Tambjamine YP1 is a pyrrole-containing natural product.
Analysis
of the enzymes encoded in the Pseudoalteromonas tunicata “tam” biosynthetic gene cluster (BGC)
identified a unique di-domain biocatalyst (PtTamH).
Sequence and bioinformatic analysis predicts that PtTamH comprises an N-terminal, pyridoxal 5′-phosphate (PLP)-dependent
transaminase (TA) domain fused to a NADH-dependent C-terminal thioester
reductase (TR) domain. Spectroscopic and chemical analysis revealed
that the TA domain binds PLP, utilizes l-Glu as an amine
donor, accepts a range of fatty aldehydes (C7–C14 with a preference for C12), and produces the
corresponding amines. The previously characterized PtTamA from the “tam” BGC is an ATP-dependent, di-domain
enzyme comprising a class I adenylation domain fused to an acyl carrier
protein (ACP). Since recombinant PtTamA catalyzes
the activation and thioesterification of C12 acid to the holo-ACP domain, we hypothesized that C12 ACP
is the natural substrate for PtTamH. PtTamA and PtTamH were successfully coupled together
in a biocatalytic cascade that converts fatty acids (FAs) to amines
in one pot. Moreover, a structural model of PtTamH
provides insights into how the TA and TR domains are organized. This
work not only characterizes the formation of the tambjamine YP1 tail
but also suggests that PtTamA and PtTamH could be useful biocatalysts for FA to amine functional group
conversion.
Enzymatic deficiencies cause the accumulation of toxic levels of substrates in a cell and are associated with life-threatening pathologies. Restoring physiological enzymes levels by injecting a recombinant version of the defective enzyme could provide a viable therapeutic option. However, these enzyme replacement therapies have had limited success, as the recombinant enzymes are less catalytically active, cause immune response and are difficult to manufacture. Moreover, the vast sequence design space makes finding enzymes with desired therapeutic properties extremely challenging. Here, we present a new enzyme engineering framework, which builds on recent advances in deep learning, variational calculus and natural language processing, to design variants of human enzymes with biochemical features comparable to the wild type protein as a way to rapidly build targeted libraries for downstream screening. We applied our method to design variants of human Sphyngosine-1-phosphate lyase (HsS1PL) as potential therapeutic treatments for nephrotic syndrome type 14 (NPHS14), and characterized their biochemical properties through extensive sequence and molecular dynamics analyses.
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