Biotin is an essential vitamin in plants and mammals functioning as the carbon
dioxide carrier within central lipid metabolism. Biotin is composed of a fused bicylic
ring system and a five carbon, carboxylic acid chain. Biotin biosynthesis in bacteria is
catalysed by a series of enzymes that use fatty acid, amino acid and sulfur-containing
substrates. In Bacillus subtilis, pimeloyl-CoA synthetase (PCAS, EC 6.2.1.14,
UNIPROT code: P53559, 29.6 kDa) is the first enzyme in the biotin biosynthetic
pathway and acts as a highly specific substrate selection gate ensuring the integrity of
the carbon chain in biotin synthesis. PCAS catalyses the synthesis of the key
acyl-thioester, pimeloyl-CoA in two steps; the first involves activation of pimelic acid
(C7 dicarboxylic acid) using ATP to give an acyl-adenylate, enzyme-bound
intermediate and pyrophosphate (PPi), and in the second step, this pimeloyl-adenylate
reacts with coenzyme A (CoASH) to form the pimeloyl-CoA thioester. This thesis
describes the results of biochemical, structural and mechanistic studies of B. subtilis
PCAS. Recombinant PCAS was prepared by expressing the B. subtilis BioW gene in
E. coli in various hexa-histidine affinity-tagged forms and the enzyme purified in high
purity and yield. Enzyme activity and kinetic constants were measured using
reverse-phase HPLC and enzyme coupled spectroscopic assays. These revealed the
enzyme to have a strict carboxylic acid specificity. In collaboration with colleagues at
the University of St. Andrews various commercial and in-house screens were used to
obtain diffraction-quality crystals suitable for X-ray crystallography. This also
included the generation of seleno-methionine (SeMet) labelled PCAS, as well as
heavy-metal derivatives. Structures of B. subtilis PCAS in complex with the substrate
pimelic acid and the pimeloyl-adenylate intermediate and product PPi were
determined at 2.04 Å and 2.34 Å resolution respectively. The B. subtilis PCAS
displays a novel 3D fold and defines a new class (Class IV) in the ANL superfamily
of adenylate forming enzymes. The enzyme is a homodimer composed of two
domains, a short N-terminus and a large C-terminal domain and the ligand-bound
structures revealed the residues potentially involved in substrate specificity and
enzyme catalysis. The enzyme uses an internal ruler composed of a number of
conserved arginine residues (Arg213, Arg227 and Arg170) to select the correct
dicarboxylic acid substrate. The X-ray structures guided the production of a number
of site directed mutants to identify residues involved in the catalytic mechanism and
stabilising the acyl-adenylate intermediate. This also allowed rational engineering of
the PCAS active site to generate mutants with altered substrate specificity. Mutant
PCAS Y211F was shown to synthesise both heptanoyl (C7) and octanoyl (C8) mono
carboxylic acid-CoA and C8 dicarboxylic-CoA thioester products, highlighting the
synthetic potential of PCAS.
The PCAS pimeloyl-CoA product is the substrate for the next enzyme in the biotin
pathway, a pyridoxal 5‟phosphate (PLP)-dependent 8-amino 7-oxononanoate synthase
(AONS). AONS catalyses the condensation of pimeloyl-CoA with L-alanine to give
AON which is converted to biotin by the action of three other enzymes. We used
genome mining to identify a putative ~66 kDa, bi-functional PCAS/AONS enzyme
with an N-terminal PCAS domain fused to C-terminal AONS domain in the organism
Corynebacterium amycolatum. A recombinant C. amycolatum PCAS/AONS fusion
protein was expressed and purified from E. coli and initial studies suggest that it
forms a functional, fused, dimeric enzyme
