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OleB from Bacterial Hydrocarbon Biosynthesis Is a β‑Lactone Decarboxylase That Shares Key Features with Haloalkane Dehalogenases
journal contribution
posted on 2017-09-05, 00:00 authored by James
K. Christenson, Serina L. Robinson, Tiffany A. Engel, Jack E. Richman, An N. Kim, Larry P. WackettOleB
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 Xanthomonas
campestris performs an unprecedented β-lactone decarboxylation
reaction, to complete cis-olefin biosynthesis. OleB
reactions monitored by 1H nuclear magnetic resonance spectroscopy
revealed a selectivity for decarboxylating cis-β-lactones
and no discernible activity with trans-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 oleABCD gene clusters, suggesting
a misannotation. OleB from X. campestris showed very
low dehalogenase activity only against haloalkane substrates with
long alkyl chains. A haloalkane substrate mimic alkylated wild-type X. campestris OleB but not OleBD114A, 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 Micrococcus luteus was demonstrated to have the same activity, stereochemical preference,
and dependence on the proposed Asp nucleophile. H218O studies with M. luteus OleBC suggested
that the canonical alkyl–enzyme intermediate of HLDs is hydrolyzed
differently by OleB enzymes, as 18O 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.
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Protein sequence analysesGram-negative bacterium Xanthomonas campestrisalkylated wild-type Xnucleophilic aspartic acidoleABCD gene clustersOleB D 114ABacterial Hydrocarbon Biosynthesistrans -β- lactonesShares Key FeaturesOleB proteinsHLD-IIIGram-positive bacterium Micrococcus luteush 2 18 O studiesbiosynthesize long-chain olefinic hydrocarbonsβ- lactone decarboxylation reactiondecarboxylating cis -β- lactonesβ- lactone decarboxylationHLDHaloalkane Dehalogenases OleBβ- lactone decarboxylases
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