divergent
OleB, found as part of a natural OleBC fusion and classified as an
HLD-III, from the Gram-positive bacterium <i>Micrococcus luteus</i> was demonstrated to have the same activity, stereochemical preference,
and dependence on the proposed Asp nucleophile. H<sub>2</sub><sup>18</sup>O studies with <i>M. luteus</i> OleBC suggested
that the canonical alkyl–enzyme intermediate of HLDs is hydrolyzed
differently by OleB enzymes, as <sup>18</sup>O is not incorporated
into the nucleophilic aspartic acid. This work defines a previously
unrecognized reaction in nature, functionally identifies some HLD-III
enzymes as β-lactone decarboxylases, and posits an enzymatic
mechanism of β-lactone decarboxylation.
2017-09-05 00:00:00
Protein sequence analyses
Gram-negative bacterium Xanthomonas campestris
alkylated wild-type X
nucleophilic aspartic acid
oleABCD gene clusters
OleB D 114A
Bacterial Hydrocarbon Biosynthesis
trans -β- lactones
Shares Key Features
OleB proteins
HLD-III
Gram-positive bacterium Micrococcus luteus
h 2 18 O studies
biosynthesize long-chain olefinic hydrocarbons
β- lactone decarboxylation reaction
decarboxylating cis -β- lactones
β- lactone decarboxylation
HLD
Haloalkane Dehalogenases OleB
β- lactone decarboxylases