Msed_0406 (A4YDT1, HBCL1_METS5) notes

Automated deep research was unavailable for this review. The notes below come from
my own reading of the cached full texts (PMID:23258541, PMID:24532060), the UniProt
record, PubMed searches (E-utilities: "Msed_0406", "Msed_0394",
"Metallosphaera sedula 4-hydroxybutyrate CoA ligase") and the RCSB PDB entry 8BIQ.
No -deep-research-*.md file was created.

Identity

• 564 aa class I adenylate-forming (ANL superfamily) acyl-CoA synthetase; Pfam
  PF00501 + PF13193; PANTHER PTHR43605:SF10 ("acyl-CoA synthetase medium chain family
  member 3"), CDD cd05972 MACS_like.
• Paralog Msed_0394 (A4YDR9, HBCL2). Msed_1353 is the conserved Sulfolobales
  acetate/propionate-CoA synthetase; Msed_1456 is the 3-hydroxypropionyl-CoA
  synthetase of the same cycle.
• Crystal structures 8BIQ (with acetyl-AMP, 2.8 A, four chains) and 8BIT; primary
  citation Capra et al. 2024 Front Catal 4 (doi:10.3389/fctls.2024.1360129), an
  engineering paper on lactam formation. No PMID was found for it, so it is not cited
  as a reference.

Biochemistry (Hawkins et al. 2013)

• Identified as the missing 4HB-CoA ligase of the 3HP/4HB cycle
  PMID:23258541; product confirmed by HPLC PMID:23258541
• Paralog comparison: similar Km for 4HB, different rates PMID:23258541. Conclusion:
  PMID:23258541 and PMID:23258541
• Substrate profile of Msed_0406: kcat/Km propionate 43,000 > butyrate 26,000 >
  valerate 7,500 > acetate/3HP 3,200 > 4HB 910 M-1 s-1 PMID:23258541; the authors call it PMID:23258541. Substrate range is C2-C5 only
  PMID:23258541
• Msed_0394 (HBCL2) prefers butyrate (kcat/Km 3,700) and valerate; 4HB is its
  poorest substrate (160) and Vmax on 4HB is ~8x lower than Msed_0406.
• Both genes are constitutively expressed (<2-fold change autotrophy vs heterotrophy),
  but in the top quartile of the transcriptome PMID:23258541.
• Open question raised by the authors PMID:23258541. No knockout exists.
• 4HB has no other known role in crenarchaeal metabolism PMID:23258541.

Pathway reconstitution (Hawkins et al. 2014)

• Msed_0406, Msed_1321, Msed_0399 and Msed_0656 were combined in vitro and converted
  4HB to acetyl-CoA PMID:24532060.
• Msed_0394 is dismissed for the cycle PMID:24532060
• The 2 mM Km for 4HB makes this a rate-limiting step PMID:24532060

Pathway context

• Cycle discovery PMID:18079405
• In the cycle, propionyl-CoA is made from 3-hydroxypropionyl-CoA (dehydratase +
  acryloyl-CoA reductase), not from free propionate, and acetyl-CoA is regenerated by
  thiolysis. So the acetate and propionate activities are not cycle steps. They are
  real in-vitro activities with no demonstrated physiological role.

GO assessment

• Physiological activity: 4-hydroxybutyrate-CoA ligase (EC 6.2.1.40, RHEA:23128). GO
  has no term (OLS/QuickGO searches for "4-hydroxybutyrate", "hydroxybutyrate-CoA
  ligase", "4-hydroxybutanoyl-CoA" returned none). Precedent: GO:0043955
  3-hydroxypropionyl-CoA synthetase activity exists for the neighbouring Msed_1456
  step. Propose a new term.
• GO:0031956 medium-chain fatty acid-CoA ligase activity is defined for C6-C12 acids.
  The IDA and IEA rows came from EC 6.2.1.2 / RHEA:48340 (butyrate, valerate). Msed_0406
  has no measured activity on C6 or longer, so GO:0031955 short-chain fatty acid-CoA
  ligase activity (fewer than 6 C; parent of GO:0050218) is the correct term. This is
  an EC-to-GO naming mismatch: EC 6.2.1.2 is named "butanoate--CoA ligase" /
  medium-chain acyl-CoA ligase with a C4-C11 range, whereas GO medium-chain means C6-C12.
• GO:0004321 fatty-acyl-CoA synthase activity is the fatty acid synthase reaction
  (EC 2.3.1.86, acetyl-CoA + malonyl-CoA + NAD(P)H), not an acyl-CoA synthetase.
  "Synthase" vs "synthetase" mix-up in the PANTHER TreeGrafter node (PTN008683229).
  Remove.
• GO:0006633 fatty acid biosynthetic process: archaeal membranes are isoprenoid ether
  lipids. The enzyme activates short acids; it plays no part in fatty acid synthesis.
  Mammalian ACSM-seeded TreeGrafter propagation. Remove.
• GO:0016020 membrane: from a predicted TM helix (105-125), inside the N-terminal ANL
  domain core. The enzyme was purified from the soluble fraction of E. coli lysates and
  crystallised without a membrane. Remove.
• Process: NEW GO:0015977 carbon fixation (as in Msed_0709); the enzyme catalyses a
  cycle step, so it passes the participation test. Reuse the proposed term name
  "carbon fixation by 3-hydroxypropionate/4-hydroxybutyrate cycle". GO:0043427 is the
  Chloroflexus glyoxylate-releasing bicycle, so it is not used.